ES2350895T3 - PREPARATION OF T GAMMA DELTA HUMAN CELLS PRESENTING ANTIGENS AND ITS USE IN IMMUNOTHERAPY. - Google Patents

Abstract

Method for the preparation of effective human antigen presenting γδ T cells comprising selecting the γδ T cells from human peripheral blood mononuclear cells, treating the selected cells with a stimulus for the induction of the antigen presenting functions and applying the antigen for its uptake and presentation by these cells.

Description

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Preparation of Tγγ cells Human antigen presenting and its use in immunotherapy.

Field of the Invention

The invention relates to a method for the Preparation of human T? cells presenting effective antigen, to prepared γδ T cells using such a method, and its use in immunotherapy, the antigen identification and competition diagnosis immune

Background of the invention The cellular components of the immune system adaptive

\hbox{y sus TCR no requieren restricción por MHC para el
reconocimiento del antígeno (véase a continuación).}

The cellular components of the immune system are divided into the cells of the innate immune system and the cells of the adaptive immune system (acquired or specific). Innate immune system cells include (among others) monocytes, granulocytes, natural cytolytic cells in peripheral blood, and mast cells, macrophages and dendritic cells (DC) in extravascular compartments that include peripheral tissues, such as skin, respiratory tract, Gastrointestinal and urogenital tracts, internal organs as well as secondary lymphatic tissues, such as the spleen, lymph nodes (LN) and Peyer's plaques (PP). The main functions of innate cells are a) provision of immediate protection by neutralizing and limiting the spread of infectious particles, and eliminating tumor cells, b) immunovigilance of healthy tissues, and c) initiation of adaptive immune responses. Adaptive immune system cells include lymphocytes, such as T and B cells. These are distinguished from innate cells by the presence of clonotypic cell surface antigen receptors, termed T cell antigen receptor (TCR) and antigen receptor. B cells (BCR). Each individual lymphocyte carries a different TCR or BCR that recognizes a particular antigen. The specificity of antigen recognition is determined by rearrangement of multiple gene segments of TCR or BCR variables during the development of T and B cells and during maturation of the affinity of the antigen at the time of the generation of effector T and B cells. . T cells not exposed to virgin antigens in peripheral blood differ from each other in the antigen selectivity of their TCRs, and individual virgin T cells expand in response to immune activation by agents containing the antigen for which they are specific . Therefore, during adaptive immune responses a group of virgin T cells with TCRs specific for the potentially infectious agent is expanded by cell proliferation, and develops into a) effector T cells for immediate involvement in the defense against the potentially infectious agent, and in b) memory T cells for long-term protection against this particular potentially infectious agent. The effector T cells have a short life, that is to say they disappear during the resolution phase of the immune response, while the memory T cells have a long life and are divided into subgroups of memory T cells according to their primary tissue residence or the routes of preferential recirculation (Moser et al ., 2004). The T cells are further divided into?? T cells and?? Cells (see below) according to the composition of the heterodimeric TCRs; the?? -TCRs are made up of? and? -protein chains, and?? -TCRs are made up of? and?-protein chains. The majority (> 80%) of all CD3 + T cells in a healthy, normal person are?? T cells. TCRs are associated with the invariable CD3 molecule that distinguishes T cells from B cells and all other types of immunocytes. Most T?? Cells recognize the antigen in a manner called restricted to the major histocompatibility complex (MHC) molecules. This contrasts the BCRs in B cells that bind directly to the nominal antigen in a manner not restricted to MHC. The term "MHC restriction" refers to the way in which TCRs recognize their antigens and involves the presentation of antigenic peptides together with MHC molecules, such as the so-called MHC-peptide complexes, in antigen presenting cells (APC), which include DC (see below). There are two main classes of MHC molecules, MHC class I (MHC-I) and MHC class II (MHC-II), which activate the TCRs of the two main subgroups of T?? Cells, T? Cells? beta CD8 + and the α? beta CD4 + cells. The TCRs in the α? CD4 + cells recognize MHC-II-peptide complexes while the TCRs in the α? CD8 + T cells recognize MHC-I-peptide complexes. In remarkable contrast, the main subgroup of γδ T cells in human peripheral blood does not express CD4 or CD8

 \ hbox {and its TCRs do not require restriction by MHC for
antigen recognition (see below).} 

Tγγ cells

Γ T cells are a differentiated subgroup of CD3 + T cells that have TCRs that are encoded in the V γ and V δ gene segments (Morita et al. , 2000; Carding and Egan, 2002) . They are further divided according to their primary residence in blood or tissues, the protein chain composition of their VγVδ-TCR and their antigen selectivity. In humans, Tγδ cells expressing the Vδ1 + -CRT chain (TV δ1 + cells) predominate in epithelial or epithelial / mucosal-associated skin tissues. airways, digestive and urogenital tracts, and various internal organs, and constitute a minor fraction (<20%) of the T? cells in peripheral blood. TCRs from δ1 + TV cells recognize lipid antigens presented by MHC-related CD1 molecules. In addition, TV δ1 + cells respond to stress-associated proteins, which include MHC and MHC-related MHC molecules, and heat shock proteins. It is believed that these provide a first-line defense against tumors and cells stressed in other ways and, in addition, are believed to contribute to healing, tissue repair and autoimmunity. In human peripheral blood of healthy individuals, δ T cells make up 2-10% of the total CD3 + T cells, and the majority (> 80%) of the Tγ δ cells of Peripheral blood are Tγ? cells that express the Vγ2V? 2 + -CRCR chain (TV ?2V ?2 +??) (Morita et al ., 2000; Carding and Egan, 2002). They are highly selective for small non-peptide antigens of mostly microbial origin and do not require antigen presentation by classical MHC molecules, as is typical for peptide-selective α? Beta cells (see above). TV γ2V δ2 + γ δ cell antigens include prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), alkylamines and metabolites of a newly discovered isoprenoid biosynthesis pathway found in most human microbial pathogens and commensal bacteria (Morita et al ., 2000; Eberl et al ., 2003). Some of these antigens of TV? 2V? 2 +?? Cells, such as IPP and alkylamines, are also released by necrotic tissue cells. The homologue of the human γ2V δ2 + γ2 cells, which carries the homologous TCRV γV with selectivity for small non-peptide antigens of microbial origin, also exists in higher primates, such as macaques , but does not exist in rodents, including mice and rabbits. It is not clear why the presence of TV? 2V? 2 +? Cells is limited to higher primates but it could be speculated that they evolved to meet the special need for cellular protection against a species-specific selection, differentiated from microbes. TV γ2V δ2 + γ δ cells rapidly expand in response to the IPP of the model antigen or microbial extracts in vitro during tissue culture of TV cells γ2V δ2 + γ δ peripheral blood, or in vivo during vaccination experiments in higher primates, such as macaques (Chen and Letvin, 2003). In addition, microbial infections in humans are frequently associated with a huge expansion of peripheral blood TV γ2V δ2 + γ δ cells, which reach levels of up to> 60% of CD3 T cells +} peripheral blood. These findings support the notion that human TV? 2V? Delta2 +? Cells play an important role in immune processes during microbial infections (Morita et al. , 2000; Carding and Egan, 2002; Chen and Letvin, 2003). Their unique selectivity for non-peptide antigens that are commonly found in microbes, including pathogens and commensal bacteria, suggests that TCRs in TV? G2V? Delta2 +? Gamma? Cells fulfill a similar function as toll-like receptors (TLR) that trigger the activation and maturation of DCs and other APCs in response to various ligands of microbial origin.

\hbox{soporta un papel para las células  T
 \gamma  \delta  en la presentación de antígeno.}

Tγ cells contribute to the elimination of pathogens by the rapid secretion of chemokines that initiate the recruitment of innate immune system cells and proinflammatory cytokines (TNF-?, IFN-?) That stimulate the presenting cells antigen and enhance bacterial destruction by granulocytes, macrophages and NK cells (Morita et al ., 2000; Carding and Egan, 2002; Chen and Letvin, 2003). They also express natural cytolytic cell receptors to destroy infected or neoplastic tissue cells. These findings support the notion that T? Cells primarily fulfill innate functions, although it is known that the secretion of proinflammatory cytokines, such as TNF-?, Also contributes to local adaptive immune responses. On the other hand, the evidence of the direct involvement of Tγ cells in adaptive immune responses is not well defined. For example, it is reported that CD1-restricted T cells induce maturation in DCs that have CD1-lipid complexes. In addition, studies in mice demonstrated a role not further explained for γδ T cells in B cell responses, and it was shown that human γδ cells regulate B cell responses during in- culture cocultures. vitro (Brandes et al ., 2003). Finally, studies in macaques showed that TV γ2V δ2 + γ cells could produce memory responses in vivo against Mycobacterium bovis antigens (Chen and Letvin, 2003). Together, these findings provide evidence that γ T cells can interact with adaptive immune system cells, such as B cells and DC cells. Most of these immunomodulatory functions were attributed to the production of cytokines by means of γδ cells or left unexplained. Importantly, none of these findings

 \ hbox {supports a role for T cells
 γ δ in the antigen presentation.} 

The function of lymphocytes is closely related to the migration potential of lymphocytes, as defined by the expression of chemokine receptors and adhesion molecules (Moser et al ., 2004). Accordingly, α? T cells are divided into a) virgin T cells that express the chemokine receptor that targets LN CCR7 but lack receptors for inflammatory chemokines, b) short-lived effector T cells that carry different combinations of chemokine receptors and inducible adhesion molecules that reflect inflammatory states at the site of infection, and c) three subgroups of long-lived, resting memory T cells. The distinction of the subgroups of T cells according to their potential migration, that is, their expression profile of cell surface chemokine receptors and adhesion molecules, correlates well with their state of differentiation and potential function in immune processes. Of importance, "profiling" of chemokine receptors and adhesion molecules is widely used for phenotypic and functional definition of leukocyte subgroups, including DC, and T and B cells (Moser et al ., 2004).

The migration properties of human peripheral blood T? Cells differ markedly from those of human peripheral blood T?? Cells (Brandes et al ., 2003). Most notably, the majority (> 80%) of the TV cells γ2V δ2 + γ δ (hereinafter referred to as "T γ cells") lack CCR7 and, therefore, are excluded from secondary lymphatic tissues, but have an inflammatory migration profile (Brandes et al ., 2003). In clear contrast, the majority (> 70%) of the α? Cells in the peripheral blood express CCR7, which is consistent with their continuous recirculation through the secondary lymphatic tissues (the spleen, the LN, the PP) in which they quickly examine APCs to determine the presence of appropriate MHC-peptide complexes. In the case of ongoing adaptive immune responses, a selection of T?? Cells is activated during contact with APCs that exhibit their analogous antigens and differentiates into effector cells negative for CCR7 with the potential to target inflammatory sites. In contrast, peripheral blood γ T cells have an inflammatory migration program for immediate tissue mobilization in response to inflammatory chemokines produced at the sites of infection. With activation, for example in response to antigens of microbial extracts or defined small non-peptide antigens (such as IPP), the migration profile in Tγγ cells rapidly changes from an inflammatory phenotype to a phenotype that targets LN, as evidenced by the decrease modulation of receptors for inflammatory chemokines and the induction of CCR7 (Brandes et al ., 2003). Unlike T?? Cells, T?? Cells are relatively uncommon in LNs, which is consistent with their differentiated mode of activation that is completely independent of the APCs that restrict the MHC present in these locations (Brandes et al. , 2003). The frequency of T? Cells increases in disease-associated NLs (notably in germinal centers), suggesting that T? Cells may contribute to the initiation of humoral responses (antibodies) and possibly other adaptive immune processes.

Together, the migration characteristics of human Tγ cells and their occasional presence in LNs suggest a role for these cells in the initiation of adaptive immune responses. However, this paper is not defines additionally and there is no evidence that T cells γ δ can function as presenting cells of antigen

Dendritic cells (DC)

DCs form a differentiated class of leukocytes, are derived from hematopoietic progenitor cells in the bone marrow and reside primarily in extravascular sites that include epithelial / mucous tissues (skin, respiratory tract and gastrointestinal / urogenital tracts, among others), and secondary lymphatic tissues (spleen, NL, PP) (Banchereau and Steinman, 1998; Steinman et al ., 2003; Banchereau et al ., 2004). In peripheral blood, DC or DC precursors make up less than 1% of mononuclear leukocytes. The different DC subgroups differ in their tissue location, as shown by way of example by interstitial DCs that mainly reside in soft tissues that border on epithelia, Langerhans (LC) cells present in the epidermis, and plasmacyte DCs with preferences to address the LN. These subgroups of DC are completely differentiated non-proliferative cells with a limited life of several days to several weeks, indicating that they are continuously replaced under steady state conditions by bone marrow derived precursors. On the contrary, human memory T cells survive for many years and are maintained through steady state (homeostatic) proliferation.

The main function of tissue-resident DCs is the uptake and processing of local antigens, their relocation by lymphatic vessels related to drainage LNs and the initiation of adaptive immune responses specific for antigen (Banchereau and Steinman, 1998; Steinman et al ., 2003; Banchereau et al ., 2004). DCs also induce tolerance when antigens are presented to T cells under tolerogenic conditions, that is, in the absence of costimulation of proinflammatory T cells. Similarly, antigen presenting B cells have been shown to induce tolerance (Zhong et al ., 1997). It is believed that the breakdown of immune tolerance against autoantigens is the frequent cause of autoimmune diseases and, therefore, tolerogenic DCs that have autoantigens are essential regulators of immune homeostasis. In healthy peripheral tissues, DCs are present in their completely differentiated but "immature" state. Immature DCs express a group of proinflammatory chemokine receptors for rapid recruitment for local infection, inflammation or tissue damage. They themselves are poorly immunogenic, that is, they cannot induce primary adaptive immune responses. On the other hand, they are experts in the uptake of antigens (through fluid-mediated pinecitic mechanisms or endocytic receptors mediated), the processing of antigens and the loading of peptides in intracellular MHC-I / II molecules and their presentation on the surface mobile. Since immature DCs generally do not express the receptor that is directed to LN CCR7, it is currently unknown how this type of DC reaches the areas of T cells in the spleen, LNs and PPs. A multitude of maturation signals, including stimuli derived from viruses or bacteria that activate toll-like receptors (TLR), inflammatory mediators derived from the host cell (interferon [IFN] - γ, tumor necrosis factor [TNF] - α, interleukin [IL] -1, prostaglandin E2 [PGE2], tissue growth factors, among others), and costimulatory T-cell molecules (CD40 / CD154 ligand), induce the "maturation" of DC (Banchereau and Steinman, 1998; Steinman et al. , 2003; Banchereau et al ., 2004). During the early phase of DC maturation, DC secretes high levels of inflammatory chemokines for increased inflammatory response by recruiting additional immature DC and innate immune system cells (monocytes, granulocytes, natural cytolytic cells). Subsequently, the inflammatory migration program is gradually replaced by a migration program that targets LN characterized by the replacement of receptors by inflammatory chemokines with CCR7. CCR7 is essential for the effective relocation of sensitized DC from peripheral tissues to drainage LNs in response to the two selective chemokines for CCR7 ELC / CCL19 and SLC / CCL21 present in the lymphatic vessels and in the T cell area of the spleen, the LN and the PP. Therefore, the expression of CCR7 marks mature or maturing DC. In addition to the properties of targeting LN, mature DCs exhibit stable cell surface expression of the MHC-I / II-peptide complexes in large quantities as well as various costimulatory molecules that are required for proper stimulation of Tα cells β-virgins (not exposed to antigens) (Banchereau and Steinman, 1998; Steinman et al ., 2003; Banchereau et al ., 2004). DCs are also called "professional" APCs because they can stimulate virgin T?? Cells during primary immune responses. Memory T cells (exposed to antigens) have a lower activation threshold and therefore respond to less stringent stimulation regimens. The functional duality of the DC that distinguishes between the two differentiation states, a) processing antigens, immature, in peripheral tissues and b) DC presenting antigens and costimulators, mature, relocated, in tissue drainage LN, is a hallmark of the physiology of DC and is closely related to local inflammation, infection and tissue damage. Finally, the result (quality and quantity) of the adaptive immune response is largely determined by the "mode" of response initiation. It is known that DC "instructs" the virgin T cells within the zone of the T cells of the LN and the PP about the type of immune response required for the elimination of pathogens (Banchereau and Steinman, 1998; Steinman et al . , 2003; Banchereau et al ., 2004). Therefore, the inflammatory environment in the tissue directly influences the maturation of the DC and, due to the relocation of the DCs, also determines the differentiation of the T cells within the drainage LNs. The different effector destinations of virgin T cell differentiation include specialized subgroups of helper T cells (type 1 cooperating T cells [Th1] producing IFN-? / TNF-?, IL-4 / IL producing Th2 cells -5 / IL-13, among others), regulatory T cells and cytolytic T cells (CTL). The effector T cells are directed to the sites of inflammation, rapidly produce effector functions (cytokine secretion, lysis / destruction of infected / tumor cells) and have a limited life. In contrast, memory T cells are the long-lived product of primary immunization and produce superior immune responses to memory antigens.

Dendritic cells in immunotherapy

DCs are "natural adjuvants," that is, they constitute the most expert cellular system for inducing protective immune responses, and, therefore, are being developed for use in human immunotherapy (Fong and Engleman, 2000; Steinman et al ., 2003 ; Schuler et al ., 2003; Figdor et al ., 2004). Potential applications include cancer treatment, vaccination against pathogens (such as human immunodeficiency virus [HIV] -1 and hepatitis C virus) and the treatment of autoimmune diseases. Current DC treatment protocols include

one.

Isolation and purification of DC precursors of patients' blood (precursors CD34 + hematopoietic bone marrow derivatives or cells CD14 + or CD11c + peripheral blood).

2.

DC generation during cell culture in vitro .

3.

In vitro antigen loading for peptide presentation by mature DC.

Four.

Treatment of patients with individual or repeated injections of DC presenting peptide. Currently, the application of DC in immunotherapy faces several problems (Fong and Engleman, 2000; Steinman et al ., 2003; Schuler et al ., 2003; Figdor et al ., 2004). In summary, DC precursors are scarce in peripheral blood and do not proliferate during in vitro culture, requiring repeated manipulation of large blood samples from patients. DCs are functionally heterogeneous and can induce opposite or undesirable effects, for example immune suppression rather than generation of effector T cells. In addition, DCs are functionally unstable and go through a pre-established sequence of irreversible differentiation stages that end in compromised ("depleted") immune functions. This causes great difficulties in generating functionally homogeneous DC preparations by in vitro manipulations. Finally, the generation of DC peptide presenters for use in immunotherapy is technically demanding, time consuming and expensive.

Summary of the invention

The present invention describes the simple isolation and in vitro preparation of human antigen presenting γδ δ cells and their use as antigen presenting cells (APCs) effective in immunotherapy. Similar to dendritic cells (DC) in potency and efficacy, human Tγ cells delta process antigens and present antigenic peptides to Tαβ cells and induce antigen-specific responses (proliferation and differentiation) in T cells. alpha? virgins. Tγ cells are relatively frequent in peripheral blood (2-10% of CD3 + T cells), they are easily purified from peripheral blood by various simple techniques, they acquire the state of "maturation" ( MHC-II expression, and adhesion molecules and essential costimulators) within 1 day of in vitro culture under simple stimulating conditions, maintain effective antigen presenting functions for 7 days or more of in vitro culture and induce marked responses of primary and secondary cooperating T cells. In addition, Tγγ cells easily expand during in vitro culture for storage and later use.

The invention relates to a method for the Preparation of human T? cells presenting effective antigen comprising selecting T cells γ δ peripheral blood mononuclear cells human, treat selected cells with a stimulus for induction of antigen presenting functions, and apply the antigen to these cells (either before, during or after the induction of antigen presenting functions), at antigen-presenting human Tγ? cells effective prepared by such a method, for use in immunotherapy and in the manufacture of a medicine for use in immunotherapy, to a method of treating tumors or diseases chronic or recurrent infectious with such T cells γ δ human antigen presenting effective, at a vaccination method against tumors or agents that induce infectious or non-infectious diseases with vaccines that select as target? T cells? and the use of such vaccines that select T? cells? in the preparation of a medicine, to a method of identification of antigens derived from novel tumors or pathogens, and to a method of diagnosis of the immune competence of patients who they use such human δ T cells presenting effective antigen.

Brief description of the figures

Figure 1 (1A and 1B). Stimulated γδ T cells have numerous antigen presentation, costimulation and adhesion molecules.

\hbox{horizontales
representan los intervalos para el 99% de las tinciones de
fondo.}

The examination of the cell surface molecules is performed by flow cytometry with Tγγ (Tγδ) cells, either freshly isolated from peripheral blood or after stimulation with IPP and in vitro culture for 1 or 7 days, either with T?? (T??) Cells after 1 day of stimulation with anti-CD3 / CD28 antibody, or with freshly isolated peripheral blood monocytes (M). The numbers above the individual point transfers refer to the time of in vitro culture; (0d) freshly isolated, (1 d) 1 or (7d) 7 days. Positivity is defined by staining with matching isotype control antibodies, and the lines

 \ hbox {horizontal
represent the intervals for 99% of the stains of
background.} 

Figure 2. The stimulated δ T cells express functional CCR7 and respond to LN chemokine SLClCCL21.

Data from Brandes et al ., 2003 are taken. A) Chemotaxis responses are examined in T? Cells after stimulation for 36 hours with IPP, and expressed as the fraction of cells (%), is say the% of total input cells, which have migrated in response to the indicated chemokines. C = Control (white). B) CCR7 expression is determined by flow cytometry, n = cell counts. Continuous and dashed lines refer to the expression of CCR7 in peripheral blood γ T cells δ stimulated with IPP (see chemotaxis) and at rest, respectively; the filled histogram represents the control staining with the isotype antibody.

Figure 3. The formation of rapid and extensive aggregates between stimulated γδ T cells and virgin Tαβ cells.

Tγγ cells are cultured stimulated for 1 day together with virgin CD4 + T cells, autologous, at the ratio of 1: 5 for 3 hours (T δ, 2 examples). As a positive and negative control, the DCs are mixed derived from mature monocytes (DC) and T?? cells stimulated for 1 day (T?), respectively, with autologous virgin α? beta cells. They are isolated all cells from the same donor to exclude antigenic interactions. Be load virgin Tα? CD4 + cells with CFSE for identification by fluorescence microscopy. Be they take the images by phase contrast and by fluorescence of living cells with an inverted microscope Zeiss-Axiovert 35 and the images are combined in overlay layers.

Figure 4. Stimulated γδ T cells induce potent TT-specific proliferation responses in resting α4 CD4 + T cells.

Tγγ cells are stimulated for 1 day in the presence of 20 µg / ml of TT, they are washed and then they are added to the specific α? beta cells to TT marked with CFSE at a ratio of 1: 2. After 4 (4d) and 6 (6d) days culture, CFSE signals in CD4 + cells are examined by flow cytometry (T? δ (+ TT)). How positive control, DC derived from monocytes mature in presence of 20 µg / ml of TT (DC (+ TT)) and are used to stimulate T?? cells at a ratio of 1:10 therein conditions that T γ? TT presenting cells. As a negative control, T?? Cells are cocultivated with T cells δ (γ δ (-TT)) and DC (DC (-TT)), which are stimulated and mature, respectively, in the absence of TT. Horizontal bars above histograms indicate the positions of CFSE signals in non-cells divided (no response); n = cell counts.

Figure 5. Proliferation of TT-specific?? CD4 + T cells requires contact with the TT? Presenting T? Cells .

TT cell preparation is done γ δ TT presenters and the responding cells marked with CFSE as well as the analysis of the data exactly such as described in figure 4. In a tissue culture system from two chambers, T? g cells are added TT presenters and TT-specific responding cells (T?? + CD4 +) to the lower chamber (B), and add the cells that respond alone to the upper chamber (A). Be separate the two chambers by a porous membrane that allows the free exchange of soluble proteins but not intact cells. Horizontal bars above histograms indicate the intervals for CFSE-labeled cells not divided.

Figure 6. TT-presenting T? Cells induce modulation by decreasing TCR in TT? CD4 + specific T?? Cells.

T cells are stimulated and mature γ δ and the DC, respectively, in the presence of increasing concentrations of TT, ranging from 0 (without TT added) up to 1,000 µg / ml and then grown with alpha? CD4 + T cells specific for TT at rest at an APC ratio: cells that respond 1: 2. After 18 hours, it determines the level of CD3 expression in T cells α? by flow cytometry and shown in% of average fluorescence intensity (MFI) compared to cells without added TT. T cell preparation is performed [gamma] delta of TT and the responding cells as well as the analysis of the data exactly as described in figure 4.

Figure 7. TT presenting δ T cells induce expression of activation markers in TT? CD4 + specific T? Cells.

The level of the markers of CD25, ICOS and OX40 activation in T?? cells CD4 + specific for TT (responding cells) by flow cytometry after culture for 5 days with T cells γ δ that are stimulated in the presence (+ TT) or absence (-TT) of 10 µg / ml of TT (see Figure 4). Histograms voids and fillings show fluorescent stains with specific and control isotype antibodies, respectively; n = cell counts The horizontal bars represent the intervals for positive marker cells, and numbers above the histograms indicate the degree of positivity, expressed as a percentage of positive cells (%) and intensity of medium fluorescence (MFI).

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Figure 8. Heterologous EBV-B cells and TT-specific CD? + T? Cells do not functionally present TT to TT-specific responding cells.

CP.EBV cells are grown, cells that respond (TT? CD4 + T? cells specific for TT) and T? δ cells (as a positive control) for 1 day in the presence of 20 µg / ml of TT, they are washed, irradiated and then they are added to the α? beta CD4 + cells specific for TT at a ratio of 1: 2. After 3 (3d) and 8 (8d) days culture, CFSE signals in CD4 + cells are examined  by flow cytometry. "Examples" describe the preparation of CP.EBV cells and those that respond, marking with CFSE and flow cytometry.

Figure 9. Stimulated γδ T cells are also effective in the processing and presentation of complex protein antigen (PPD).

Tγ? Cells are stimulated / matured and DC in the presence (+ PPD) or absence (-PPD) of 20 µg / ml of PPD and are analyzed to determine the induction of proliferation in α? beta CD4 + cells specific for autologous PPD Resting. As an additional negative control, T cells are cultured α? beta CD4 + specific for PPD in the absence of Tγ or DC cells but in the presence of 20 µg / ml of PPD. The experimental system is identical to the one used in Figure 4. "Examples" describe the preparation of T cells δ δ and DC, CFSE labeling of T cells α? beta CD4 + specific for PPD and cytometry of flow.

Figure 10. Tγδ cells loaded with TSST-1 induce proliferation in autologous virgin α? CD4 + T cells.

Stimulated Tγδ cells (Tγδ), stimulated Tαβ CD4 + cells (Tαβ), mature DC (DC) and freshly isolated blood monocytes (M) are loaded with 10 ng / ml TSST-1 and mixed with freshly isolated CFSE labeled α? beta CD4 + cells at a ratio of 1: 5. After 4 days of culture, proliferation responses in V? 2 + -?? (V? 2) T cells are determined by flow cytometry. The vertical and horizontal lines on point transfers define the intervals for the TV? 2 + -?? Cells and the divided cells; the upper left and lower left quadrants show the divided V? 2 + cells and the divided V? 2 neg cells, respectively, and the upper right and lower right quadrants show the V? 2 + cells undivided and undivided Vβ2 neg cells; the numbers refer to the percentage of total cells present within the individual quadrants. Cell preparation, loading with TSST-1 and flow cytometry are performed according to
"Examples."

Figure 11. Tγ? Cells loaded with TSST-1 demonstrate potent APC functions (TSST-1 titration).

Tγδ (T) cells are loaded δ) and T?? (T??) cells, dendritic cells (DC) and monocytes (M) with concentrations increasing TSST-1 and mixed with T cells α? beta CD4 + virgins labeled with CFSE at a rate of 1: 5. After 4 days of culture, the number of T cells is determined V? 2 + -?? Divided by culture well (T V? 2 +) by flow cytometry (see upper left quadrants in figure 10). The antigen presenting cells tested are the shown in Figure 10 and, in addition, T?? cells stimulated that are grown for 7 days and then loaded with variable concentrations of TSST-1 (T γ δ 7d). Each data point plus error bar represents the mean ± standard deviation (SD) of values duplicates of 2 separate experiments and the data are representative of 3 independent experiments. The preparation of cells, loading with TSST-1 and cytometry of Flow are performed according to "Examples".

Figure 12. Tγ? Cells loaded with TSST-1 demonstrate potent APC functions (APC titration).

Stimulated γδ T cells are loaded and mature DC with either 1 µg / ml or 100 ng / ml of TSST-1 and undergo various dilutions (from 1: 5 to 1: 200) of APC: T?? cells (APC: T cells γ δ or mature DC) for the induction of proliferation in virgin Tα? CD4 + cells. The preparation of cells, loading with TSST-1 and cytometry of Flow are performed according to "Examples".

Figure 13 (13A and 13B). Tγδ cells loaded with TSST-1 induce differentiation of helper T cells into virgin Tα? CD4 + cells.

Tγ? Cells are mixed stimulated, mature T?? or DC cells loaded with 10 ng / ml TSST-1 with α? T cells Virgin CD4 + and proliferation cells are examined that they respond Vβ2 + as described in Figure 10. After 4 days of culture, expression of the marker of the CD45RO memory and the degree of cell division by cytometry of flow in cells that respond Vβ2 +. After 21 days of culture, when the cells return to a non-proliferative state, at rest, the cells are stimulated by PMA / ionomycin and are examine by flow cytometry to determine production of intracellular cytokines IL-4 e IFN-? (Figure 13B). The numbers in the upper left, upper right and lower right quadrants define the fraction (percentage of total cells) of Th2 cells, Th0 and Th1 generated, respectively, and the numbers above punctual transfers refer to the APC reason with regarding the cells that respond to the start of the cultures cell phones. Cell preparation, loading with TSST-1 and the determination of the production of Cytokines are described in "Examples."

Figure 14. The induction of proliferation in virgin T?? CD4 + cells by T?? Cells loaded with TSST-1 is cell contact dependent.

Stimulated γδ T cells are loaded with 100 ng / ml TSST-1 and T cells are cultured α? beta CD4 + virgins labeled with CFSE alone (A) or together (B) with γ T cells at an APC ratio: cells which respond from 1: 5. Figure 5 describes the system of two chamber culture and analysis of cytometry data from flow with CFSE.

Figure 15. Tγδ cells induce responses of primary CD8 + T cells.

Leukocyte responses mixed with T cells α8 CD8 + virgins and T cells? stimulated with heterologous IPP (circles), DC derived from monocytes, matured with LPS (squares) or T cells α? beta stimulated with superantigens (triangles) a decreasing reasons (representative of 6 experiments) of APC: cells that respond. The responses of proliferation in cells that respond with CD8 + labeled with CFSE by flow cytometry data analysis as it is described in figure 5.

Figure 16. Tγδ cells induce differentiation of virgin CD8 + T cells into cytotoxic T cells.

CD8 + T cells derived from mixed leukocyte responses, as described in the Figure 15, after 14 days of culture to determine the activity cytolytic The cytotoxicity assay included effector cells, ie CD8 + T cells derived from leukocyte responses mixed, and CFSE labeled target cells. The mixture of target cells contained true target cells (T cells Heterologous CD4 +) and negative control target cells (cells Autologous CD4 + T) at a ratio of 1: 1. The cells were marked true and negative control target with different CFSE concentrations before mixing in order to differentiate between the two subgroups of target cells.

(A) Flow cytometric analysis of the mixture of target cells in the absence of effector cells shows Negative control cell (C) and cell populations true target (T).

(B) After 12 h of T cell coculture CD8 + and target cells at 30: 1, 10: 1, 3: 1 and 1: 1 ratios, are evaluated the degree of lysis of target cells by measuring the decrease in number of the population of true target cells (arrow). The numbers next to the arrows refer to the percentage of specific destruction and are related to the loss of CFSE high fluorescence cell counts (target cells true) compared to control cell counts with low fluorescence of CFSE.

Left column: T cells were derived CD8 + from leukocyte responses mixed with γ T cells stimulated with IPP such as APCs.

Right column: T cells were derived CD8 + from leukocyte responses mixed with DC mature like the APC.

Figure 17. Tγδ cells express high levels of DEC205 (CD205) and CD11b endocytic cell surface proteins.

Freshly γ T cells were analyzed isolated from peripheral blood (T?) and immature DC (DC) to determine the expression of CD205 and CD11b by flow cytometry as described in Figure 7. The vertical and horizontal lines in the transfer figure point define the intervals for the cells that express CD205 or CD11b, and the upper right quadrants in the figure represent double positive cells for CD205 and CD11b; the numbers they refer to the percentage of total cells present within of the individual quadrants. Positivity is defined by staining with matching isotype control antibodies, and lines horizontal or vertical represent the intervals for 99% of The background stains.

Detailed description of the invention

The flow chart of the scheme summarizes the method of the invention for the preparation of effective antigen-presenting human T γ δ cells (TV γ2V δ 2 + γ δ cells, hereinafter referred to herein) "T γ cells") by isolation and in vitro treatment of T γ cells from human peripheral blood. Although protocols for the isolation and in vitro proliferation of Tγγ cells as such are known, the particular combination of stimulation and application of antigens or loading of peptides has not been described. The starting material is human peripheral blood that is treated by differential centrifugation or immunoabsorption to give expanded or freshly isolated γδ T cells, respectively, at high purity. Short-term stimulation (for example 24 hours) in combination with the application of antigens or the loading of peptides results in γ-T cells with effective antigen presentation function for use in immunotherapy.

to)

Tγ cells delta easily expand during peripheral blood lymphocyte (PBL) culture in the presence of isopentenyl pyrophosphate (IPP) or other low molecular weight non-peptide compounds with selectivity for Tγ cells such as It is listed below in this document (Morita et al ., 2000; Eberl et al ., 2003). IPP or other low molecular weight non-peptide compounds with selectivity for T? Cells are also used to induce antigen presenting functions in selected T? Cells as described hereinbelow. After 10-21 days in culture, the vast majority of live expanded cells are Tγδ cells (Vγ2Vδ2 +), which are separated from dead cells by centrifugation with Ficoll-Paque and used immediately for the generation of antigen presenting cells (APC) or stored in liquid nitrogen. Alternatively, as described in "Examples," T? Cells are isolated directly from peripheral blood mononuclear cells (PBMC) by positive selection.

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Scheme

T cell preparation γ δ human antigen presenting effective

one

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b)

Be stimulate freshly isolated γδ T cells or expanded over a short period (for example from 12 to 96 hours) with selective γ-T cell compounds (Vγ2V δ2 +) or phytohemagglutinin (PHA) or others stimuli as listed below herein document for the induction of antigen uptake, of the presentation and molecule expression function costimulators

C)

The Tγγ cells are treated before, during or after stimulation (b) with antigens which results in T cells γ δ antigen presenting. This application of antigens includes a variety of independent approaches, such as (among others) the addition of complex extracts or proteins defined from tumors, microbes or cells infected with virus or DNA / RNA encoding such proteins derived from pathogens, either in the form of purified nucleic material or packaged in attenuated expression vectors or viruses for transfection / transduction of T cells δ and the endogenous expression and processing of antigens derived from microbes, pathogens or tumor cells.

d)

In instead of adding antigen (in the form of protein or DNA / RNA) to γ T cells during the stimulation procedure for short term (combinations of b and c), these stages can be performed sequentially Then the T cells γ δ stimulated are "loaded" for a period short time with defined peptide antigens that do not require intracellular proteolytic processing.

In addition to IPP, the non-peptide antigens of low molecular weight alternatives with selectivity for T cells γ δ (V γ 2 V δ 2 +) considered in the step (a) are pyrophosphate of (E) -4-hydroxy-3-methyl-but-2-enyl  (HMB-PP), ethyl pyrophosphate (EPP), pyrophosphate farnesyl (FPP), dimethylalkyl phosphate (DMAP), pyrophosphate dimethylalkyl (DMAPP), ethyl adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), isopentenyl adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), pyrophosphate 3-formyl-1-butyl (TUBAg 1), X-pyrophosphate (TUBAg 2), triphosphate 3-formyl-1-butyl-uridine  (TUBAg 3), triphosphate 3-formyl-1-butyl deoxythymidine (TUBAg 4), monoethylalkylamines, allyl pyrophosphate, pyrophosphate of crotoílo, triphosphate of dimethylalkyl-γ-uridine, triphosphate crotoyl- γ-uridine, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, iso-amylamine and bisphosphonates containing nitrogen. The IPP is preferably applied as presented by B cells or substitutes.

The positive selection is made, for example, adding antibodies against Human γ2V δ2-TCR to cells human peripheral blood mononuclear, for example incubation of 1-3 hours, followed by cell sorting magnetic Alternatively, a positive selection can be made adding antibodies against Human VγVδ-TCR to cells human peripheral blood mononuclear (T cell selection pan- \ gamma \ delta) followed by classification magnetic cell

Selective T-cell compounds γ δ (V γ 2 V δ 2 +) useful as stimuli for the induction of antigen presenting functions in the step (b) are IPP and other non-peptide compounds that are listed above for step (a), for example pyrophosphate from 4-hydroxy-3-methyl-but-2-enyl  or other related microbial metabolites. Alternatively, the PHA or other substitutes are useful as stimuli for induction of antigen uptake, presentation function and expression of costimulatory molecules.

If desired, cells can be irradiated stimulated in order to inhibit the proliferation of T cells γ δ.

Examples of antigens applied as proteins in step (c) and / or (d) are those currently used in immunotherapy protocols that employ DC as antigen presenting cells and are related to therapeutic targets, for example tumor cells, infectious agents. (microbes including viruses, bacteria, yeasts and parasites) and toxins associated with pathogens. Such antigens include (but are not limited to) tumor-associated antigens (tumor-specific cell surface, structural and metabolic proteins, and the like); virus-associated antigens (enzyme, metabolic, structural and envelope proteins encoded by viruses, for example HIV gp120 proteins of different viral clades, HIV Tat, HIV proteases, for example enzymatic, structural, metabolic and envelope proteins derived from hepatitis virus, influenza virus, human cytomegalovirus, polio virus, rabies virus, herpes virus, among others); bacteria-associated antigens (enzymatic, metabolic, structural and cell-wall proteins encoded by bacteria, and the like, for example those derived from Mycobacteria (for example M. tuberculosis , M. leprae ), Listeria monocytogenes , Pneumococci , Staphylococci (for example S aureus ), Streptococci (for example S. pyogenes , S. pneumoniae ), Vibrio cholerae , Clostridium tetani , among others); yeast and fungal associated antigens (enzymatic, metabolic, structural and cell wall proteins encoded by yeasts / fungi, and the like, for example those derived from Candida albicans , Aspergillus fumigatus , among others); and toxins derived from pathogens (bacterial enterotoxins, for example staphylococcal enterotoxins, toxic shock syndrome toxin, tetanus toxins, among others). Examples of antigens applied as proteins also include those related to bacteria that cause an increase in resistance to antibiotic treatment and those microorganisms that cause life-threatening diseases worldwide (for example Plasmodia (for example P. falciparum , P. vivax , P Malariae ), Leishmania , Trypanosoma , Entamoeba , Schistosoma , Filaria , among others). The antigens applied in step (c) in the form of RNA / DNA include those currently used in blood and tissue cell transfection protocols and include (but are not limited to) those that code for tumor cell proteins, infectious agents (microbes) including viruses, bacteria, yeasts and parasites) and toxins associated with pathogens, as listed above.

"Effective" as used in "effective human antigen presenting γ T cells" means that the antigen presenting functions of such cells are comparable to the corresponding antigen presenting functions of the DC. "Effective" is for example at least 10% as effective as with DC under comparable conditions, the difference being a result of different cell morphology, which includes the cell form and area
superficial.

Tγδ cells stimulated by short term evenly express the CCR7 chemokine receptor, which is a prerequisite for APCs to address the lymph nodes and Peyer's plaques and therefore is a critical factor for the initiation of immune responses adaptive T γ peripheral blood cells in resting do not express MHC-II molecules and therefore cannot present peptide antigens in a way tolerogenic This "security" issue differentiates γ T cells from other APCs, such as DCs and B cells, with known tolerance induction properties. The applications of the presenting T? Cells presenting antigen include, among others, induction and / or improvement of immune responses against tumors in the treatment of patients with tumors and against microbes and viruses in the treatment of patients with chronic or competent infections Inappropriate immune In addition, T?? Cells antigen presenters can be used for the identification of antigens derived from tumors or otherwise from novel pathogens with immunogenic properties marked for potential application in immunotherapy Finally, the application of the cells is described T γ δ to monitor immune competence adaptive (immune competence status) of individuals immunosuppressed (among others).

The invention is now described in more detail. with reference to the attached figures, which provide extensive evidence of the effectiveness of the method of the invention.

Figure 1 describes the expression of molecules of cell surface in Tγγ cells, immediately after peripheral blood isolation or after Stimulation and culture for 1 or 7 days. For the comparison, the same surface molecules are also examined cell in T?? cells stimulated for 1 day and Freshly isolated monocytes. Cell surface molecules shown in figure 1 include MHC-II; the CD80 and CD86 costimulatory molecules, the two selective ligands for the CD28 receptor present in virgin T cells and of memory; CD70, the ligand for CD27; CD40, the ligand receptor of CD154 / CD40 as well as adhesion molecules CD54 / ICAM-1, CD11a / αL-integrin, and CD18 / β2-integrin, which are involved in the cell-cell contact These data show that freshly isolated γδ T cells have levels moderate adhesion molecules but lack molecules of MHC-II and costimulators that are essential for activation and differentiation of virgin T cells. For him on the contrary, the short-term (1 day) stimulation of T cells γ δ results in very high levels of expression of MHC-II molecules and costimulators and also enhance the expression of adhesion molecules. Of importance, and with the exception of CD40 and CD54, the levels of these are maintained cell surface molecules or are further enhanced during the cultivation of Tγγ cells for 7 days. In clear contrast, the same series of molecules (with the exception of CD86 in monocytes) are expressed moderately or absent in T?? cells and monocytes.

The numbers in table 1 summarize the results of an extensive investigation of the expression of the molecules of cell surface in the δ T cells and, for the comparison, in mature T?? cells, monocytes and DC. The list includes the examples shown in Figure 1 as well as additional cell surface molecules. The numbers indicate average expression levels, expressed as the average of the average fluorescence intensity (MFI), and standard deviations corresponding (DE). The remarkable positivity of the MHC-II (HLA-DR) molecules, costimulators and adhesion in γ T cells activated that resembles mature DC but far exceeds differentiates the levels observed in the α? beta cells activated and monocytes.

Figure 2 and Table 2 correspond to previous results published by Brandes et al ., 2003, and are included in this case to describe the additional characteristics of activated human γδ cells that are relevant to this invention. Table 2 lists the expression levels of chemokine receptors that clearly differ between newly isolated peripheral blood γδ cells and stimulated γδ T cells. Tγ cells in peripheral blood express chemokine receptors and adhesion molecules (shown in Figure 1) for rapid recruitment to sites of inflammation and infection while short-term activation of T cells gamma δ partially inhibits these inflammatory migration properties (i.e., decrease modulation of CCR2 and CCR5) and instead rapidly induces CCR7 expression to effectively target the T cell areas of the spleen, LNs and PP

TABLE 1 Levels of surface molecule expression cell in human γ T cells

2

TABLE 2 Expression of chemokine receptors in cells T γ human δ

4

Figure 2 illustrates the migration properties of activated γδ T cells in the short term, as assessed by in vitro chemotaxis analysis (Brandes et al ., 2003). The data clearly demonstrates the responsiveness to the CCR7 ligand SLC / CCL21 and the markedly reduced responsiveness to the CCR5 (and CCR1, CCR3) RANTES / CCL5 ligand in activated γδ cells. The data also demonstrate that changes in the level of cell surface chemokine receptors are directly reflected in the migration responses to the corresponding chemokines.

Together, the results summarized in Figures 1-2 and Tables 1-2 underline the fact that T? Cells stimulated but not at rest (freshly isolated) from peripheral blood express many essential factors, including adhesion molecules and costimulators, and chemokine receptors, required to target LN and the stimulation of virgin T cells. The activation-induced modulation of these migration-related parameters and with APC compares well with the activation-induced changes that occur in DC (Banchereau and Steinman, 1998; Steinman et al ., 2003; Banchereau et al . , 2004). In addition, these data imply that stimulated human Tγ cells act as potent APCs comparable to those
DC

The formation of cell clusters Fast, extensive and antigen independent is characteristic of DC-T cell interactions and is a requirement prior to antigen-dependent stimulation and differentiation of virgin T cells. Figure 3 documents that stimulated Tγγ cells also induce huge cell clusters with virgin CD4 + T cells (at rest) within 3 hours of cultivation. Of importance, you are clusters are formed in the absence of antigen, suggesting that adhesion molecules and costimulators are responsible for this effect. The formation of groupings mediated by T cells γ δ is at least as strong as that observed with DC mature In contrast, activated α? Beta cells for 1 day or freshly isolated monocytes (not shown) are much less  effective, which is consistent with the reduced levels of Adhesion molecules and costimulators in these cells. These data provide additional evidence that T cells γ δ activated by short term have a function of Powerful APC.

Activated γδ T cells can capture, process and present antigen to trigger responses in the antigen specific δ? CD4 + T cell lines. Figure 4 shows the proliferation of tetanus toxoid (TT) specific α? Beta CD4 + T cells in response to TT-presenting T? Cells, stimulated by short-term or, as a control, presenting DC of TT. The TT? CD4 + T? Cell line is derived from the same donor that provided the?? Cells. Tα? CD4 + cells specific for TT at rest are loaded with CFSE and proliferation of the responding cells is determined by measuring the reduction of CFSE signals in cultured cells by flow cytometry (see " Examples "). During cell division, CFSE content is distributed in the two daughter cells so that each round of cell division is characterized by a 50% reduction in CFSE signals. This type of analysis allows the determination of a) fractions of cells that do not respond to those that proliferate (input / maximum versus reduced CFSE signals), b) cell subgroups with differentiated rounds of cell division, and c) the fraction of cells at the beginning of the experiment that have responded to the APC. This information cannot be obtained by performing the 3 H-thymidine incorporation assay, which is an alternative method for determining cell proliferation. Figure 4 shows that T? Cells can induce proliferation in TT-specific CD4 + T cells and that this response is completely dependent on the antigen, since T? Cells that have been stimulated in the absence of TT are inactive. This further shows that between day 4 and day 6 of culture the CD4 + T cells continue to proliferate, as evidenced by further reduction of CFSE signals (left shift of fluorescence signals in the figure 4). Of importance, γδ T cells are potent in a similar manner to mature DCs in this response, demonstrating that short-term stimulated γδ T cells have potent presentation functions.
antigen.

Figure 5 illustrates the fact that the TT-specific proliferation responses require contact cell between the T? -tata cells presenting TT and TT? CD4 + specific T?? cells that respond (cells that respond). The responding cells that are separated by a porous membrane from the cocultures that contain Tγ-presenting TT cells and cells They respond do not proliferate. Porous membranes prevent cell exchange but do not prevent mediator exchange soluble between the culture compartments. Therefore, the results in figure 5 also show that cytokines and the growth factors produced during the co-cultivation of TT presenting δ T cells and cells that respond have no effect on the proliferation of cells that They respond that they are grown separately.

The activation of the TCR is accompanied by the internalization of the TCR, which results in the regulation by decrease of the TCR of cell surface and auxiliary molecules, such as CD3. Figure 6 documents that TT? Presenting T cells induce modulation by decreasing TCR in TT-specific responding cells (see Figure 4), as assessed by the loss of cell surface CD3. Accordingly, T?? Cells have the ability to capture and process TT and present TT-derived peptides in the form of MHC-II-peptide complexes to TT-specific responding cells in a manner sufficient for binding to the TCR. Since peripheral blood T? Cells? Activated but not at rest express cell surface MHC-II molecules, the antigen presentation function is closely associated with the activation of?? Cells. Figure 6 also shows that TCR decrease regulation is a function of the density of TT peptides in stimulated γδ T cells, which is controlled by varying the amount of TT added during stimulation of Tγ cells. \delta. Obviously, the more MHC-II-peptide complexes derived from TT occur in stimulated γδ T cells, the more TCR in the responding cells will couple and regulate by decrease. The different potency in the modulation by decrease of TCR between the Tγ and DC cells is probably due to notable differences in cell morphology, since the cellular surface area in mature antigen presenting DCs is> 10 times greater than in stimulated δ T cells (Miller et al .,
2004).

In addition to proliferation, TT-presenting δ T cells induce expression of activation markers in TT-specific CD4 + T cells. Figure 7 shows the regulation by increase in the cell surface of cells that respond to T cell activation markers, including CD25, ICOS and CD134 / OX40, which are de novo expressed or potentiated in response to TCR activation. As in the induction of T-cell proliferation, the high level expression of these activation markers is completely dependent on the presentation of TT peptides by short-term stimulated γ-T cells, since these activation markers do not they are induced with Tγγ cells, which are stimulated in the absence of TT.

Figure 8 shows that heterologous Epstein Barr virus (EBV) immortalized B cells and autologous responding cells lack the TT presentation function. A protocol for the stimulation of γδ T cells includes the use of irradiated IPP presenting cells that are either autologous B cells or, for the convenience of in vitro experimentation , B cell lines with EBV, is say, the heterologous CP.EBV line. Figure 8 demonstrates that TT-treated CP.EBV cells do not induce proliferation of TT-specific CD? + T? Cells, indicating that CP.EBV material did not contribute to proliferative responses labeled obtained with the TT? peptide presenting T? cells. Also, Figure 8 illustrates the function of deficient TT presentation of the T?? Cell line itself.

Similar effects are obtained to those shown in Figures 4-8 with TT with the purified protein derivative (PPD) of complex / undefined antigen of Mycobacterium tuberculosis . Figure 9 shows proliferation responses of PPD-specific α? CD4 + cells after 4 days or 8 days of culture after stimulation with PPD peptide-presenting δ T cells or, alternatively, DC PPD peptide presenters such as APC. Again, the experiments are performed under autologous conditions, that is, the APCs are derived, (Tγ and DC cells) and the responding cells (Tα? CD4 + T cell line specific for PPD) from the same donor. As documented for TT, proliferation responses are specific for PPD and do not differ substantially between Tγ and DC cells. Also, the α? T cell line itself does not induce proliferation in PPD-specific responsive cells. At equal concentrations of antigen, T? And DC cells induce more vigorous responses with TT compared to PPD, which is due to differences in complexity between TT (M_ {r} [TT]: 150 kDa ) and PPD (M_ {r} [PPD]: ≥10,000 kDa). Complex antigens, such as PPD or whole microorganisms, are highly diverse in the repertoire of antigenic peptides with the consequence that individual APCs have discrete MHC-peptide complexes at low levels. Accordingly, modulation by decrease of TCR is much less evident in CD4 + T cells specific for PPD, cloned, during co-culture with PPC-presenting APCs than what is observed in the TT system (Figure
6).

Together, the experiments with cells that respond specific to TT and specific to PPD document the finding of the present invention that T cells γ stimulated (but not at rest) derived from Human peripheral blood have antigen uptake functions, antigen presentation and T cell stimulation powerful The potency and effectiveness of these T cell functions γ δ are notable and equal to those obtained with the DC

The characteristic of effective APCs, such as DCs, is their ability to induce primary adaptive immune responses that involve the stimulation of virgin T cells (no experience with antigens) and their differentiation into antigen-specific effector T cells with the ability to produce cytokines or destroy target cells (Banchereau and Steinman, 1998; Steinman et al ., 2003; Banchereau et al ., 2004). Fully differentiated memory T cells have reduced activation thresholds, and TCR activation in the absence of costimulation is sufficient to initiate effector functions. The experimental results shown in the following figures demonstrate that stimulated Tγγ cells also have effective antigen presenting functions comparable to those of DC. Thus, instead of autologous, newly isolated α? CD4 + T? Cell lines, autologous newly isolated?? CD4 + cells are used as responsive cells power-
them.

Figure 10 shows the degree of proliferation in virgin Tα? CD4 + cells, labeled with CFSE in response to γδ T cells stimulated by short term, loaded with toxin from toxic shock syndrome (TSST-1) and mature DC loaded with TSST-1, instead of T?? Cells stimulated loaded with TSST-1 or fresh monocytes isolates loaded with TSST-1. He TSST-1 binds to the molecules of MHC-II in APCs and is selective for α? -TCR containing the chain V? 2. 4-10% of CD3 + T cells from Peripheral blood are Vβ2 + and respond with high affinity to the APC presenters of TSST-1. As it demonstrates in Figure 10, the loaded δ? cells with TSST-1 they are APC experts in the induction of proliferation of virgin V? 2 + T cells, as evidence by reducing the CFSE signals, and this response is much more prominent in the α? beta cells CD4 + virgins carrying the V? 2 <+> - TCR than those carrying the V? Neg -TCR. Finally, after the completion of cell expansion, most of the T cells of Resulting memory express V? 2 + - TCR (see also table 3). Importantly, the responses of proliferation are equal to those obtained with DC loaded with TSST-1 Under these conditions, monocytes or T?? cells loaded with TSST-1 are completely inactive, indicating that the level of presentation and / or costimulation of TSST-1 in these cells is not enough to induce primary T cell responses effective.

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TABLE 3 Tα? And cell expansion Th cell differentiation during long culture term (21 days)

5

6

In addition to costimulatory molecules, the parameters that largely determine the kinetics and the degree of proliferation in virgin Tα? CD4 + cells are the density of MHC-II-TSST-1 complexes in APCs and the reason between APC and responding cells. Figure 11 shows the proliferation of cells that respond to Vβ2 + virgins in response to increasing concentrations of TSST-1 that are used to load the different types of APC. Prior to the addition to virgin Tα? CD4 + cells, APCs are washed to remove excess TSST-1 (see "Examples"). The list of autologous APCs includes γδ T cells stimulated for 1 day or 7 days, mature DC, freshly isolated monocytes and Tα? Cells stimulated for 1 day. The δ T cells stimulated for 1 day induce T cell proliferation responses at concentrations with TSST-1 loading of only 1 ng / ml and are equally effective as DCs for maximum responses. The approximately 10 times greater potency of DCs may be due to their greatly enlarged surface area (Miller et al ., 2004), which allows more frequent and extensive contacts with the responding cells (see also Figure 6). Of importance, substantial proliferation responses are still obtained with stimulated γδ T cells that expand during culture for 7 days before loading with TSST-1. Obviously, Tγγ cells maintain APC functions for prolonged periods of time, which is in accordance with the observed preservation of adhesion and costimulatory molecules (see also Figure 1 and Table 1). Monocytes and T?? Cells are> 100 times less potent than T?? Cells. These data demonstrate that stimulated γ T cells have effective antigen presenting functions.

In the experiment shown in Figure 12, in time of the TSST-1 titration during the loading of APC, the ratio of APC is varied with respect to the cells that virgins respond between 1: 5 and 1: 200 while maintaining the TSST-1 load concentration at 100 ng / ml or 1 \ mug / ml. Proliferation responses of the Virgin α? CD4 + V? 2 + T cells as in Figure 11. There is no obvious difference between T cells δ δ and DC stimulated for 1 day, and at the dilution of Higher APC (1: 200) proliferation responses still oscillate between 28-40% of maximum responses. These data further illustrate the competence of T cells γ δ as effective APCs.

Primary immune responses involve the differentiation of virgin CD4 + T cells into polarized Th1, Th2 or Th0 cells capable of producing type 1 (IFN-?), Type 2 (IL-4) or cytokines type 0 (IFN-? + IL-4), respectively. Virgin CD4 + T cells have the ability to differentiate into any one of these polarized Th cells. The polarization of T cells is determined by the costimulatory environment provided by APCs at the time of sensitization of virgin T cells. Effective APCs not only induce the proliferation of virgin T cells but also support their differentiation into effector cells. Figure 13 demonstrates that stimulated δ T cells can fully present TSST-1 in the appropriate costimulation context for the generation of effector Th cells. Four days after sensitization with γδ T cells loaded with TSST-1, most of the virgin CD4 + T cells ββ2 + have responded by proliferation and expression of the CD45RO memory marker . After 21 days of culture, most cells return to a resting state, uniformly express CD45RO and consist of β2 + TV cells (table 3). Importantly, most cells produce typical cytokines either from Th1, Th2 or Th0 cells, and Th1 polarization is further enhanced by increasing the ratio of γδ T cells loaded with TSST-1 and cells that answers up to 1: 1. Again, the most prominent effect observed with CD may be due to the morphological criteria discussed above (Miller et al ., 2004) (see also Figures 6, 9). In contrast, non-selective activation of antigen (phytohemagglutinin) does not result in selective expansion of TV? 2 + cells (table 3), and stimulated T?? Cells do not induce CD45RO expression and the proliferation of virgin Tα? CD4 + cells (Figure 13).

As seen with T cells α? beta CD4 + specific for TT and specific for PPD  (see for example Figure 5), the induction of responses in Virgin α4 CD4 + T cells is completely dependent on cell-cell contact with Tγγ cells loaded with TSST-1 (figure 14). Stimulated γδ T cells induce specific antigen responses marked on T cells γ? CD4 + virgins and support their proliferation and differentiation in a typical way for effective APCs.

CD4 + T cells recognize complexes of MHC class II-peptide in APCs while CD8 + T cells recognize class MHC complexes I-peptide in the APC. MHC class molecules I express themselves ubiquitously in blood and tissue cells; therefore, all cells in the body are target cells potentials for CD8 + T cells. Of importance, the CD8 + T cells are crucial participants in the defense against viral infections and tumors and, frequently, the satisfactory vaccination depends on the generation of T cells Cytotoxic, antigen selective CD8 +. Figure 15 documents that V δ2 + T cells are highly APC potent in the induction of CD8 + T cell responses Primary Untouched CD8 + T cells, virgins such as cells that respond in proliferation assays that contain γ T cells stimulated with mature IPP, DC or cells Tα [beta] stimulated with superantigen such as APC (all of the same donor). Tγγ cells coincide completely or are even better than DC in induction of the proliferation of CD8 + T cells, and T cells α? are lower APCs. Figure 16 documents that Tγγ cells induce cell differentiation T cytotoxic effectors. The data illustrates that T cells γ δ are indistinguishable from the DCs in the drive of differentiation of virgin CD8 + T cells in cells Cytotoxic T, alloantigen specific. Together, the T δ2 + T cells stimulated with TCR induce responses labeled proinflammatory drugs in both the α? beta cells CD4 + as virgin CD8 + in a manner similar to APC "professional".

Tγ? Cells also express endocytic receptors, such as lectin type C DEC-205 (CD205) and the CD11b integrin subunit with selectivity for multiple protein and non-protein ligands. It is known that CD205 and CD11b are highly expressed in DC (Banchereau and Steinman, 1998; Steinman et al ., 2003; Banchereau et al ., 2004). Figure 17 documents that T? Cells, like DC, intrinsically express high levels of these endocytic receptors. In analogy with the novel methods of supplying DC antigens, these data demonstrate that antigens, including protein vaccines for tumors and infectious agents, can also target Tγ cells to process and present antigens in vivo.

These experimental details show that the method of the invention for the preparation of T cells γ δ human effective antigen presenters that comprises selecting T cells δ from cells human peripheral blood mononuclear, treat cells selected with a stimulus for the induction of functions antigen presenters, and apply the antigen to the cells Stimulated provides APC comparable to DC.

In particular, the invention relates to a such method for the preparation of T cells γ δ human antigen presenting effective in the that the selection of the δ T cells is made by magnetic cell classification with antibodies against human VγVδ-T cell receptors. Alternatively, T cell selection is performed γ δ culturing peripheral blood lymphocytes freshly isolated in the presence of low non-peptide compounds structurally defined molecular weight that induces expansion selective of γδ cells expressing chains of the V? 2V? 2 + - T cell receptor, for example in presence of IPP, for example as it occurs in B cells or substitutes.

The invention also relates to the method particular in which the stimulus for the induction of functions effective antigen presenters is a non-peptide compound of low molecular weight or a substitute or phytohemagglutinin, and the particular method in which the antigen is applied in the form of defined proteins, mixtures of undefined proteins, extracts gross or enriched tumor and infected cells, for example wherein the applied antigen is a pathogen derived peptide or of tumor cell, or a protein derived from pathogen that is applies in the form of DNA or RNA that encodes it under conditions that allow endogenous expression of said protein derived from pathogens, in particular in the form of purified DNA or RNA or a insertion vector containing such DNA or RNA. If the antigen is in the form of a DNA or RNA the conditions of so that said DNA or RNA can be properly expressed. The application of an antigen may occur before, during or after the stimulation of Tγγ cells for induction of antigen presenting functions. If an antigen is applied as a peptide (protein fragment), its application can also  be after stimulation in a separate stage called "load of peptide. "

Effective antigen-presenting human T? Cells prepared according to the invention as described hereinbefore may be used in immunotherapy. Such use may be similar to the known use of DC in immunotherapy, thereby overcoming the disadvantages of the use of DC such as peripheral blood shortage, inability to proliferate in vitro , heterogeneity and functional instability.

In particular, T cells can be used γ δ human effective antigen presenting prepared according to the invention as described above in this document for the manufacture of a medicine (pharmaceutical composition) for use in immunotherapy.

The present invention also relates to pharmaceutical compositions comprising T cells γ δ human effective antigen presenting prepared according to the invention as described above in this document, and which can be used especially in the treatment of the diseases mentioned above and to continued in this document. They are especially preferred compositions for parenteral administration, such as intravenous, intramuscular, subcutaneous, mucosal or submucosa, to humans. The compositions comprise the cells together with a pharmaceutically acceptable carrier. The Dosage of the cells of the invention depends on the disease that will be treated, and of age, weight and individual status, individual pharmacokinetic data and mode of administration. The pharmaceutical compositions comprise from about 0.01% to about 50% of the cells of the invention. Unit dosage forms are, for example, ampoules or vials

Preference is given to the use of isotonic aqueous suspensions. The pharmaceutical compositions may comprise excipients, for example preservatives, stabilizers, wetting and / or emulsifying agents, solubilizers, salts for regulating osmotic pressure and / or buffers and are prepared in a manner known per se , for example by means of mixing procedures. conventional. The manufacture of injectable preparations is usually carried out under sterile conditions, such as filling, for example, in ampoules or vials, and sealing the containers.

The invention further relates to a method of treatment of tumors or chronic infectious diseases or recurring in which human T? cells are injected Effective antigen presenters in a patient who needs them. In particular, the method involves single or repeated applications of said Tγγ cells, for example compositions pharmaceuticals that contain them, intradermally, subcutaneously, intravenous, mucosa or submucosa.

In the method of treating tumors, they are used Tγγ cells, which have been stimulated in the presence of defined tumor proteins or tumor cell extracts gross (not defined) or, alternatively, that have been obtained using treatment with recombinant RNA / DNA technologies that routinely use for transfection or cell transduction live blood or tissue. Preferably, if the Tumor peptides defined for direct loading into molecules of cell surface MHC, then such peptides are added to the stimulated γδ T cells are incubated, washed and They are used immediately for therapy. It is important to emphasize that the number of T? cells used per administration as well as the route and frequency of T cell administration γ δ depend on the induction efficiency of the immune response with the individual tumor antigen used thus such as the type and location of the tumor present in the patient individual. Preferred protocols are, for example, 1-20 x 10 6 cells / 0.5-2 ml by administration with 1 to 6 successive administrations with the same or smaller amounts of cells at two week intervals at two months.

In the disease treatment method chronic or recurrent infectious, T cells are used γ δ that have been stimulated in the presence of agents defined infectious, preferably in an attenuated form, or Gross infected cell extracts (undefined) or, alternatively, they have been obtained using treatment with recombinant RNA / DNA technologies that are routinely used to transfection or transduction of blood or tissue cells live The preferred treatment protocol corresponds to that described previously.

The invention further relates to a method of vaccination against tumors or chronic infectious diseases or recurring in which human T? cells are injected effective antigen presenters, to which they have been applied non-infectious and non-tumor antigens, in a patient who is going to get vaccinated For vaccination purposes, they apply procedures described above herein document but in which δ T cells are used at that non-infectious and non-tumor antigens have been applied. The Preparation of antigen-presenting δ T cells of vaccine and administration of these cells for vaccination of patients against tumor antigens or infectious agents follow the description of tumor immunotherapy (see previously). Preferred protocols are those currently employed in vaccination treatments based on DC. The immune status of such treated patients, that is, the quality (efficacy, kinetics, etc.) of the vaccine responses, as described above.

The invention further relates to another method of vaccination (prophylactic or therapeutic) against tumors or vaccination against agents that induce infectious diseases or non-infectious, which includes the administration of vaccines targeting γT cells to individuals. Preferably, such vaccines targeting T cells γ δ are hybrid compounds composed of agents of vaccine and molecules targeting Tγγ cells. The molecules targeting T? cells are antibodies or specific ligands for endocytic T-cell receptors δ δ, which include but are not limited to CD11b and CD205. Vaccine agents are proteins or related molecules against to which immune protection is desired.

Administration of cell-directed vaccines Tγγ involves repeated treatment of individuals with vaccines targeting δ T cells by injections, oral administrations or any other protocol of vaccine supply that provides immune protection optimal

The invention further relates to a method of identifying antigens derived from novel tumors or pathogens comprising selecting Tγγ cells from human peripheral blood mononuclear cells, treating the selected cells with a stimulus for induction of the presenting functions of effective antigen, apply fractions of a mixture of undefined proteins, or crude or enriched extracts of tumor and infected cells, or libraries of RNA or DNA derived from tumors and infectious agents, test for in vitro activation of T cells \ Autologous alpha? beta and compare the activation results of different antigen fractions.

In this method, Tγγ cells are used as in vitro selection tools for the identification of novel and improved antigens for use in therapeutic and prophylactic vaccinations. Isolation, selection and stimulation of γδ T cells is performed as described in the method of preparing effective antigen presenting human γδ T cells. In the antigen application stage, fractions of crude antigen preparations (for example cell extracts or undefined mixtures) or RNA / DNA libraries derived from tumors and infectious agents are used. Then such δ T cells prepared for detection of the activation of the virgin Tα? Cells of the same donor, ie autologous Tα? Cells, are tested. The readings in these in vitro immune response assays are the proliferation and production of cytokines in T?? Cells, or any other simple measurement of T?? Cells activation. The culture conditions are preferably those described above for the responses of the?? T cells versus T?? Cells presenting TSST-1 in Figures 10-14 and in the "Examples". When the results of activation of different fractions of antigens are compared, the responses of the improved?? Cells indicate that the sources of antigens are "enriched" in terms of immunogenicity. The corresponding "enriched" fractions are further treated, and the entire cycle of experimental steps is repeated with the additionally fractionated antigens. Finally, repeated fractionation of the sources of "enriched" antigens (fractionation of the DNA or protein library) leads to individual proteins with maximum immunostimulatory functions. Such novel proteins can also be manipulated by proteolytic cleavage, and consequently the cleavage mixtures are analyzed to detect the generation of (small) immunogenic peptides for direct loading into the APCs.

The invention further relates to a method of diagnosing the immune competence of a patient comprising selecting Tγγ cells from peripheral blood mononuclear cells of the patient, treating the selected cells with a stimulus for induction of the presenting functions of effective antigen, apply the antigen for which immune competence is to be determined and test for in vitro activation of autologous α? beta cells.

In this method, the δ T cells and their impact on the antigen-specific memory Tα? Cells of the same donor are used as in vitro tools for the diagnosis of a patient's immune competence with respect to a particular antigen and for the determination of whether a vaccination has been satisfactory. Isolation, selection and stimulation of γδ T cells is performed as described in the method of preparing effective antigen presenting human γδ T cells. In the antigen application stage, the particular antigen for which the immune competence is to be determined is applied to the stimulated Tγ cells. The culture conditions and the readings for the determination of the responses of the?? T cells ( in vitro immune response assays ) are preferably those described above for the responses of the?? Cells against T? Cells? gamma δ presenters of TSST-1 in Figures 10-14 and the "Examples". The decisive difference is that memory T?? Cells are monitored instead of virgin T?? Cells to detect enhanced immune responses (proliferation, cytokine production, etc.) during stimulation with T cells? gamma δ antigen presenting. Successful immunotherapy (vaccination) leads to the generation of antigen-specific memory / effector T cells, which will provide many times improved immune responses during in vitro monitoring of the immune status compared to virgin Tα? Beta cells. These assays are preferably performed with mass? (? Unfractionated)?? Cells, since antigen-specific memory?? Cells are highly enriched in successfully vaccinated individuals.

Examples Abbreviations

DC

dendritic cell

LN

lymph node

PP

peyer plates

TCR

T cell antigen receptor

BCR

B cell antigen receptor

MHC

major histocompatibility complex

APC

antigen presenting cell

cells V δ1 + T

T cells γ δ expressing chains of V δ1 + - TCR

cells V γ2V δ2 + T

T cells γ δ expressing chains of V γ2V δ2 + - TCR

IPP

isopentenyl pyrophosphate

TT

Clostridium tetani tetanus toxin

PPD

purified protein derivative of Mycobacterium tuberculosis

TSST-1

Staphylococcus aureus toxic shock syndrome toxin 1

PHA

phytohemagglutinin

IFN-?

interferon- γ

TNF-?

tumor necrosis factor?

IL

interleukin

FACS

 Flow cytometry activated by fluorescence

MFI

Medium fluorescence intensity

FROM

Standard error

CFSE

succinimidyl ester of diacetate carboxyfluorescein.

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1. Isolation and cell generation Tγγ cells

Human peripheral blood mononuclear cells (PBMC) are isolated from leukocyte blood layers of donors treated with heparin or fresh blood by centrifugation with Ficoll-Paque according to conventional protocols (Brandes et al ., 2003). Of the PBMC, T? Cells with antibodies against human V? V? -TCR are positively selected using the Miltenyi Biotec magnetic cell sorting system. In this way, 50 ml of fresh blood routinely provide 2-5 x 10 6 cells with a purity of 98-99% T? Cells.

Virgin T? \ Beta cells

T?? CD4 + or T cells are isolated Virgin CD8 +, intact, (98-99% purity) at from PBMC by magnetic cell sorting negative with specific antibodies against VγVδ-TCR, CDlc, CD14, CD16, CD19, CD25, CD45RO, CD56, HLA-DR plus CD4 or CD8, respectively, followed by flow cytometry activated by fluorescence of cells that have negative staining for these markers.

Tα? Beta cell lines

T cells are positively selected ?? CD4 + of PBMC with antibodies against CD4 human by magnetic cell sorting. T cells α? CD4 + are stimulated with irradiated PBMC (30 Gy), autologous presenters of TT or PPD, at a ratio of 1: 100 in the first and 1:10 in the following cycles, and they expand in between which contains IL-2. The specificity of the antigen by a CFSE-based proliferation assay (see a continued) after three cycles of selection and expansion antigenic

B cells

B cells are isolated by classification negative magnetic cell of PBMC. B cells either use directly for stimulation of T cells? (see below), or B cell lines are generated by VEB induced transformation following the protocols conventional and then used for T cell stimulation γ δ.

Monocytes

Monocytes are isolated by classification positive magnetic cell of PBMC with antibodies against Human CD14, and the rigorous washing of separation columns before elution of magnetically trapped cells gives as result of enrichment of CD14 high monocytes.

DC

Monocyte-derived DCs are generated by culturing high CD14 cells in medium containing 10% FCS in the presence of IL-4 (10 ng / ml) and GM-CSF (25 ng / ml). After 6-7 days of culture, most cells are immature, as evaluated by cell surface staining for HLA-DR, CD1a, CD14, CD80, CD83, CD86 and CCR7. DC maturation is induced by additional culture for 8 hours in the presence of 100 ng / ml of LPS (from Salmonella abortus equi ) (Langenkamp et al ., 2000). DC maturation is confirmed by flow cytometry staining for cell surface DC maturation markers HLA-DR, CD80, CD83, CD86 and CCR7.

2. Stimulation of T cells Tγγ cells

50 µM isopentenyl pyrophosphate (IPP) presented by B cell lines transformed with autologous or heterologous EBV or primary B cells at a dilution of 1:10 is used to activate resting (newly isolated) Tγ cells? as described (Brandes et al ., 2003). Tγ? Cells are cultured in medium supplemented with 8% human serum in the presence of IL-2 (20 or 200 IU / ml).

T? \ Beta cells

T?? Cells are activated positively selected on antibody coated plates anti-CD3 (OCT3) 10 µg / ml plus antibody anti-CD28 (28.2) 250 ng / ml or, alternatively, with forbol 12-myristate 13-acetate (PMA) 10 ng / ml plus ionomycin 1 µg / ml or, alternatively, with PHA 1 µg / ml, and grown in medium supplemented with serum 8% human in the presence of IL-2 (200 IU / ml).

3. Marked with succinimidyl ester of diacetate carboxyfluorescein (CFSE)

Cells are washed with PBS - and labeled with 2.5 µM CFSE (Molecular Probes, Eugene, OR) in PBS - supplemented with 1% FCS for 4 min. at room temperature. The labeling is stopped by repeated washing of the cells with PBS - ice cream supplemented with 5% FCS. They are used immediately CFSE labeled cells in activation assays and cell proliferation

4. In vitro antigen presentation assays Presentation of antigen to T cell lines α?

Blood δ T cells are activated with the B cell line with heterologous EBV presenting IPP and irradiated (100 Gy) CP-EBV (see above) for 24 to 60 hours in the presence of TT 10-20? Mug / ml (Bern Biotech, Bern, Switzerland) or PPD 20 µg / ml (Statens Serum Institut Copenhagen, Denmark). Monocyte-derived DCs are cultured with TT or PPD for the same period of time and mature during the last 8 hours (see above). After irradiation (26 and 40 Gy for Tγ and DC cells, respectively) and intense washing, these APCs are used to stimulate clones of CD4 + specific T?? Cells for TT and PPD at a ratio of 1: 5 (if not stated otherwise). At various culture time points, the responding cells are examined by flow cytometry to determine the expression of activation markers (HLA-DR, ICOS, and CD25), internalization of TCR (loss of cell surface CD3) and proliferation cell (reduction in fluorescence signals with
CFSE).

Sensitization of the α? Beta CD4 + cells virgins

After stimulation of the T? Cells and the T?? Cells or after the maturation of the monocyte-derived DCs or after the isolation of monocytes from the PBMCs, these potential APCs are pulsed for 1 hour at 37 ° C with various concentrations of toxic shock syndrome toxin (TSST-1) (Toxin Technology, Sarasota, FL). Then, the potential APCs are irradiated with 12 Gy (or 40 Gy for DC), washed and thoroughly mixed (routinely at a ratio of 1: 5) with virgin Tα? Beta CD4 + cells labeled with CFSE Generally, 96-well round bottom plates contain 8 x 10 3 APC and 4 x 10 4 responsive cells labeled with CFSE in culture medium without exogenous cytokines. Cell proliferation is analyzed after 4 days by cytometry of
flow.

In Th cell differentiation assays, 100 IU / ml of IL-2 is added on day 5 of culture, and the cells are expanded for the next 10-16 days until the responding cells stop proliferating and return to a resting state (Langenkamp et al ., 2000). On day 21, the differentiation of Th cells is examined by measuring the production of intracellular cytokines. Cells are stimulated for 6 hours with PMA / ionomycin in the presence of 10 µg / ml Brefeldin (Sigma-Aldrich), then fixed with 2% paraformaldehyde, permeabilized with 0.5% saponin in PBS containing FCS at 2%, stained with antibodies against IL-2, IFN-γ, IL-4 and Vβ2-TCR, and analyzed by flow cytometry.

Sensitization of α? Beta CD8 + cells virgins

In mixed leukocyte responses, T cells γ δ stimulated with IPP, irradiated, T cells α-beta activated with superantigen or DC matured with LPS they are co-cultured with virgin α8 CD8 + T cells marked with heterologous CFSE. Generally, bottom plates Round 96 wells contain 4 x 10 4 cells that respond labeled with CFSE and APC in the range of 4 x 10 4 cells at 4 cells per well, corresponding to APC ratios: cell which responds from 1: 1 to 1: 10,000. Cell proliferation is analyzed after 6 days of culture by flow cytometry. The generation of CD8 + effector cells, after 14 days of mixed leukocyte responses with α? T cells Virgin CD8 +, in a cytolytic assay, which involves the co-culture for 12 h with a mixture of CD4 + T cells heterologous (true targets) and autologous (negative control) labeled with high (1 µM) and low (0.05 µM) doses of CFSE, respectively, followed by flow cytometry that analyzes the CFSE signals. Reduction in target cell counts true indicates specific destruction of antigen by cells CD8 + effectors while reducing the counts of Negative control cells indicates the destruction of target cells not specific for CD8 + effector cells.

5. Culture media

The medium used from the beginning to the end is RPMI 1640 supplemented with 2 mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, penicillin / streptomycin 50 µg / ml, 5 x 10-5M 2-mercaptoethanol and FCS al 10% (Hyclone Laboratories, Logan, UT, or GIBCO BRL) or serum 8% human (Swiss Red Cross, Bern, Switzerland). It is produced Human recombinant IL-2 using the system expression based on myeloma.

6. Flow cytometry Cell preparation

The cells were washed twice in PBS - ice cream supplemented with 2% FCS and 0.01% sodium acid. After block for 10 min. with human immunoglobulin 10 mg / ml, it incubate the cells sequentially for 20 min. on ice with primary antibodies specific for various cellular proteins or matching isotype control antibodies, washed, and in case of unlabeled primary antibodies, incubated additionally with secondary reagents labeled with labels fluorescence. After the final wash, the fluorescence is measured associated to cells with a FACSCalibur device (Becton Dickinson, San José, CA), and the recorded data is analyzed again using the CellQuestPro software (Becton Dickinson).

Antibodies

Antibody Source: Mouse AcM anti-CD1a (HI149), CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8α), CD11b (D12), CD14 (M \ PhiP9), CD16 (3G8), CD19 (HIB19), CD20 (2H7), CD25 (M-A251), CD40 (5C3), CD45RA (HI100), CD45RO (UCHL-1), CD50 (TU41), CD54 (HA58), CD56 (B159), CDw70 (Ki-24), CD80 (L307.4), CD83 (HB15e), CD86 (2331; FUN1), CD134 (L106), CD205 (MG38), HLA-DR (G46-6), pan-VγVδ-TCR (11F2), IL-2 (MQ1-17H12), IL-4 (8D4-8), IFNγ (B27), and IL-10 (No. 20705A) of BD PharMingen, San Diego, CA; Mouse ACM anti-CD1a (Nal / 34-HLK) and CD19 (HD37) of DAKO Diagnostics, Glastrup, Sweden; Mouse acm anti-TCRV? 2 (MPB2D5) from Immunotech, Marseille, France; Anti-CD138 mouse AcM (B-B4) from Diaclone, Besançon, France; AcM Leinco anti-CD102 (B-T1) Technologies, San Luis, MO; Anti-CD11a mouse AcM (TS1-22) and CD18 (TS1-18) of R. Pardi, Milan, Italy; Mouse anti-ICOS AcM (F44) from R.A. Kroczek, Berlin, Germany; Rat mAb anti-CCR7 (3D12) by M. Lipp, Berlin, Germany. These are used for flow cytometric analysis or isolation mobile.

The following secondary Ac are used, Conjugates and Ac control: goat anti-mouse IgG conjugated to RPE of Sigma-Aldrich, St. Louis, MO; Jackson's RPE conjugated anti-rat ass IgG ImmunoResearch Laboratories, West Grove, PA; streptavidin (SA) conjugated with RPE as well as with RPE-Cy5 of DAKO; SA conjugated with APC of BD PharMingen; Mouse control IgG1 (MOPC21) of Sigma-Aldrich; Other Ac control matching isotype of BD PharMingen.

7. Immunotherapy of tumors or infections chronic / recurring

For the preparation of stimulated tumor antigen-presenting T? Cells, 50-150 ml of peripheral blood are extracted from patients with tumors (or patients with chronic / recurrent infections, see below) and isolation of the cells is performed. Tγγ cells and antigen loading as described above. Alternatively, such freshly isolated γδ T cells are expanded by in vitro culture under conditions of VγVδ2 + TCR stimulants (see above methods of activation of γδ T cells with IPP) in presence of 20-1000 IU / ml of IL-2 and then stored in liquid nitrogen for later use in the preparation of tumor? presenting antigen (or vaccine, see below). Importantly, instead of defined vaccine / tumor proteins, many other ways of delivering antigen to the Tγ cells for the preparation of APCs are possible, including (among other things) the addition of tumor cell extracts. gross (undefined) or infected cell extracts (see below) or, alternatively, the treatment of γδ T cells by recombinant RNA / DNA technologies that are routinely used for transfection or transduction of living blood or tissue cells . Also, if the tumor peptides (or vaccine, see below) defined for direct loading onto the cell surface MHC molecules are known, then such peptides at 0.1-10 µg / ml are added to T cells? delta stimulated at 1-10 x 10 6 cells / ml, which are then incubated at 20-37 ° C for a short period of time, washed twice with isotonic phosphate buffered saline and used immediately for therapy. It is important to emphasize that the number of δ T cells used per administration as well as the route and frequency of administration of T γ cells depend on the efficacy in inducing the immune response by the tumor antigen (or vaccine, see below) individual used as well as the type and location of the tumor present in the individual patient. The protocols follow those currently used in DC-based immunotherapies (Fong and Engleman, 2000; Steinman et al ., 2003; Schuler et al ., 2003; Figdor et al ., 2004), ie 1-20 x 10 6 } cells / 0.5-2 ml per administration with 1 to 6 successive administrations with the same or lower amounts of cells at intervals of two weeks to two months.

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Claims (24)

1. Method for the preparation of T cells γ δ human effective antigen presenters that comprises selecting T cells δ from cells human peripheral blood mononuclear, treat cells selected with a stimulus for the induction of functions antigen presenters and apply the antigen for its uptake and presentation by these cells. 2. Method according to claim 1, wherein T γ? cell selection is performed by magnetic cell classification with antibodies against receptors of human T-VγVδ cells by conventional protocols 3. Method according to claim 1, wherein T cell selection is performed? culturing freshly isolated peripheral blood lymphocytes in the presence of structurally low molecular weight non-peptide compounds defined that induce the selective expansion of T cells γ δ expressing T cell receptor chains V γ 2 V δ 2 +. 4. Method according to claim 3, wherein the structurally low molecular weight non-peptide compound defined is isopentenyl pyrophosphate. 5. Method according to claim 1, wherein the stimulus for the induction of the presenting functions of Antigen is a low molecular weight non-peptide compound. 6. Method according to claim 5, wherein the structurally low molecular weight non-peptide compound defined is isopentenyl pyrophosphate. 7. Method according to claim 5, wherein the structurally low molecular weight non-peptide compound defined is pyrophosphate 4-hydroxy-3-methyl-but-2-enyl. 8. Method according to claim 1, wherein the stimulus for the induction of the presenting functions of antigen is phytohemagglutinin. 9. Method according to claim 1, wherein The applied antigen is in the form of defined proteins, mixtures of undefined proteins, or raw or enriched extracts of tumor or infected cells. 10. Method according to claim 1, wherein the applied antigen is a peptide derived from pathogens or from tumor cells. 11. Method according to claim 9, wherein The antigen is a protein derived from pathogens, which is applied in form of DNA or RNA that encodes it under conditions that allow the endogenous expression of said pathogen derived proteins. 12. Method according to claim 11, wherein the DNA or RNA is purified DNA or RNA or an insertion vector that It contains such DNA or RNA. 13. Human Tγ cells effective antigen presenters prepared by the method according to claim 1. 14. Use of human Tγ? Cells effective antigen presenters for the manufacture of a medication for use in immunotherapy or vaccination. 15. Use of vaccines targeting γδ T cells for the manufacture of a medicament for use in immunotherapy or vaccination or recurrent infectious tumors or diseases, wherein said vaccines targeting γδ T cells comprise an antigen that it is captured and presented by human γ T cells in order to present
antigen. 16. Use according to claim 15, wherein the use implies single or repeated applications of said T cells γ δ intradermally, subcutaneously, intramuscular, intravenous, mucosa or submucosa. 17. Use according to claim 16, wherein the application involves injection into a patient who is going to get vaccinated 18. Use according to the claims 15-17, wherein said medicament comprises a vaccine that is a hybrid compound composed of vaccine agents and molecules targeting Tγγ cells. 19. Use according to claim 18, wherein said hybrid compound comprises specific antibodies or ligands  for endocytic receptors in γ T cells. 20. Use according to claim 18, wherein said hybrid compound comprises specific antibodies or ligands  for CD1 1 b and CD205. 21. Use according to claim 18, wherein said vaccine agents are proteins against which it is desired Immune protection 22. Use according to claim 15, wherein the administration of vaccines targeting δ T cells It involves repeated oral administrations. 23. Method of identifying antigens associated with tumors or novel pathogen derivatives comprising selecting Tγγ cells from human peripheral blood mononuclear cells, treating the selected cells with a stimulus for induction of antigen presenting functions, applying fractions of a mixture of undefined proteins, or crude or enriched extracts of tumor or infected cells, or libraries of RNA or DNA derived from tumors and infectious agents, tested for in vitro activation of virgin Tα? beta cells autologous, and select protein samples with increased antigenicity. 24. Method of diagnosing the immune competence of a patient that comprises selecting Tγ cells from peripheral blood mononuclear cells of the patient, treating the selected cells with a stimulus for induction of antigen presenting functions, applying the antigen for which the immune competence is to be determined, and tested for in vitro activation of autologous α? beta cells.