WO2025165945A1 - Compositions et procédés pour détecter un état de rejet de greffe et le traiter - Google Patents

Compositions et procédés pour détecter un état de rejet de greffe et le traiter

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WO2025165945A1
WO2025165945A1 PCT/US2025/013716 US2025013716W WO2025165945A1 WO 2025165945 A1 WO2025165945 A1 WO 2025165945A1 US 2025013716 W US2025013716 W US 2025013716W WO 2025165945 A1 WO2025165945 A1 WO 2025165945A1
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expression pattern
rejection
detected
expression
igd
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David Mark ROTHSTEIN
Aravind Cherukuri
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University of Pittsburgh
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University of Pittsburgh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/10—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1488—Methods for deciding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • Hie present disclosure relates to compositions and methods for the detection and treatment of transplant rejection.
  • biomarkers have been developed mostly based on transcript expression of various genes in peripheral blood or allograft-derived cell free DNA. Unfortunately, almost all have been tested in select groups of patients where confounding diagnoses like BK infection, that also lead to renal inflammation, have been excluded. Some biomarker studies have only examined patients retrospectively with clinical AR whereas, few have examined patients with subclinical rejection. In these select patient groups, the commercially available biomarkers uniformly have reasonably high negative predictive values of 80-86% with one study reporting 98%. This is expected given the relatively low prevalence of AR on biopsy. However, accuracy is relatively poor for the most common type of mild AR (Banff 1A) and improves for more severe rejection, which is unlikely to be clinically silent. None have good positive predictive values - especially for surveillance biopsies (13-48%). Thus, currently available biomarkers are not useful in identifying patients who have mild or subclinical AR.
  • biomarkers of transplant rejection that can be used in methods for detection and/or treatment of such rejection, including mild and subclinical AR.
  • a method of detecting rejection status of a transplant in a subject comprising obtaining a sample comprising B cells from the subject, detecting an expression pattern of the B cells, comparing the detected expression pattern to a control expression pattern; wherein a transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control no rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control rejection expression pattern and/or wherein a lack of transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control no rejection expression pattern.
  • the B cells are identified by detecting an expression of a B cell identification marker.
  • the B cell identification marker is selected from a group consisting of CD19, CD20, CD79alpha, CD79beta, FcRL5, FcRL4, CD138, and B cell receptor or a component of the B cell receptor complex. In some embodiments, the B cell identification marker is CD 19.
  • the expression pattern comprises expression data for two or more of thirteen polypeptides, or polynucleotides encoding the two or more of thirteen polypeptides, and wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3.
  • the detected expression pattern consists of expression data for a B cell identification marker, CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the detected expression pattern comprises expression data for two or more of twenty-one polypeptides, or polynucleotides encoding the two or more of twenty-one polypeptides, and wherein the twenty -one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the detected expression pattern consists of expression data for a B cell identification marker, TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the detected expression pattern comprises expression data for CD19, CD38, and CD24. In some embodiments, the detected expression pattern comprises expression data for CD19, CD27, and CD21. In some embodiments, the detected expression pattern comprises expression data for CD19, CD38, and CD73. In some embodiments, the detected expression pattern comprises expression data for CD19, CD38, and CD23. In some embodiments, the detected expression pattern comprises expression data for CD19, CD24, and CD73. In some embodiments, the detected expression pattern comprises expression data for CD19, CD24, and CD21. In some embodiments, the detected expression pattern comprises expression data for CD19, CD25, and IgD. In some embodiments, the detected expression pattern comprises expression data for CD19, CD73, and IgM.
  • the detected expression pattern comprises expression data for CD 19, CD73, and IgD. In some embodiments, the detected expression pattern comprises expression data for CD19, CD39, and CD25. In some embodiments, the detected expression pattern comprises expression data for CD 19, CD39, and CD73. In some embodiments, the detected expression pattern comprises expression data for CD19, CD73, and LAG3. In some embodiments, the detected expression pattern comprises expression data for CD19, CD73, and CD10. In some embodiments, the detected expression pattern comprises expression data for CD19, CD23, and CD73. In some embodiments, the detected expression pattern comprises expression data for CD19, CD21, and CD9.
  • the detection expression pattern comprises CD19 bnght , CD39 dim , CD80 dim , CD23 dim , CD73 neg , IgM dim , CD21 neg , CD27 neg , CD24 pos , IgD dim and CD38 neg .
  • the detection expression pattern comprises CD19 intennediate , CD39 bngbt , CD80 neg , HLA-II bright , CD23 bright , CD73 bright , IgM dim , CD2 intermedia,e , CD27 neg , CD24 dim , IgD intermedia,e , CD38 dim , and CD9 dim .
  • the expression pattern is a surface expression pattern.
  • detecting the surface expression pattern comprises flow cytometry.
  • the detected expression pattern and the control expression pattern are obtained using a method comprising t-distributed stochastic neighbor embedding (t-SNE).
  • t-SNE t-distributed stochastic neighbor embedding
  • the statistically significant difference in the detected expression pattern and the control expression pattern is at least 0.05%. In some embodiments, the statistically significant difference in the detected expression pattern and the control expression pattern is at least 0.1%.
  • the transplant is a kidney, a liver, a lung, a heart, a pancreas, an intestine, multi-visceral, a uterus, a vascularized composite allograft, a pancreatic islet, a stem cell, or a neuronal cell.
  • the transplant is an organ.
  • the organ is a kidney.
  • the transplant rejection is indicated.
  • the method further comprises obtaining a biopsy of the transplant.
  • the transplant rejection is an acute transplant rejection.
  • the transplant rejection is a subclinical transplant rejection.
  • the transplant rejection is a clinical transplant rejection.
  • the subject is a human.
  • the sample is a blood sample.
  • the method further comprising administering to the subject a treatment for the transplant rejection.
  • the treatment is an immunosuppressive therapy.
  • kits for detection of rejection status of a transplant in a subject wherein the kit is used, to obtain a sample comprising B cells from the subject; and to detect an expression pattern of the B cells; and comparing the detected expression pattern to a control expression pattern; wherein a transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control no rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control rejection expression pattern and/or wherein a lack of transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control no rejection expression pattern.
  • the B cells are identified by detecting expression of a B cell identification marker.
  • the B cell identification marker is selected from a group consisting of CD19, CD20, CD79alpha, CD79beta, FcRL5, FcRL4, CD138, and B cell receptor or a fragment thereof.
  • the B cell identification marker is CD 19.
  • the detected expression pattern comprises expression data for two or more of thirteen polypeptides, or polynucleotides encoding the two or more of thirteen polypeptides, and wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the detected expression pattern comprises expression data for two or more of twenty-one polypeptides, or polynucleotides encoding the two or more of twenty-one polypeptides, and wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the kit comprises an antibody or ligand specific for a B cell identification marker, TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and/or CD80.
  • the kit comprises an antibody or ligand specific for B cell identification marker, CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10 and/or LAG3.
  • Figure 1 shows representative flow cytometry plots of human peripheral blood mononuclear cells (PBMC), identifying B cells for subsequent t-SNE analysis.
  • PBMC peripheral blood mononuclear cells
  • CD19 is used as a “pan-B cell” marker to identify all B cells.
  • CD3 is a T cells marker used to exclude T cells helping to distinguish B cells for the analysis.
  • FIG. 2 shows concatenated t-distributed stochastic neighbor embedding (t-SNE) analysis of B cells data from 28 renal transplant patients undergoing surveillance biopsies based on simultaneous analysis of B cells using a pan-B cell marker such as CD 19, plus the following 21 additional markers expressed on the surface of subpopulations of B cells: TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • t-SNE plot individual B cells fall into regions based on similarities in their relative distribution of these markers.
  • the subpopulation regions labeled G1-G7 were determined empirically.
  • the Left panel shows the t-SNE analysis for all patients.
  • the center panel shows the t-SNE analysis from the 14 patients whose biopsies showed no rejection (NR).
  • the Right panel shows the t-SNE analysis from the 14 patients whose biopsies showed revealed subclinical acute rejection (AR).
  • Figure 3A is a bar graph showing the percentage of B cells in the G7 region identified in Figure 2, divided into patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in G7 between NR and AR patients is highly significant.
  • Figure 3B shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in G7 predicting the presence of acute rejection on biopsy.
  • the area under the curve (AUC) is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.89 with a confidence interval (CI) of 0.75-1.0.
  • an optimal cut-off for differentiating between AR and NR can be set at 15.5% (shown in Fig 3A).
  • the percent of cells in G7 in any given patient predicts acute rejection with a sensitivity (Sens) of 93%, a specificity (Spec) of 86%, and with a positive predictive value (PPV) of 87% and a negative predictive value (NPV) of 92%.
  • Figure 4A is a bar graph showing the percentage of B cells in the G5 region identified in Figure 2, in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/triangles. The differences in percent of cells in G5 between NR and AR patients is highly significant.
  • Figure 4B shows a ROC curve for the percentage of cells in G5 predicting the lack of acute rejection on biopsy. The AUC for G5 for predicting no rejection is 0.85 with a Sensitivity of 86%, Specificity of 79%, PPV 80% and NPV 85% at an optimized cut-off of 0.7% of B cells in G5 (Fig 3A).
  • Figure 5 shows relative expressions of various surface markers in each of the B cell subpopulations (G1-G7) identified in Figure 2, left panel (total patient population), as visualized on a Heatmap map.
  • Figure 6 shows t-SNE analysis of CD19 plus the 21 B cell surface markers for 28 patients as discussed in relation to Figure 2 but having different subpopulation regions assigned than in Figure 2.
  • the NR is no rejection.
  • AR is subclinical acute rejection.
  • the percentages refer to the average percent of B cells in each region in AR and NR patients.
  • Figure 7 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD19 plus one additional marker from the panel of 21 B cell surface markers (CD19 + CD24).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 8(A-O) shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD19 + two B cell surface markers CD19 + CD38 + CD24 as shown in Figure 8A, CD19 + CD27 + CD21 as shown in Figure 8B, CD19 + CD38 + CD73 as shown in Figure 8C, CD19 + CD38 + CD23 as shown in Figure 8D, CD 19 + CD24 + CD73 as shown in Figure 8E, CD 19 + CD24 + CD21 as shown in Figure 8F, CD19 + CD25 + IgD as shown in Figure 8G, CD19 + CD73 + IgM as shown in Figure 8H, CD19 + CD73 + IgD as shown in Figure 81, CD19 + CD39 + CD25 as shown in Figure 8J, CD19 + CD39 + CD73 as shown in Figure 8K, CD19 + CD73 + LAG3 as shown in Figure 8L, CD19 + CD73 + CD10 as shown in Figure 8M, CD19 + CD23 + CD73
  • Figure 9 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + three B cell surface markers (CD19 + CD24 + CD38 + CD27).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 10 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + four B cell surface markers (CD19 + CD24 + CD38 +CD27 + CD21).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 11 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + five B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 12 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + six B cell surface markers (CD19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 13 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + seven B cell surface markers (CD19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 14 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + eight B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 15 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + nine B cell surface markers (CD19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25 + CD9).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 16 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + ten B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25 + CD9 + IgD).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 17 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + eleven B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25 + CD9 + IgD + IgM).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 18 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + twelve B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25 + CD9 + IgD + IgM + CD10).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 19 shows t-SNE analysis of B cell data from the 28 renal transplant patients discussed in relation to Figure 2 based on simultaneous analysis of CD 19 + thirteen B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25 + CD9 + IgD + IgM + CD10 + EAG3).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 20A is a bar graph showing the percentage of B cells in the Pl region of Figure 19 in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in Pl between NR and AR patients is highly significant.
  • Figure 20B shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in Pl predicting the presence of acute rejection on biopsy.
  • the area under the curve (AUC) is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.89 with a confidence interval (CI) of 0.76-1.0.
  • FIG. 21 A is a bar graph showing the percentage of B cells in the P3 region of Figure 19 in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in P3 between NR and AR patients is highly significant.
  • Figure 21B shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in P3 predicting the presence of acute rejection on biopsy.
  • the area under the curve (AUC) is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • AUC is 0.94 with a confidence interval (CI) of 0.87-1.0. From this curve an optimal cut-off for differentiating between AR and NR can be set at 31.34% (shown in Fig 21 A).
  • the percent of cells in Pl in any given patient predicts acute rejection with a sensitivity (Sens) of 86%, a specificity (Spec) of 93%, and with a positive predictive value (PPV) of 92% and a negative predictive value (NPV) of 87%.
  • Figure 22A is a bar graph showing the percentage of B cells in the P4 region of Figure 19 in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in P4 between NR and AR patients is highly significant.
  • Figure 22B shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in P4 predicting the presence of acute rejection on biopsy.
  • the area under the curve (AUC) is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.88 with a confidence interval (CI) of 0.74-1.0.
  • an optimal cut-off for differentiating between AR and NR can be set at 17.74% (shown in Fig 22A).
  • the percent of cells in P4 in any given patient predicts acute rejection with a sensitivity (Sens) of 93%, a specificity (Spec) of 86%, and with a positive predictive value (PPV) of 87% and a negative predictive value (NPV) of 92%.
  • Figure 23 shows relative expressions of various surface markers in each of the B cell subpopulations (P1-P5) shown in Figure 19 as visualized on a Heatmap map.
  • Figure 24(A-B) shows individual t-SNE analyses of B cell data from each of the individual 28 renal transplant patients discussed in relation to Figure 19 based on simultaneous analysis of fourteen B cell surface markers (CD 19 + CD24 + CD38 +CD27 + CD21 + CD39 + CD23 + CD73 + CD25 + CD9 + IgD + IgM + CD10 + LAG3).
  • A 14 patients with no rejection (NR).
  • B 14 patients with acute rejection (AR).
  • Figure 25A is a bar graph showing the percentage of B cells in the P2 region of Figure 19 in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in P2 between NR and AR patients is highly significant.
  • Figure 25B shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in P2 predicting the presence of acute rejection on biopsy.
  • the area under the curve (AUC) is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.82 with a confidence interval (CI) of 0.65-0.98.
  • an optimal cut-off for differentiating between AR and NR can be set at 2.59% (shown in Fig 25A).
  • the percent of cells in P2 in any given patient predicts acute rejection with a sensitivity (Sens) of 71%, a specificity (Spec) of 86%, and with a positive predictive value (PPV) of 83% and a negative predictive value (NPV) of 75%.
  • Figure 26A is a bar graph showing the percentage of B cells in the P5 region of Figure 19 in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in P5 between NR and AR patients is highly significant.
  • Figure 26B shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in P5 predicting the presence of acute rejection on biopsy.
  • the area under the curve (AUC) is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.90 with a confidence interval (CI) of 0.78-1.0.
  • an optimal cut-off for differentiating between AR and NR can be set at 12.6% (shown in Fig 26A).
  • the percent of cells in P5 in any given patient predicts acute rejection with a sensitivity (Sens) of 79%, a specificity (Spec) of 86%, and with a positive predictive value (PPV) of 85% and a negative predictive value (NPV) of 80%.
  • Figure 27A shows t-SNE analysis of B cell data from the 28 renal transplant patients based on simultaneous analysis of CD19 + two B cell surface markers (CD19 + CD24 + CD27).
  • NR is no rejection.
  • AR is subclinical acute rejection.
  • Figure 27B is a bar graph showing the percentage of B cells in the KI region of Figure 27 A in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in KI between NR and AR patients is highly significant.
  • Figure 27C shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs. specificity for the percentage of cells in KI predicting the presence of acute rejection on biopsy.
  • ROC Receiver Operator Characteristic
  • the area under the curve is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.73 with a confidence interval (CI) of 0.54- 0.92.
  • CI confidence interval
  • From this curve an optimal cut-off for differentiating between AR and NR can be set at 8.54% (shown in Fig 27B).
  • the percent of cells in KI in any given patient predicts acute rejection with a sensitivity (Sens) of 71%, a specificity (Spec) of 64%, and with a positive predictive value (PPV) of 57% and a negative predictive value (NPV) of 71%.
  • Figure 28A shows t-SNE analysis of B cell data from the 28 renal transplant patients based on simultaneous analysis of CD19 + two B cell surface markers (CD19 + CD24 + CD73). There are two subpopulation groups A1-A2. NR is no rejection. AR is subclinical acute rejection.
  • Figure 28B is a bar graph showing the percentage of B cells in the A2 region of Figure 28A in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in A2 between NR and AR patients is significant (at a cut-off of p ⁇ 0.1 for statistical significance).
  • Figure 28C shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs.
  • ROC Receiver Operator Characteristic
  • the area under the curve is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.70 with a confidence interval (CI) of 0.51-0.9.
  • CI confidence interval
  • From this curve an optimal cut-off for differentiating between AR and NR can be set at 20.84% (shown in Fig 28B).
  • the percent of cells in A2 in any given patient predicts acute rejection with a sensitivity (Sens) of 86%, a specificity (Spec) of 57%, and with a positive predictive value (PPV) of 73% and a negative predictive value (NPV) of 65%.
  • Figure 29A shows t-SNE analysis of B cell data from the 28 renal transplant patients based on simultaneous analysis of CD19 + two B cell surface markers (CD19 + CD21 + CD39). There are two subpopulation groups H1-H2. NR is no rejection. AR is subclinical acute rejection.
  • Figure 29B is a bar graph showing the percentage of B cells in the H2 region of Figure 29A in patients who have no rejection (NR) vs. those with acute rejection (AR). Each individual patient is represented by small black square/rhombus. The differences in percent of cells in H2 between NR and AR patients is significant (at a cut-off of p ⁇ 0. 1 for statistical significance).
  • Figure 29C shows a Receiver Operator Characteristic (ROC) curve plotting sensitivity vs.
  • ROC Receiver Operator Characteristic
  • the area under the curve is a measure of predictive value of the marker (with 1.0 being 100% sensitive and specific).
  • the AUC is 0.68 with a confidence interval (CI) of 0.48-0.89.
  • CI confidence interval
  • From this curve an optimal cut-off for differentiating between AR and NR can be set at 6.5% (shown in Fig 29B).
  • the percent of cells in H2 in any given patient predicts acute rejection with a sensitivity (Sens) of 71%, a specificity (Spec) of 71%, and with a positive predictive value (PPV) of 71% and a negative predictive value (NPV) of 71%.
  • Sens sensitivity
  • Spec specificity
  • PPV positive predictive value
  • NPV negative predictive value
  • a method of detecting rejection status of a transplant in a subject that includes obtaining a sample comprising B cells from the subject, detecting an expression pattern of the B cells, and comparing the detected expression pattern to a control no rejection expression pattern and/or a control rejection expression pattern, wherein a transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control no rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control rejection expression pattern.
  • lack of rejection is indicated by a statistically significant difference in the detected expression pattern and the control rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control no rejection expression pattern.
  • compositions such as kits for detecting rejection status of a transplant in a subject.
  • a cell includes a plurality of cells, including mixtures thereof.
  • administering refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra- articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
  • antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • antibody or “antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Antibodies may also be derived from mammals in the Camelidae family, such as camels, llamas, and alpacas. They are also known as single-domain antibodies (sdAbs) or nanobodies. Camelid antibodies are made up of two identical heavy chains, and lack light chains and the CHI region and are smaller than other antibodies.
  • antibody fragment refers to a portion of a full-length antibody, that includes the target, or antigen, binding or variable region.
  • antibody fragments include Fab, Fab', F(ab')2 and Fv fragments.
  • the “antibody fragment” is a compound having qualitative biological activity in common with a full-length antibody.
  • antibody fragment with respect to antibodies, includes Fv, F(ab) and F(ab’)2 fragments.
  • An “Fv” fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer).
  • variable domains interact to define a target binding site on the surface of the VH-VL dimer.
  • the six CDRs confer target binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for a target
  • Single-chain Fv or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding.
  • the Fab fragment contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • B cell refers to what is known in the art as a “B lymphocyte” and is a type of white blood cell.
  • the B cell is a plasma cell.
  • the B cell is a memory B cell.
  • the B cell is a regulatory B cell.
  • the B cell is identified as a B cell based on its expression of CD19.
  • the B cell is identified as a B cell based on other markers including, but not limited to: CD20; CD79alpha, CD79beta, the B cell receptor, FcRL5, FcRL4, CD138, signaling molecules specific for B cells amongst lymphocytes (e.g.
  • B cells can be identified prior to, simultaneously with, or after detecting the B cell expression pattern. In some embodiments, the B cell is identified as a B cell prior to detecting the B cell expression pattern
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • the term “expression” refers to either or both “gene expression” and “protein expression.” “Gene expression” refers to the process by which polynucleotides are transcribed into mRNA and “protein expression” refers to the process by which mRNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. “Gene overexpression” refers to the overproduction of the mRNA transcribed from the gene, at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample.
  • Protein overexpression includes the overproduction of the protein product encoded by a gene at a level that is at least about 1.5 times higher, at lease about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample.
  • surface expression refers to the process by which polypeptides are translocated to the surface of a cell such that at least a portion of the polypeptide is located at the exterior of the cell surface.
  • “Surface overexpression” includes an increase in the amount of a particular polypeptide at the exterior surface of a cell, at a level that is at least 5% higher, 10% higher, 20% higher, 30% higher, 40% higher, 50% higher, 60% higher, 70% higher, 80% higher, 90% higher, 100% higher, 1.5 times higher, 2.0 times higher, 2.5 times higher, 5 times higher, or 10 times higher than the surface expression level detected in a control sample.
  • the term “expression pattern” refers to the levels of expression of more than one, or a group, of polypeptides or polynucleotides.
  • a “detected expression pattern” is the expression pattern of the subject’s B cells.
  • a “control expression pattern” is either a B cell expression pattern associated with transplant rejection (“control rejection expression pattern”) or a B cell expression pattern associated with no rejection (“control no rejection expression pattern”).
  • control rejection expression pattern a B cell expression pattern associated with transplant rejection
  • control no rejection expression pattern a B cell expression pattern associated with no rejection
  • the association with rejection and/or no-rejection is determined through analysis of B cell expression data from an appropriate cohort (a cohort presenting with rejection or a cohort presenting with no rejection) using t-distributed stochastic neighbor embedding (t-SNE).
  • the t-SNE control expression pattern data is represented in a two- or three- dimensional plot.
  • the compared expression patterns are limited to correlating subpopulation groups identified on t-SNE plots.
  • the control rejection expression pattern predicts acute rejection vs. no rejection with a sensitivity (Sens) of at least 71%, a specificity (Spec) of at least 79%, and with a positive predictive value (PPV) of at least 80% and/or a negative predictive value (NPV) of at least 75%.
  • the control no rejection expression pattern predicts acute rejection vs.
  • a “protein”, “polypeptide”, or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids.
  • the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide.
  • This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like.
  • glycosylation e.g., the addition of a saccharide
  • acetylation e.g., the addition of a saccharide
  • phosphorylation e.g., phosphorylation, and the like.
  • a polypeptide and/or protein is defined as a polymer of amino acids, typically of length >100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a polypeptide containing 20-100 amino acids is generally considered a peptide or a short polypeptide.
  • a peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • the peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N- terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at
  • glycation Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine).
  • polysialylation e.g., the addition of polysialic acid
  • glypiation e.g., glycosylphosphatidylinositol (GPI) anchor formation
  • hydroxylation e.g., hydroxylation
  • iodination e.g., of thyroid hormones
  • phosphorylation e.g., the addition of a
  • percent identity refers to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.
  • a desired response is treatment of a patient who is having transplant rejection.
  • a desired response is pre-emptive treatment, and/or more close surveillance of a patient who is likely to have future transplant rejection.
  • a desired response is reduction or prevention of a future transplant rejection.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • pharmaceutically effective amount include that amount of a compound or compounds such as an immunosuppressive therapy that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated.
  • the therapeutically effective amount will vary depending on the immunosuppressive compound or compounds, the disorder or conditions and its severity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and/or diet of the subject to be treated.
  • statically significant difference refers to p ⁇ 0.1, and more preferably, p ⁇ 0.05.
  • statically significant sameness refers to p ⁇ 0.1, and more preferably, p ⁇ _0.05.
  • subject is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.
  • surface expression refers to the process by which polypeptides are translocated to the surface of a cell such that at least a portion of the polypeptide is located at the exterior of the cell surface. It should be understood that “surface expression” does not include secretion of a polypeptide by a B cell as in, for example, a B cell’s secretion of a cytokine or antibody.
  • t-distributed stochastic neighbor embedding or “t-SNE” refers herein to a nonlinear dimensionality reduction algorithm.
  • the t-SNE analytical parameters include iterations: 2000.
  • the t-SNE parameters include perplexity: 50.
  • the t-SNE parameters include learning rate 48787.
  • the term “transplant” can refer to vascularized composite allografts, organs, bodily tissues, or cells.
  • vascularized composite allografts are face, fingers, hands, arms, toes, feet, and legs.
  • organs are kidney, lung, liver, heart, pancreas, intestines, and uterus.
  • bodily tissues are multi- visceral transplant tissue, and uterine tissue.
  • cells are pancreatic islets, stem cells, neuronal cells, and genetically modified cells.
  • treat include partially or completely alleviating, mitigating or reducing the intensity of one or more attendant signs or symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition.
  • Treatments according to the invention may be applied palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of a transplant rejection), during early onset (e.g., upon initial signs and symptoms of a transplant rejection), or after an established development of a transplant rejection. Prophylactic administration can occur for several days to years intending to reduce future rejection episodes.
  • a method of detecting rejection status of a transplant in a subject that includes obtaining a sample comprising B cells from the subject, detecting an expression pattern of the B cells, and comparing the detected expression pattern to a control rejection expression pattern and/or a control no rejection expression pattern, wherein a transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control no rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control rejection expression pattern and/or wherein a lack of transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control no rejection expression pattern.
  • B cells are identified as B cells prior to, simultaneously with, or after detecting the expression pattern of the B cell.
  • the B cells are identified as B cells by detecting a pan B cell marker.
  • the B cell identification marker selected from a group consisting of CD19, CD20, CD79alpha, CD79beta, FcRL5, FcRL4, CD 138, and B cell receptor or a fragment thereof.
  • the pan B cell marker is CD19.
  • the expression pattern comprises expression data for two or more of thirteen polypeptides, or polynucleotides encoding the two or more of thirteen polypeptides, and wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the detected expression pattern consists of expression data for CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the detected expression pattern comprises expression data for two or more of twenty-one polypeptides, or polynucleotides encoding the two or more of twenty-one polypeptides, and wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the detected expression pattern consists of expression data for TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the detected expression pattern comprises expression data for CD38 and CD24.
  • the detected expression pattern consists of expression data for CD27 and CD21.
  • the detected expression pattern comprises expression data for CD38 and CD73.
  • the detected expression pattern consists of expression data for CD38 and CD23.
  • the detected expression pattern comprises expression data for CD24 and CD73.
  • the detected expression pattern consists of expression data for CD24 and CD21. In some embodiments, the detected expression pattern comprises expression data for CD25 and IgD. In some embodiments, the detected expression pattern consists of expression data for CD73 and IgM. In some embodiments, the detected expression pattern comprises expression data for CD73 and IgD. In some embodiments, the detected expression pattern consists of expression data for CD39 and CD25. In some embodiments, the detected expression pattern comprises expression data for CD39 and CD73. In some embodiments, the detected expression pattern consists of expression data for CD73 and LAG3. In some embodiments, the detected expression pattern comprises expression data for CD73 and CD10. In some embodiments, the detected expression pattern consists of expression data for CD23 and CD73. In some embodiments, the detected expression pattern comprises expression data for CD21 and CD9.
  • the expression pattern of the B cells comprises expression data one or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for two or two or more of twenty-one polypeptides, wherein the twenty - one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for five or five or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for six or six or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for seven or seven or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for eight or eight or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for nine or nine or more of twenty-one polypeptides, wherein the twenty- one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for ten or ten or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for eleven or eleven or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for twelve or twelve or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD 10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for thirteen or thirteen or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells comprises expression data for fourteen or fourteen or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern of the B cells consists of expression data for TNFR2, CD19, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the method can further comprises detecting CD 19 or another B cell identification marker.
  • the method comprises detecting expression data for one or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for two or two or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3.
  • the expression pattern of the B cells comprises expression data for three or three or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for four or four or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data five or five or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for six or six or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for seven or seven or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for eight or eight or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for nine or nine or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for ten or ten or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for eleven or eleven or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3.
  • the expression pattern of the B cells comprises expression data for twelve or twelve or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the method can further comprises detecting CD 19 or another B cell identification marker.
  • the expression pattern of the B cells consists of expression data for CD 19, CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • detecting the expression pattern of CD 19 can occur prior to or concurrently with detection of the expression pattern of the remaining B cell polypeptides described herein.
  • CD 19 is detected before the expression pattern of one or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80 is detected.
  • the expression pattern is a surface expression pattern.
  • transplant rejection refers to acute rejection and chronic rejection.
  • the transplant rejection is acute.
  • the transplant rejection is chronic.
  • the acute transplant rejection can be severe, moderate, mild, clinical or subclinical.
  • the transplant rejection can be mediated by antibodies, or by T cells, or other immune cells.
  • the transplant rejection is an acute organ transplant rejection. Acute transplant rejection commonly occurs days to over one year after a transplant.
  • the transplant rejection is a chronic transplant rejection. Chronic transplant rejection commonly occurs or continues after about six months following a transplant. In certain aspects, the chronic transplant rejection is mild.
  • the chronic transplant rejection is subclinical [0081]
  • the “transplant” can be a transplant of vascularized composite allografts, organs, bodily tissues, or cells.
  • the transplant rejection is an acute organ transplant rejection.
  • the transplant rejection is a subclinical organ transplant rejection.
  • the transplant rejection is a mild organ transplant rejection.
  • the transplant rejection is a chronic organ transplant rejection.
  • the organ can be any organ, and in some embodiments is a kidney, liver, lung, heart, small bowel, multi- visceral, pancreas, limb or face (composite tissue allografts).
  • the organ is a kidney, liver or lung.
  • the organ is a kidney. In other embodiments, the organ is a liver. In other embodiments, the organ is a lung. In other embodiments the transplant may be cellular comprised of pancreatic islets, neuronal cells, stem cells or genetically modified cells.
  • Classification as “acute,” “chronic,” “subclinical,” “clinical,” “antibody-mediated” or “T cell mediated” of kidney transplants can be achieved using the Banff Classification of Allograft Pathology, a classification system known to those of ordinary skill in the art (Appendix 1).
  • “acute” rejection of kidney transplants is characterized by tubulitis, interstitial inflammation, glomerulitis, peritubular capillaritis and arteritis as defined in the Banff Classification.
  • chronic rejection of kidney transplants is characterized by tubular atrophy, interstitial fibrosis, transplant glomerulopathy, multilayering of peritubular capillary (PCT) basement membranes and transplant arteriopathy as defined in the Banff Classification.
  • a subclinical rejection is scored as less than a Banff grade 1A.
  • the subject has no or a minor increase in creatine levels.
  • the sample is a blood sample.
  • the B cells may be obtained from allograft biopsies, bronchial washings (bronchioalveloar lavage), urine or other excretions or secretions.
  • the B cells can be naive B cells, memory B cells, plasma B cells, and any combination thereof.
  • the subject is a human.
  • the methods of the present disclosure include detecting a B cell expression pattern on B cells in the sample.
  • the B cells may be live, fixed, or cryopreserved.
  • the B cell expression pattern is a B cell surface expression pattern.
  • the B cell expression pattern is a pattern of relative expression of a group of B cell polypeptides such as CD 19 or another B cell specific marker plus two or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • a group of B cell polypeptides such as CD 19 or another B cell specific marker plus two or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the B cell expression pattern is a pattern of relative expression of a group of B cell polypeptides such as CD 19 or another B cell identification marker plus five or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • a group of B cell polypeptides such as CD 19 or another B cell identification marker plus five or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • control rejection expression pattern is either a B cell expression pattern associated with transplant rejection (“control rejection expression pattern”) or a B cell expression pattern associated with a lack of rejection (“control no rejection expression pattern”).
  • control rejection expression pattern is either a B cell expression pattern associated with transplant rejection (“control rejection expression pattern”) or a B cell expression pattern associated with a lack of rejection (“control no rejection expression pattern”).
  • control rejection expression pattern is either a B cell expression pattern associated with transplant rejection (“control rejection expression pattern”) or a B cell expression pattern associated with a lack of rejection (“control no rejection expression pattern”).
  • control rejection expression pattern is either a B cell expression pattern associated with transplant rejection (“control rejection expression pattern”) or a B cell expression pattern associated with a lack of rejection (“control no rejection expression pattern”).
  • control no rejection expression pattern is determined through analysis of B cell expression data from an appropriate cohort (a cohort presenting with rejection or a cohort presenting with no rejection) using t-distributed stochastic neighbor embedding (t-SNE).
  • data is analyzed using other dimensionality reduction algorithms (such as, for example, Principal component analysis (PCA), Independent component analysis (ICA), Low variance filter, High correlation filter, Isomap, or Singular value decomposition).
  • PCA Principal component analysis
  • ICA Independent component analysis
  • Low variance filter Low variance filter
  • High correlation filter High correlation filter
  • Isomap High correlation filter
  • Singular value decomposition Singular value decomposition
  • the t-SNE control expression pattern data is represented in a two- or three-dimensional plot. Non-limiting examples of t-SNE plots are shown in Figures 2 and 6-19.
  • the compared expression patterns are limited to correlating subpopulation groups identified on t-SNE plots. Accordingly, a control rejection expression pattern can be as shown in a t-SNE plot in its entirety or a portion of a t-SNE plot.
  • control expression pattern is a subpopulation of a t-SNE plot and the detected expression pattern to which it is compared is a correlating subpopulation of a t-SNE plot, wherein “correlating” refers to having the same spatial location and boundaries in a t-SNE plot wherein the t-SNE plots were generated using the same t-SNE parameters.
  • the G7 subpopulation in each panel of Figure 2 correlates with the other G7 subpopulations in the other panels of Figure 2.
  • the P2 subpopulation in each panel of Figure 19 correlates with the other P2 subpopulations in the other panels of Figure 19.
  • the control rejection expression pattern is that shown in the right panel of Figure 2 or one or more the Gl, G2, G3, G4, G5, G6 or G7 subpopulations in the right panel of Figure 2.
  • the control rejection expression pattern is or comprises that shown in the G7 subpopulation in the right panel of Figure 2.
  • the control no rejection expression pattern is that shown in the G7 subpopulation of the center panel of Figure 2.
  • the control rejection expression pattern is that shown in the right panel of Figure 19.
  • the control no rejection expression pattern is that shown in the center panel of Figure 19.
  • the control rejection expression pattern is that shown in one or more of the Pl, P3 and P4 subpopulations shown in the right panel of Figure 19.
  • the control no rejection expression pattern is that shown in one or more of the Pl, P3 and P4 subpopulations shown in the center panel of Figure 19.
  • an “expression pattern” refers to the expression levels of a group of polypeptides or polynucleotides and can include values for expression, no expression, and any level of expression.
  • expression values are obtained using flow cytometry methods.
  • the expression values are obtained using a t-distributed stochastic neighbor embedding (t-SNE) analysis of flow cytometry data.
  • t-SNE stochastic neighbor embedding
  • the t-SNE surface expression values can range broadly but the algorithm places cells in a given region according to their relative brightness of each marker.
  • surface marker fluorescence may range from -3,000 to 600,000, wherein ranges below the background fluorescence of isotype and fluorochrome controls are referred to as “negative” or “neg,” ranges above the background fluorescence of isotype and fluorochrome controls are referred to as “positive” or “pos,” ranges between about 40% and 70% compared to the brightest population are referred to as “dim,” and ranges between about greater than 70% of the brightest population are referred to as “bright.” In some embodiments, “neg,” “dim,” “pos” and “bright” correspond with the results shown in Figure 5.
  • the expression pattern comprises the expression level of 20 or less of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80. In some embodiments, the expression pattern comprises the expression level of 15 or less of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern comprises the expression level of 12 or less of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80. In some embodiments, the expression pattern comprises the expression level of 10 or less of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern comprises the expression level of 5 or less of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the method comprises detecting expression data for one of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for two or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for three or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for four or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data five or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3.
  • the expression pattern of the B cells comprises expression data for six or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for seven or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for eight or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3.
  • the expression pattern of the B cells comprises expression data for nine or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for ten or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the expression pattern of the B cells comprises expression data for eleven or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3.
  • the expression pattern of the B cells comprises expression data for twelve or less of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the B cell expression pattern comprises the expression level of CD 19, CD39, CD80, CD23, CD73, IgM, CD21, CD27, CD24, IgD and CD38.
  • detecting the expression pattern of CD19 can occur prior to or concurrently with detection of the expression pattern of the remaining B cell markers described herein.
  • CD19 expression is detected before the expression pattern of any combination of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the expression pattern corresponds to the G7 group shown in Figure 2.
  • the expression level correlates with the Gl, G2, G3, G4, G5 or G6 group shown in Figure 2.
  • the expression pattern comprises CD19 bngbt , CD39 d,ra , CD80 dim , CD23 dim , CD73 ncg , IgM dim , CD21 ncg , CD27 ncg , CD24 pos , IgD dim and CD38 ncg as determined by t-SNE.
  • the surface expression pattern corresponds to group G5 and in some embodiments comprises CD19 bright , CD39 dim , CD80 dim , CD23 dim , CD73 neg , IgM dim , CD21 neg , CD27 neg , CD24 pos , IgD dim and CD38 neg and correlates with the results shown in Figure 5.
  • the expression pattern comprises CD19 udermediate , CD39 bngbt , CD80 neg , HLA-II bnght , CD23 bright , CD73 bright , IgM dim , CD2 intermediate , CD27 neg , CD24 dim , i g D inte TM ediate , CD38 dim , and CD9 dim as determined by t-SNE.
  • the B cell expression pattern comprises the expression level of two to twelve of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of two to eleven of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of two to ten of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • the B cell expression pattern comprises the expression level of two to nine of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of two to eight of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of two to seven of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of two to six of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • the B cell expression pattern comprises the expression level of three to twelve of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of three to eleven of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of three to ten of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • the B cell expression pattern comprises the expression level of three to nine of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level three to eight of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of three to seven of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of three to six of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • the B cell expression pattern comprises the expression level of five to twelve of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of five to eleven of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of five to ten of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • the B cell expression pattern comprises the expression level of five to nine of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of five to eight of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of five to seven of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39. In some aspects, the B cell expression pattern comprises the expression level of five or six of CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • detecting the expression pattern of CD19 can occur prior to or concurrently with detection of the expression pattern of the remaining B cell markers described herein.
  • the expression pattern of CD 19 is detected before the expression pattern of one or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80 is detected.
  • the B cell expression pattern is a B cell surface expression pattern. In some embodiments, the B cell expression pattern is a relative B cell expression pattern. In some embodiments, the B cells are live. [0097] In some embodiments, the CD 19 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 1633, Entrez Gene: 930, Ensembl:
  • the CD19 polypeptide comprises SEQ ID NO: 1. In some embodiments, the CD19 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1.
  • the CD39 polypeptide is also known as NTPDase-1 encoded by the ENTPD1 gene.
  • the CD39 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 3363, NCBI Gene: 953, Ensembl: ENSG00000138185, OMIM: 601752, and UniProtKB: P49961.
  • the CD39 polypeptide comprises SEQ ID NO:2.
  • the CD39 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:2, or a polypeptide comprising a portion of SEQ ID NO:2.
  • the CD80 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 1700, NCBI Gene: 941, Ensembl: ENSG00000121594, OMIM: 112203, and UniProtKB: P33681.
  • the CD80 polypeptide comprises SEQ ID NOG.
  • the CD80 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NOG, or a polypeptide comprising a portion of SEQ ID NOG.
  • the CD23 polypeptide is also known as the FC Epsilon Receptor II encoded by the FCER2 gene.
  • the CD23 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 3612, NCBI Gene: 2208, Ensembl: ENS G00000104921, OMIM: 151445, and UniProtKB: P06734.
  • the CD23 polypeptide comprises SEQ ID NOG.
  • the CD23 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NOG, or a polypeptide comprising a portion of SEQ ID NOG.
  • the CD73 polypeptide is also known as 5 '-Nucleotidase Ecto and encoded by the NT5E gene.
  • the CD73 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 8021, NCBI Gene: 4907, Ensembl: ENSG00000135318, OMIM: 129190, and UniProtKB: P21589.
  • the CD73 polypeptide comprises SEQ ID NOG.
  • the CD73 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NOG, or a polypeptide comprising a portion of SEQ ID NOG.
  • the CD21 polypeptide is also referred to as the Complement C3d Receptor 2 encoded by the CR2 gene.
  • the CD21 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 2336, NCBI Gene: 1380, Ensembl: ENSG00000117322, OMIM: 1120650, and UniProtKB: P20023.
  • the CD21 polypeptide comprises SEQ ID NO:6. In some embodiments, the CD21 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:6, or a polypeptide comprising a portion of SEQ ID NO:6.
  • the CD27 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 11922, NCBIGene: 939, Ensembl: ENSG00000139193, OMIM: 186711, and UniProtKB: P26842.
  • the CD27 polypeptide comprises SEQ ID NO:7.
  • the CD27 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:7, or a polypeptide comprising a portion of SEQ ID NO:7.
  • the CD24 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 1645, NCBI Gene: 100133941, Ensembl: ENSG00000272398, OMIM: 600074, and UniProtKB: P25063.
  • the CD24 polypeptide comprises SEQ ID NO:8.
  • the CD24 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:8, or a polypeptide comprising a portion of SEQ ID NO:8.
  • the CD38 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 1667, NCBI Gene: 952, Ensembl: ENSG00000004468, OMIM: 107270, and UniProtKB: P28907.
  • the CD38 polypeptide comprises SEQ ID NO:9.
  • the CD38 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:9, or a polypeptide comprising a portion of SEQ ID NO:9.
  • the TNFR2 polypeptide is also referred to as the TNF Receptor Superfamily Member IB encoded by the TNFRSF1B gene.
  • the TNFR2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 11917, NCBI Gene: 7133, Ensembl: ENSG00000028137, OMIM®: 191191, UniProtKB/Swiss-Prot: P20333.
  • the TNFR2 polypeptide comprises SEQ ID NO: 10.
  • the TNFR2 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 10, or a polypeptide comprising a portion of SEQ ID NO: 10.
  • the LAG3 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 6476, NCBI Gene: 3902, Ensembl: ENSG00000089692, OMIM®: 153337, UniProtKB/Swiss-Prot: P18627.
  • the LAG3 polypeptide comprises SEQ ID NO:11.
  • the LAG3 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 11, or a polypeptide comprising a portion of SEQ ID NO:11.
  • the IgM polypeptide is also referred to as the Immunoglobulin Heavy Constant Mu encoded by the IGHM gene.
  • the IgM polypeptide is that identified in one or more publicly available databases as follows: HGNC: 5541, NCBI Gene: 3507, Ensembl: ENS G00000211899, OMIM®: 147020, UniProtKB/Swiss-Prot: P01871.
  • the IgM polypeptide comprises SEQ ID NO: 12.
  • the IgM polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 12, or a polypeptide comprising a portion of SEQ ID NO: 12.
  • the CD9 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 1709, NCBI Gene: 928, Ensembl: ENS G00000010278, OMIM®: 143030, UniProtKB/Swiss-Prot: P21926.
  • the CD9 polypeptide comprises SEQ ID NO: 13.
  • the CD9 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 13, or a polypeptide comprising a portion of SEQ ID NO: 13.
  • the CD10 polypeptide is also referred to as the Membrane Metalloendopeptidase encoded by the MME gene.
  • the CD10 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 7154, NCBI Gene: 4311, Ensembl: ENS G00000196549, OMIM®: 120520, UniProtKB/Swiss-Prot: P08473.
  • the CD10 polypeptide comprises SEQ ID NO: 14.
  • the CD10 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 14, or a polypeptide comprising a portion of SEQ ID NO: 14.
  • the IgD polypeptide is also referred to as the Immunoglobulin Heavy Constant Delta encoded by the IGHD gene.
  • the IgD polypeptide is that identified in one or more publicly available databases as follows: HGNC: 5480, NCBI Gene: 3495, Ensembl: ENSG000002U898, OMIM®: 147170, UniProtKB/Swiss-Prot: P01880.
  • the IgD polypeptide comprises SEQ ID NO: 15.
  • the IgD polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 15, or a polypeptide comprising a portion of SEQ ID NO: 15.
  • the PDL1 polypeptide is also referred to as the CD274 Molecule encoded by the CD274 gene.
  • the PDL1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 17635, NCBI Gene: 29126, Ensembl: ENSG00000120217, OMIM®: 605402, UniProtKB/Swiss-Prot: Q9NZQ7.
  • the PDL1 polypeptide comprises SEQ ID NO: 16.
  • the PDL1 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 16, or a polypeptide comprising a portion of SEQ ID NO: 16.
  • the TIGIT polypeptide is that identified in one or more publicly available databases as follows: HGNC: 26838, NCBI Gene: 201633, Ensembl: ENSG00000181847, OMIM®: 612859, UniProtKB/Swiss-Prot: Q495A1.
  • the TIGIT polypeptide comprises SEQ ID NO: 17.
  • the TIGIT polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 17, or a polypeptide comprising a portion of SEQ ID NO: 17.
  • the HLA Class II polypeptide is that identified in one or more publicly available databases as follows: HGNC: 4948, NCBI Gene: 3123, Ensembl: ENSG00000196126, OMIM®: 142857, UniProtKB/Swiss-Prot: P01911.
  • the HLA Class II polypeptide comprises SEQ ID NO: 18.
  • the HLA Class II polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 18, or a polypeptide comprising a portion of SEQ ID NO: 18.
  • the TIM-1 polypeptide is also referred to as the Hepatitis A Virus Cellular Receptor 1 encoded by the HAVCR1 gene.
  • the TIM-1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 17866, NCBI Gene: 26762, Ensembl: ENSG00000113249, OMIM®: 606518, UniProtKB/Swiss- Prot: Q96D42.
  • the TIM-1 polypeptide comprises SEQ ID NO: 19.
  • the TIM-1 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 19, or a polypeptide comprising a portion of SEQ ID NO: 19.
  • the TIM-4 polypeptide is also referred to as the T Cell Immunoglobulin And Mucin Domain Containing 4 encoded by the TIMD4 gene.
  • the TIM-4 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 25132, NCBI Gene: 91937, Ensembl: ENSG00000145850, OMIM®: 610096, UniProtKB/Swiss-Prot: Q96H15.
  • the TIM-4 polypeptide comprises SEQ ID NO:20.
  • the TIM-4 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:20, or a polypeptide comprising a portion of SEQ ID NQ:20.
  • the PD1 polypeptide is also referred to as the Programmed Cell Death 1 encoded by the PDCD1 gene.
  • the PD1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 8760, NCBI Gene: 5133, Ensembl: ENSG00000188389, OMIM®: 600244, UniProtKB/Swiss-Prot: Q15116.
  • the PD1 polypeptide comprises SEQ ID NO:21.
  • the PD1 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:21, or a polypeptide comprising a portion of SEQ ID NO:21.
  • the CD25 polypeptide is also referred to as the Interleukin 2 Receptor Subunit Alpha encoded by the IL2RA gene.
  • the CD25 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 6008, NCBI Gene: 3559, Ensembl: ENS G00000134460, OMIM®: 147730, UniProtKB/Swiss-Prot: P01589.
  • the CD25 polypeptide comprises SEQ ID NO:22.
  • the CD25 polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:22, or a polypeptide comprising a portion of SEQ ID NO:22.
  • the methods of detection can allow a determination of who to biopsy (or obtain other diagnostic tissue or fluid sample from the patient) or who needs or does not need a biopsy. Therefore, in some embodiments of the method of detection, no rejection is detected and no biopsy or diagnostic sample is needed from the subject. The methods herein can therefore prevent unnecessary biopsy or sampling from the subject.
  • detection of the expression pattern may be used to rule out transplant rejection and avoid biopsy or treatment.
  • this might comprise a decrease in B cells in subpopulation G7 as shown in Figure 3, or an increase in cells in G5 as shown in Figure 4 when the polypeptides are TNFR2, CD 19, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD8O.
  • rejection is detected and the method further comprises obtaining a biopsy or diagnostic sample from the subject.
  • the biopsy is a transplant biopsy.
  • the transplant is an organ.
  • the diagnostic sample is a blood sample.
  • the diagnostic sample is a bronchoalveloar lavage.
  • rejection is detected and the method further comprises one or more of a functional test, bronchioalveolar lavage, cardiac catheterization and blood measurement after detection of the transplant rejection.
  • the method of treating a transplant rejection in a subject comprises obtaining a sample comprising B cells from the subject, detecting an expression pattern of the B cells, and comparing the detected expression pattern to a control rejection expression pattern and/or a control no rejection expression pattern, wherein a transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control no rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control rejection expression pattern, and administering to the subject a treatment for the detected transplant rejection.
  • the methods of treatment include obtaining a biopsy or diagnostic sample from the subject prior to treatment and after detection of the transplant rejection.
  • the method further comprises obtaining a biopsy or diagnostic sample from the subject.
  • the biopsy is a transplant biopsy.
  • the diagnostic sample is a blood sample.
  • the diagnostic sample is a bronchoalveloar lavage or cardiac catheterization.
  • the detected B cell expression pattern can comprise any combination of B cell polypeptides or polynucleotides described herein and can be any as described herein as indicating a rejection.
  • the method of treating a transplant rejection in a subject comprises a) obtaining a sample comprising B cells from the subject, and b) detecting a B cell expression pattern comprising identification marker plus one or more of twenty-one polypeptides, or polynucleotides encoding the two or more of twenty-one polypeptides, wherein the twenty-one polypeptides are TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80, comparing the detected expression pattern to a control rejection expression pattern and/or a control no rejection expression pattern and identifying an indication of transplant rejection, and c) administer
  • the method of treating a transplant rejection in a subject comprises a) obtaining a sample comprising B cells from the subject, and b) detecting a B cell expression pattern comprising CD 19 or another B cell identification marker plus two or more of thirteen polypeptides, or polynucleotides encoding the one or more of thirteen polypeptides, wherein the thirteen polypeptides are CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3, wherein detection of the expression pattern is a detection of transplant rejection, and c) administering to the subject a treatment for the detected transplant rejection.
  • detecting the expression pattern of CD 19 can occur prior to or concurrently with detection of the expression pattern of the remaining B cell markers described herein.
  • the expression pattern of CD19 is detected before the expression pattern of one or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80 is detected.
  • the expression pattern is a surface expression pattern.
  • the transplant is an organ.
  • the organ is a kidney.
  • the two or more of twenty-two polypeptides are CD39, CD80, CD23, CD73, IgM, CD21, CD27, CD24, IgD and CD38.
  • the expression pattern comprises CD39 dim , CD80 dim , CD23 dim , CD73 neg , IgM dim , CD21 neg , CD27 neg , CD24P OS , IgD dim and CD38 neg .
  • the expression pattern comprises CD39 bnght , CD80 neg , HLA- II bright , CD23 bright , CD73 bright , IgM dim , CD2 in,eraiediate , CD27 neg , CD24 dira , igD in,e TM ediate , CD38 dim , and CD9 dim .
  • the two or more of thirteen polypeptides are CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39.
  • the B cell identification marker is CD19 and its expression is CD19 bnght or CD19 m,ermedia,e .
  • the polypeptides are TNFR2, CD19, LAG3, CD27, CD21, IgM, CD9, CD 10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80, and the detected expression pattern has a statistically significant sameness to the expression pattern depicted in Figure 2, right panel, using the t-SNE parameters described herein.
  • the polypeptides are TNFR2, CD19, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80, and the detected expression pattern has a statistically significant difference to the expression pattern depicted in Figure 2, center panel, using the t-SNE parameters described herein.
  • the polypeptides are CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39 and the detected expression pattern has a statistically significant sameness to the expression pattern depicted in Figure 19, right panel, using the t-SNE parameters described herein.
  • the polypeptides are CD38, IgD, CD27, CD21, IgM, LAG3, CD9, CD10, CD24, CD73, CD25, CD23, and CD39 and the detected expression pattern has a statistically significant difference to the expression pattern depicted in Figure 19, center panel, using the t-SNE parameters described herein.
  • the subject is a human and/or where the sample is a blood sample.
  • the treatment administered to the subject can be any known to those of skill in the art.
  • the treatment is an immunosuppressive therapy.
  • the treatment comprises administration of a therapeutically effective composition that is an immunosuppressive therapy.
  • the treatment is intended to reduce the likelihood of future transplant rejection or reduce the severity of future transplant rejection.
  • kits for detecting rejection status of a transplant in a subject wherein the kit is used to obtain a sample comprising B cells from the subject and to identify an expression pattern of the B cells and comparing the detected expression pattern to a control rejection expression pattern and/or a control no rejection expression pattern, wherein a transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control no rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control rejection expression pattern and/or wherein a lack of transplant rejection is indicated by a statistically significant difference in the detected expression pattern and the control rejection expression pattern or a statistically significant sameness of the detected expression pattern and the control no rejection expression pattern.
  • the kit comprises compositions for the detection of expression of any detected expression pattern described herein.
  • the kit comprises compositions for detection of expression of two or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80 in or on B cells in a sample obtained from the subject.
  • the kit comprises compositions for detection of expression of CD19 or another B cell specific marker plus two or more of CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD 10, and LAG3 in or on B cells in a sample obtained from the subject.
  • the compositions for detection comprise antibodies specific for the two or more B cell polypeptides described herein.
  • the antibodies are labeled.
  • the labels are fluorescent. More specifically, in some kit embodiments, the kit comprises labeled antibodies specific for two or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIG1T, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the kit comprises labeled antibodies specific for two or more of CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the kit comprises labeled antibodies specific for CD19, CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the compositions for detection comprise polynucleotide sequences specific for two or more B cell polynucleotides described herein. More specifically, in some kit embodiments, the kit comprises polynucleotides specific for polynucleotides that encode two or more of TNFR2, LAG3, CD27, CD21, IgM, CD9, CD10, CD38, IgD, PDL1, CD39, TIGIT, CD24, CD73, CD25, CD23, HLA Class II, TIM-1, TIM-4, PD1, and CD80.
  • the kit comprises polynucleotides specific for polynucleotides that encode two or more of CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the kit comprises polynucleotides specific for CD 19, CD24, CD38, CD27, CD21, CD39, CD23, CD73, CD25, CD9, IgD, IgM, CD10, and LAG3.
  • the kit further comprises a visual representation of a control no rejection expression pattern and/or a control rejection expression pattern as described herein. In some embodiments, the kit further comprises data representing a control no rejection expression pattern and/or a control rejection expression pattern as described herein. In some embodiments, the kit comprises information regarding statistically significant differences and/or statistically significant sameness as described herein.
  • kits describes a wide variety of bags, containers, carrying cases, and other portable enclosures which may be used to carry and store solid substances, liquid substances, and other accessories necessary to detect transplant rejection. Such kits and their contents along with any applicable procedures may be used to provide access to detection of transplant rejection in accordance with the teachings of the present disclosure.
  • the kit comprises a sample collection device, a centrifuge tube, one or more fluorescently conjugated antibody(s), one or more control sample(s), a nucleic acid detection probe, a staining buffer and a flow-cytometer, single cell isolation reagents, or a combination thereof.
  • the sample collection device is selected from a group comprising of a tourniquet, an alcohol swab, a needle, a syringe, a blood collection tube, a dissection tool, a biopsy punch device, a biopsy needle, a biopsy syringe, a specimen container, or a combination thereof.
  • the transplant of the kit embodiments can be any as described herein.
  • the transplant is a kidney, liver, lung, heart, pancreas, intestines, multi-visceral, uterus, vascularized composite allograft, pancreatic islet, stem cell, or neuronal cell.
  • the transplant is an organ.
  • the organ is a kidney.
  • the transplant rejection is an acute transplant rejection.
  • the transplant rejection is a subclinical transplant rejection.
  • the transplant rejection is a clinical transplant rejection.
  • the subject is a human.
  • the sample is a blood sample.
  • B Cell Surface Marker Staining Human PBMCs were rapidly thawed in a 37°C water bath. Thereafter, 10 mL of pre-warmed PBS-0.5% BSA is added to 1 mL of the thawed PBMCs ( ⁇ 5- 10 X 106 cells/ml). The cells were then centrifuged at 350g (1350 rpm) for 10 minutes with high brake at room temperature. The pellet obtained was then resuspended in 10 mL of PBS-0.5% BSA, then centrifuged at 350g for 10 min at 4°C.
  • the cell pellet was then resuspended in PBS-0.5% BSA at a final concentration of 2.5million cells/ml in a BD falcon tube and centrifuged for 5 min at 350g and high brake. These 2.5million cells (1ml) were used for subsequent staining.
  • a viability stain was performed using LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation (ThermoFisher Scientific, #L34966). For each sample, 1 pL of LIVE/DEAD reagent was used in 1 mL of PBS to re-suspend cell pellet and then the samples were incubated on ice and covered in darkness (with aluminum foil) for 20 minutes.
  • each sample was then washed in 2 mis of PBS-0.5% BSA at 350g (1350 rpm) for 5 minutes with high brake at 4°C. Prior to centrifugation, an aliquot of cells was removed to create a single color ‘LIVE/DEAD’ control.
  • the antibody cocktail was prepared as shown in Table 2. The cell pellet was resuspended in 200 pl of the antibody cocktail and incubated on ice and covered in darkness for 45 minutes. Thereafter, each sample was washed twice in 1ml of PBS-0.5% BSA at 350g (1350 rpm) for 5 minutes with high brake at 4°C.
  • a fixation step was performed for each sample by resuspending the cell pellet of each sample using 250 pl of Fixation/Permeabilization Solution on ice and covered by aluminum foil for 10 min. After fixation, each sample was washed in 2 mis of PBS-0.5% BSA at 350g (1350 rpm) for 5 minutes with high brake at 4°C. The final cell pellet was then resuspended in 100 pl of PBS and flow cytometry was performed immediately for analysis using Cytek Aurora spectral flow cytometer.
  • the G7 subpopulation that was significantly increased in patients with acute rejection (AR) is characterized as: CD19 bnght , CD39 dim , CD80 dmi , CD23 dmi , CD73 neg , IgM dim , CD21 neg , CD27 neg , CD24? OS , IgD dim , CD38 neg .
  • These markers can be used to distinguish G7 from other B subpopulations.
  • Histologic evidence of acute tissue injury including 1 or more of the following:
  • Circulating donor-specific antibodies (DSA to HLA or other antigens). C4d staining or expression of validated transcripts/classifiers as noted above in criterion 2 may substitute for DSA.
  • Morphologic evidence of chronic tissue injury including 1 or more of the following:
  • Grade IA Interstitial inflammation involving >25% of non-sclerotic cortical parenchyma (i2 or i3) with moderate tubulitis (t2) involving 1 or more tubules, not including tubules that are severely atrophic.
  • Grade IB Interstitial inflammation involving >25% of non-sclerotic cortical parenchyma (i2 or i3) with severe tubulitis (t3) involving 1 or more tubules, not including tubules that are severely atrophic.
  • Grade IIA Mild to moderate intimal arteritis (vl), with or without interstitial inflammation and/or tubulitis
  • Grade IIB Severe intimal arteritis (v2), with or without interstitial inflammation and/or tubulitis
  • Grade III Transmural arteritis and/or arterial fibrinoid necrosis involving medial smooth muscle with accompanying mononuclear cell intimal arteritis (v3), with or without interstitial inflammation and/or tubulitis
  • Grade I A Interstitial inflammation involving >25% of sclerotic cortical parenchyma (i-IFTA2 or i- IFTA3) AND > 25% of total cortical parenchyma (ti2 or ti3) with moderate tubulitis (t2 or t-IFTA2) involving 1 or more tubules, not including severely atrophic tubules; other known causes of i-IFTA should be ruled out
  • Grade IB Interstitial inflammation involving >25% of sclerotic cortical parenchyma (i-IFTA2 or i- IFTA3) AND > 25% of total cortical parenchyma (ti2 or ti3) with severe tubulitis (t3 or 1-1FTA3) involving 1 or more tubules, not including severely atrophic tubules; other known causes of i-IFTA should be ruled out
  • Grade II Chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell inflammation in fibrosis and formation of neointima). This may also be a manifestation of chronic active or chronic ABMR or mixed ABMR/TCMR
  • SEQ ID NO: 18 HLA Class II; HLA-II

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Abstract

La présente invention concerne des compositions et des procédés pour détecter un état de rejet d'une greffe chez un sujet, consistant à détecter un motif d'expression de cellule B chez le sujet et à comparer le motif d'expression détecté à un motif d'expression témoin, une différence statistiquement significative entre les deux motifs indiquant le rejet de la greffe. L'invention concerne également un procédé pour traiter un rejet de greffe et un kit pour détecter un état de rejet de greffe chez un sujet.
PCT/US2025/013716 2024-01-30 2025-01-30 Compositions et procédés pour détecter un état de rejet de greffe et le traiter Pending WO2025165945A1 (fr)

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Publication number Priority date Publication date Assignee Title
US10222374B2 (en) * 2010-04-08 2019-03-05 Univeersity of Pittsburgh—Of the Commonwealth System of Higher Education B-cell antigen presenting cell assay

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Publication number Priority date Publication date Assignee Title
US10222374B2 (en) * 2010-04-08 2019-03-05 Univeersity of Pittsburgh—Of the Commonwealth System of Higher Education B-cell antigen presenting cell assay

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHERUKURI ARAVIND, ROTHSTEIN DAVID M.: "Regulatory and transitional B cells: potential biomarkers and therapeutic targets in organ transplantation", CURRENT OPINION IN ORGAN TRANSPLANTATION, RAPID SCIENCE PULBISHERS, PHILADELPHIA, PA, US, vol. 27, no. 5, 1 October 2022 (2022-10-01), US , pages 385 - 391, XP093344812, ISSN: 1087-2418, DOI: 10.1097/MOT.0000000000001010 *
ELIAS CHARBEL, CHEN CHUXIAO, CHERUKURI ARAVIND: "Regulatory B Cells in Solid Organ Transplantation: From Immune Monitoring to Immunotherapy", TRANSPLANTATION, WILLIAMS AND WILKINS, GB, vol. 108, no. 5, 1 May 2024 (2024-05-01), GB , pages 1080 - 1089, XP093344811, ISSN: 0041-1337, DOI: 10.1097/TP.0000000000004798 *

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