WO2014138093A1 - Modulating th17 responses - Google Patents

Abstract

Disclosed herein are methods for treating diseases, disorders, or conditions associated with Th17 cell activity that include chronic infections, tumors, inflammatory and autoimmune diseases. Exemplary methods of treatment include administering nitric oxide and/or cGMP signaling pathway modulators. Inhibition of NOS2 or cGMP/cGK signaling abolishes the novo induction of the TH17 cells. Our data indicates that NO is required for the induction and stability of humna Th 17 responses.

Description

MODULATING TH17 RESPONSES

GOVERNMENT RIGHTS

[0001] This invention was made with government support under 1P01 CA132714;

1F30 CA165410; and 5T32 CA082084 awarded by the United States National Institutes of Health. The United States government has certain rights in the invention.

BACKGROUND

[0002] The immune system plays important roles in a variety of pathological conditions, states, and diseases. There is a need for technologies that permit understanding and/or control of immune system activity.

SUMMARY

[0003] The present invention encompasses the recognition that control of Thl7 responses can benefit treatment of a variety of diseases, disorders, and/or conditions. Among other things, the present invention defines roles played by nitric oxide (NO) and/or cyclic GMP (cGMP) in modulating Thl7 responses. Among other things, the invention provides therapeutic strategies that control and/or regulate Thl7 activity, for the treatment of certain diseases, disorders, and/or conditions.

[0004] In some embodiments, the present invention provides improvements for treatments of diseases, disorders, or conditions, the improvement comprising modulating Thl7 responses (e.g., by administration of an NO modulator and/or a cGMP modulator) during the treating. Alternatively or additionally, in some embodiments, the present invention provides compositions and/or methods for administration of a Thl7 modulator (e.g., an NO modulator and/or a cGMP modulator) together (e.g., concomitantly with) one or more other active agents.

[0005] Among other things, the present invention provides novel and unexpected findings demonstrating that myeloid derived suppressor cells (MDSCs), known to suppress CD8+ T cell responses, actively promote Thl7 differentiation of human CD4+ T cells. As described herein, the present invention demonstrates that MDSC-driven Thl7 induction depends on nitric oxide (NO), produced by MDSCs and induced in CD4+ T cells by MDSC- associated/released IL-ip/IL-6/TGFpi/IL-23. Moreover, synthetic NO donors show high activity in the induction of Thl7 cells in several MDSC-independent models.

[0006] In some embodiments, the invention provides methods of manipulating a Thl7 response to a disease, disorder, or condition, comprising steps of administering an agent that modulates Thl7 cell number or activity, wherein the agent comprises nitric oxide or a mediator of nitric oxide synthesis, levels, or signaling.

[0007] In some embodiments, the invention provides methods of manipulating a Thl7 response to a disease, disorder, or condition, comprising steps of administering an agent that modulates cyclic guanosine monophosphate (cGMP)/cyclic GMP dependent protein kinase (cGK) signaling.

[0008] In some embodiments the disease, disorder, or condition is selected from cancer, autoimmune disease, inflammatory disease, and infectious disease.

[0009] In some embodiments, the methods comprise administering one or more cytokines.

[0010] In some embodiments, cytokines are selected from IL-Ιβ, IL-6 or other STAT3 inducer, IL-23, TGF-βΙ, IL-21 and combinations thereof.

[0011] In some embodiments, the invention provides methods for increasing Thl7 cell number developed from naive, effector, and/or memory CD4+ T cell precursors.

[0012] In some embodiments, the invention provides methods for increasing IL-17A production , level, or secretion by Thl7 cells.

[0013] In some embodiments, one or more agents do not significantly alter detectable

IFN-γ production or levels.

[0014] In some embodiments, one or more agents increase Thl7 cell number or activity.

In some embodiments one or more agents, comprises a nitric oxide agonist, a nitric oxide donor, a nitric oxide stabilizer, an inhibitor of nitric oxide catabolism, a nitric oxide synthase stimulator, or substrate for nitric oxide production.

[0015] In some embodiments, one or more agents decrease Thl7 cell number or activity.

In some embodiments, one or more agents comprises a nitric oxide synthase inhibitor. In some embodiments, an agent is a selective nitric oxide synthase 2 (NOS2) inhibitor. In some embodiments, an agent decreases the number of myeloid-derived suppressor cells (MDSCs) and/or decreases the production of nitric oxide from MDSCs.

[0016] In some embodiments, the invention provides methods for administering to a subject suffering from a Thl7-associated disease, disorder, or condition a therapeutic regimen that inhibits NO production or signaling. In some embodiments, a Thl7-associated disease, disorder or condition is or comprises Thl7-malignant tumors. In some embodiments, a Thl7- associated disease, disorder or condition is or comprises a Thl7-mediated inflammatory process. In some embodiments, a Thl7-associated disease, disorder or condition is or comprises a Thl7- mediated autoimmune process. In some embodiments, a therapeutic regimen comprises one or more NOS inhibitors; cGMP/cGk inhibitors, and/or combinations thereof.

[0017] In some embodiments, the invention provides methods for administering to a subject suffering from a Thl7-susceptible disease, disorder, or condition, a therapeutic regimen that stimulates or supports NO production or signaling.

[0018] In some embodiments, a Thl7-susceptible disease, disorder or condition is or comprises a TH17-susceptible tumor or infection. In some embodiments, a Thl7-susceptible disease, disorder or condition is or comprises a chronic infection.

[0019] In some embodiments, the invention provides methods for identifying and/or characterizing therapeutic agents. In some embodiments, the methods comprise steps of contacting a system in which presence or level of Thl7 activity is detectable with a test therapeutic agent to be characterized; and detecting a difference in the presence or level of Thl7 activity that correlates with presence or level of the test therapeutic agent as compared with a comparable reference condition, wherein the test therapeutic agent is identified and/or characterized as a Thl7 inhibitory agent if the detected difference is or comprises a decrease in the presence or level of Thl7 activity, and identified and/or characterized as a Thl7 stimulatory agent if the detect difference is or comprises an increase in the presence or level of Thl7 activity. In some embodiments, a comparable reference condition lacks the test therapeutic agent. In some embodiments, a comparable reference condition includes a reference agent with known effect on presence or level of Thl7 activity. [0020] In some embodiments, the invention provides methods of providing results analyzing presence or level of Thl7 activity in a sample from a patient suffering from or susceptible to a proliferative condition selected from the group consisting of tumors,

inflammatory disorders, autoimmune disorders, and infections, which results indicate elevated presence or level of Thl7 activity relative to a reference; and administering a therapeutic regimen that modulates NO production or signaling. In some embodiments, the step of administering comprises administering an agent selected from NOS inhibitors, cGMP-specific inhibitors; and/or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A illustrates selective induction of IL-17A and Rorc (encoding RORyt) gene expression (but not FoxP3 or T-bet) in anti-CD3/CD28-expanded naive CD4+ T cells by tumor-isolated MDSCs (mean ± SD from 6 patients), as compared to control CD1 lb+ cells (mean ± SD from 3 healthy donors). MDSCs did not induce de novo differentiation of FoxP3+ Tregs from naive precursors.

[0022] Figure IB illustrates IL-17A production levels and percentages of IL-17A⁺ cells

(mean ± SD from n donors), and representative intracellular staining (IL-17A vs. IFN-γ, right) in naive CD4⁺ T cells (n=6 healthy donors) or OvCa-infiltrating CD4⁺ TILs (n=3 patients) stimulated with anti- CD3/CD28 antibodies in the presence of cancer-isolated MDSCs or control CDl lb⁺ cells.

[0023] Figure 1C depicts a comparison of IL-17A production by naive vs. memory

CD4⁺ T cells (mean ± SD from n=4 healthy donors) stimulated with either anti-CD3/CD28 antibodies or TNF-a-matured allogeneic DCs in the presence of MDSCs or control CD 1 lb⁺ cells.

[0024] Figures 1D-E illustrate the critical role of NOS in MDSC-driven induction of

Thl7 cells. Inhibition of the percentage of IL-17A⁺ (but not IFN-y⁺) CD4⁺ T cells (D) in anti-CD3/CD28/MDSC-expanded naive CD4⁺ T cells (D), by specific inhibitors of NOS (L- NMMA) and COX2 (celecoxib), but not IDO-, ARG-, or IL- 10 inhibitors are depicted in Figure ID. Figure IE illustrates the inhibition of the percentage of IL-17A secretion in anti- CD3/CD28/MDSC-expanded CD4⁺ TILs, by specific inhibitors of NOS (L-NMMA) and COX2 (celecoxib), but not IDO-, ARG-, or IL-10 inhibitors. The data (mean ± SD) from one representative experiment (performed in replicates: D, triplicate cultures; E, quadruplicate cultures). The results were confirmed in 3 independent experiments using different

patients/healthy donors.

[0025] Figure IF illustrates representative staining demonstrating specific inhibition of

IL-17A⁺ (but not IFN-y⁺) CD4⁺ T cells in co-cultures of anti-CD3/CD28-expanded CD4⁺ TILs and MDSCs by specific inhibitors of COX2 (celecoxib) and NOS (L-NMMA).

[0026] Figure 1G depicts the decrease in relative gene expression of IL-17A and

Rorc, induced in anti-CD3/CD28-expanded naive CD4⁺ T cells cultured in the presence of MDSCs, by a specific inhibitor of NOS (L-NMMA). The graphs present themean ± SD from one representative experiment (triplicate cultures) of two (using different patients/healthy donors), that both yielded similar results.

[0027] Figure 1H illustrates the correlation between the relative gene expression of

NOS2 and IL-17A in 7 d ex vivo anti-CD3/CD28-expanded cultures of OvCa ascites cells from 10 OvCa patients (n=10, r²=0.7692, p<0.001).

[0028] Figure II illustrates IL-17A production in anti-CD3/CD28-expanded cultures of naive or memory CD4⁺ T cells in the presence of MDSCs, with or without specific inhibitors of NOS2 (1400W) or cGMP (ODQ). Data (mean ± SD) from one representative experiment (triplicate cultures). The results were confirmed in 3 independent experiments using different patients/healthy donors, ns: P>0.05; * P<0.05; ** P<0.01; *** PO.001.

[0029] Figure 2A illustrates that increased N0₂ ^~ levels in co- cultures of CD4⁺ TILs

(mean ± SD from 4 patients) with tumor-isolated MDSCs (as compared to blood CD1 lb⁺ cells) is abrogated in the presence of NOS inhibitors ADMA and L-NMMA.

[0030] Figure 2B demonstrates that MDSCs spontaneously express Thl7-driving IL-Ιβ,

IL-6, and TGF-βι (left), whereas IL-23 (right) requires CD40L stimulation (similar data was obtained with CD40L-expressing J558 cells or CD4⁺ T cells; unpublished data). Data (mean ± SD) from 3 experiments involving MDSCs from 3 different patients. [0031] Figure 2C illustrates that specific induction of IL-17A (but not IFN-γ) production by anti-CD3/CD28- stimulated bulk CD4⁺ T cells from healthy donors, cultured in the absence or presence of Thl (200U/ml rhIL-12, 200ng/ml aIL-4-Ab), Thl7 (20ng/ml rhIL-Ιβ, 50ng/ml rhIL-6, lOng/ml rhIL-23), and T_reg (5ng/ml TGF-βι, 10 nM 9-cis retinoic acid)-driving cytokines, with lower (physiologic) concentrations of exogenous NO donor (DETA-NONOate). Statistically significant differences (compared to the absence of DETA- NONOate) are indicated. CD4⁺ T cells activated in the presence of Thl- and T_reg-driving cytokines did not produce detectable IL-17A. Percentage of FoxP3⁺ cells in control cultures were (-): 10.4%, Thl : 12.7%, Thl7: 5.2%, and T_reg: 41.2%. The graphs present the mean ± SD from one representative experiment (triplicate cultures) of two (healthy donors), that both yielded similar results.

[0032] Figure 2D illustrates that non-specific suppression of the CD4⁺ T cells differentiating in the Thl-, T_reg-, and Thl7-driving conditions by high concentrations (>100 μΜ) of DETA-NONOate. Proliferation of CFSE-labeled anti-CD3/CD28-stimulated bulk CD4⁺ T cells cultured in the absence or presence of Thl, Thl 7, and T_reg-driving cytokines and supplemented with increasing concentrations of DETA-NONOate. Statistically significant differences compared to conditions in the absence of the NO donor are indicated. The graph presents the mean ± SD from one representative experiment (triplicate cultures) of two

(different healthy donors), that both yielded similar results.

[0033] Figure 2E and F depict the relative gene expression of IL-17A (log scale) and

Rorc (log scale), and secretion of IL-17A by naive (E) or naive and memory (F) CD4⁺ T cells, expanded with anti-CD3/CD28 antibodies in the absence or presence of Thl7-driving cytokines and NO donor (DETA-NONOate). The graphs present the mean ± SD from one

representative experiment (E: triplicate cultures; F: quadruplicate cultures). The results were confirmed in 3 independent experiments using cells of different healthy donors. * P<0.05; ** P<0.01; *** PO.001.

[0034] Figure 3 A illustrates comparative induction of NOS2 (left) and IL-17A (right) gene expression in naive and memory CD4⁺ T cells (mean ± SD from 3 healthy donors) stimulated with anti-CD3/CD28 antibodies in the absence or presence of Thl7-driving cytokines.

[0035] Figure 3B illustrates dose dependent induction of NOS2 gene expression in naive

CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies in the presence of increasing concentrations of NO donor (DETA-NONOate) and Thl7-driving cytokines. The graph presents the mean ± SD from one representative experiment (performed with triplicate cultures). The results were confirmed in 3 independent experiments using different healthy donors.

[0036] Figure 3C demonstrates dose dependent induction of NOS2 gene expression in bulk CD4⁺ T cells, stimulated with anti-CD3/CD28 antibodies and Thl7-driving cytokines (high: 20 ng/ml IL-Ιβ, 50 ng/ml IL-6, 10 ng/ml IL-23; low: 25x dilution). The graph presents the mean ± SD from one representative experiment (triplicate cultures). The results were confirmed in 3 independent experiments using different healthy donors.

[0037] Figure 3D illustrates the induction of intracellular NO S2 protein (left and middle: representative data, right: mean ± SD from n=3 healthy donors) in CD3 -gated bulk CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies in the presence of Thl7-driving cytokines and NO donor (DETA-NONOate). Data in the right panel is represented as the fold change of the mean fluorescence intensity (MFI) over the isotype control.

[0038] Figure 3E illustrates the induction of intracellular NO (DAF-FM staining;

representative experiment, left; mean ± SD from n=3 healthy donors, right) in CD3 -gated bulk CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies in the presence of Thl7-driving cytokines and NO donor (DETA-NONOate), which is inhibited by the presence of general NOS inhibitor (L-NMMA) or NOS2-specific inhibitor (1400W). Data in the right panel is expressed as the fold increase of DAF-FM MFI over CD4⁺ T cells cultured in the absence of Thl7- driving cytokines and NO donor. When not otherwise indicated, statistically significant differences compared to the absence of NO inhibitors are shown.

[0039] Figure 3F depicts IL-17A secretion by naive CD4⁺ T cells stimulated with Thl7- driving cytokines is inhibited by general NOS inhibitor (L-NMMA) or NOS2-specific inhibitor (1400W). The graph presents the mean ± SD from one representative experiment (quadruplicate cultures). The results were confirmed in 3 independent experiments using different healthy donors.

[0040] Figure 3G illustrates induction of NOS2 (left, mean ± SD from 4 healthy donors) gene expression (NOS1 m NA is undetectable and NOS3 mR A is not induced, data not shown) correlates with the IL-17A production (right, mean ± SD from 3 healthy donors) in bulk CD4⁺ T cells by the individual Thl7-inducing factors IL-Ιβ, IL-6, IL-23, and/or TGF-βι. * P<0.05; ** P<0.01; *** PO.001.

[0041] Figure 4 A demonstrates inhibition of endogenous NO suppresses the induction of

IL-17A and other Thl7 markers. Relative gene expression of IL-17A, IL-17F, and IL-23R in bulk CD4⁺ T cells, expanded with anti-CD3/CD28 in the absence or presence of Thl7-driving cytokines and general NOS inhibitor (L- NMMA). The graphs present the mean ± SD from a representative experiment (triplicate cultures) of two (using different patients/healthy donors), that both yielded similar results.

[0042] Figure 4B illustrates regulation of (left) surface IL-23R expression on naive and memory CD4⁺ T cells (mean ± SD from 4 healthy donors) activated with anti-CD3/CD28 in the presence of NOS inhibitor (L-NMMA) or NO donor (DETA- NONOate). (Right):

Relative gene expression of IL-23R and IL-2R (right) in naive CD4⁺ T cells in the presence of increasing concentrations of NO donor and Thl7-driving cytokines. Statistically significant differences compared to the absence of DETA-NONOate and Thl7-driving cytokines are indicated. The graphs present the mean ± SD from a representative experiment (performed with triplicate cultures). The results were confirmed in 3 independent experiments using different healthy donors.

[0043] Figure 4C depicts IL-17A production by naive CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies in the absence or presence of cGMP inhibitor (ODQ, left) or supplemented with cGMP analogue (Br- cGMP, right) in the absence or presence of Thl7- driving cytokines. The graphs present the mean ± SD from a representative experiment (triplicate cultures). The results were confirmed in 3 independent experiments using different healthy donors. [0044] Figure 4D illustrates IL-17A (left) or IFN-γ (right) production by OvCa- isolated

CD4⁺ TILs (mean ± SD from 5 patients), expanded with anti-CD3/CD28 antibodies and re- stimulated in the absence or presence of NOS inhibitor (L-NMMA) or cGMP inhibitor (ODQ) for 48 h (statistically significant differences compared to the absence of inhibitors are indicated).

[0045] Figure 4E illustrates IL-17A production by in vz^'tro-generated Thl7 cells

(generated in 8 d cultures of CD4⁺ T cells stimulated with anti-CD3/CD28 in the presence of Thl7-driving cytokines), pretreated or not for 48 h with NOS inhibitor (L-NMMA) or cGMP inhibitor (ODQ). The data are shown as mean ± SD from 4 healthy donors. Statistically significant differences compared to condition in the absence of inhibitors are indicated, ns:

P>0.05; * P<0.05; ** P<0.01; *** PO.001.

[0046] Figure 5 A demonstrates representative intracellular staining for IL-17A and IFN- γ in anti-CD3/CD28-activated day 8 cultures of bulk OvCa ascites cells.

[0047] Figure 5B illustrates OvCa-isolated CD 1 lb⁺ cells.

[0048] Figure 5C depicts MDSCs, identified as CDl lb⁺CD33⁺CD34⁺CD14⁺CD15^~HLA-

DR^low cells {see Obermajer et al 2011; for complete phenotype).

[0049] Figure 5D illustrates the represented 8.9-50.0% of ascites cells (mean 24.2%, n=7) and expressed MDSC-associated suppressive factors, i.e. IL-4Ra, NOS2, ARG1, IL-10, and COX2, compared to control blood-isolated CDl lb⁺ cells (n=7 different patients (MDSCs); n=5 different healthy donors (control CDl lb⁺ cells).

[0050] Figure 5E illustrates relative expression of Thl7-driving IL-Ιβ, IL-6, IL-23pl9, and IL-12/23p40, and expression of TGF-βι and IL-10 in freshly isolated MDSCs vs. control

CDl lb⁺ cells (n=7 different patients, MDSCs; n=5 different healthy donors, control CDl lb⁺ cells). Graphs present the mean data of n different patients/donors (as indicated) ± SD.

***P<0.001.

[0051] Figure 6 A depicts intracellular staining (IL-17A vs. IFN-γ) of bulk (naive and memory) CD4⁺ T cells expanded [with anti-CD3/CD28 antibodies, immature (i)DCs, or TNF-a- matured (m)DCs] in the presence of MDSCs, control CDl lb⁺ cells, or Thl7-driving cytokines. Representative data from one of 3 independent experiments. [0052] Figure 6B illustrates IL-17A secretion by naive or memory CD4 T cells, expanded with anti-CD3/CD28 antibodies in the absence or presence of Thl7-driving cytokines (high: 20 ng/ml IL-Ιβ, 50 ng/ml IL-6, 10 ng/ml IL-23; low: 25x dilution. The graph presents the mean ± SD from one representative experiment (performed with triplicate cultures). The results were confirmed in 3 independent experiments using different donors. *P<0.05; **P<0.01.

[0053] Figure 7 illustrates induction of Thl7 responses by ovarian cancer-associated

MDSCs is dependent on CD40L-mediated activation of MDSCs by CD4⁺ T cells. IL-17A levels in co-cultures of MDSCs or control CDl lb⁺ cells with healthy donor blood-isolated bulk (naive and memory) CD4⁺ T cells in the absence or presence of CD40L blocking antibody (2 μg/ml). The data shown as mean ± SD from one representative experiment (performed with triplicate cultures). The results were confirmed in 3 independent experiments using different patients/healthy donors. * * P<0. 01.

[0054] Figure 8 A demonstrates representative intracellular staining of IL-17A and IFN-γ in memory CD4⁺ T cells (anti-CD3/CD28- stimulated) co-cultured with MDSCs or control CDl lb⁺ cells in the absence or presence of NO inhibitor (L-NMMA) or a synthetic NO donor (DETA-NONOate). Plots are representative of 3 independent experiments. Figure 8B illustrates IL-17A production in cultures of anti-CD3/CD28-stimulated naive CD4 T cells (left) or TILs (right) in the presence of MDSCs or control CDl lb⁺ cells with or without NO donor (DETA-NONOate). The data (mean ± SD) from one representative experiment (performed with triplicate cultures). The results were confirmed in 3 independent experiments using different patients/donors. *P<0.05; **P<0.01.

[0055] Figure 9 illustrates selective induction of Thl7-related factors (but not Thl,

Th2, or T_reg markers) by low doses of exogenous NO (analogous to the levels produced by MDSC; see Fig. 2A). Relative expression of IL-17F (log scale), GAT A3, FoxP3, and T- bet in naive CD4⁺ T cells, stimulated with anti-CD3/CD28 antibodies in the absence or presence of increasing concentrations of exogenous NO (NO donor DETA-NONOate) and Thl7-driving cytokines (20 ng/ml IL-Ιβ, 50 ng/ml IL-6, 10 ng/ml IL-23). Data expressed as the ratios of the individual gene expression to HPRT1. Statistically significant differences compared to the absence of DETA-NONOate and Thl7-driving cytokines are indicated. Representative data (triplicate cultures; mean ± SD) from one of 3 independent experiments with cells of different donors. *P<0.05; ***P<0.001; ns: P>0.05.

[0056] Figure 10 demonstrates selective induction of NOS2 (see Fig. 3 G), but not NOS1 and NOS3, during the cytokine- driven induction of Thl7 cells. Relative expression of NOS1 (undetectable) and NOS3 (NOS2 expression and IL-17A secretion are shown in Fig. 3 G) in bulk (naive and memory) CD4⁺ T cells (mean ± SD from 4 healthy donors) in the presence of IL-Ιβ, IL-6, IL-23, and TGF-βι alone or incombination. Statistical comparisons to the untreated cells are shown. Ns: not significant (P>0.05); nd: not detectable.

[0057] Figure 11A illustrates gene expression of baseline NOS2 in human (left) and mouse (right) naive and memory CD4⁺ T cells expanded with anti-CD3/CD28 antibodies (mean ± SD from n=3 different donors, human; n=3 different donors, mouse).

[0058] Figure 1 IB illustrates gene expression of NOS2 in mouse naive and memory

CD4⁺ T cells expanded with anti-CD3/CD28 antibodies in the absence or presence of Thl7- driving cytokines (mean ± SD from n=3 C57BL/6 mice). ** P<0.01 ns: P>0.05.

DEFINITIONS

[0059] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used.

[0060] Activating agent: As used herein, the term "activating agent" refers to an agent whose presence or level correlates with elevated level or activity of a target, as compared with that observed absent the agent (or with the agent at a different level). In some embodiments, an activating agent is one whose presence or level correlates with a target level or activity that is comparable to or greater than a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known activating agent, e.g., a positive control).

[0061] Administration: As used herein, the term "administration" refers to the administration of a composition to a subject. Administration may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.

[0062] Agent : The term "agent" as used herein may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. As will be clear from context, in some

embodiments, an agent can be or comprise a cell or organism, or a fraction, extract, or component thereof. In some embodiments, an agent is agent is or comprises a natural product in that it is found in and/or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some

embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, siR As, shRNAs, DNA/RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, small molecules, etc. In some embodiments, an agent is or comprises a polymer. In some embodiments, an agent is not a polymer and/or is substantially free of any polymer. In some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an agent lacks or is substantially free of any polymeric moiety.

[0063] Analog: As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog a substance that can be generated from the reference substance by chemical manipulation of the reference substance. In some embodiemnts, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance. [0064] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

[0065] Antagonist: As used herein, the term "antagonist" refers to an agent that i) inhibits, decreases or reduces the effects of another agent; and/or ii) inhibits, decreases, reduces, or delays one or more biological events. Antagonists may be or include agents of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity. An antagonist may be direct (in which case it exerts its influence directly upon its target) or indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, for example so that level or activity of the target is altered).

[0066] Approximately: As used herein, the term "approximately" and "about" is intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art as appropriate to the relevant context. In certain embodiments, the term

"approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0067] Associated with: Two events or entities are "associated" with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0068] Biologically active: As used herein, the phrase "biologically active" refers to a substance that has activity in a biological system (e.g., in a cell (e.g., isolated, in culture, in a tissue, in an organism), in a cell culture, in a tissue, in an organism, etc.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. It will be appreciated by those skilled in the art that often only a portion or fragment of a biologically active substance is required (e.g., is necessary and sufficient) for the activity to be present; in such circumstances, that portion or fragment is considered to be a "biologically active" portion or fragment.

[0069] Binding: It will be understood that the term "binding", as used herein, typically refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

[0070] Binding agent: In general, the term "binding agent" is used herein to refer to any entity that binds to a target of interest as described herein. In many embodiments, a binding agent of interest is one that binds specifically with its target in that it discriminates its target from other potential binding partners in a particular interaction contct. In general, a binding agent may be or comprise an entity of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc). In some embodiments, a binding agent is a single chemical entity. In some embodiments, a binding agent is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions. For example, those skilled in the art will appreciate that in some embodiments, a binding agent may comprise a "generic" binding moiety (e.g., one of biotin/avidin/streptaviding and/or a class-specific antibody) and a "specific" binding moiety (e.g., an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety. In some embodiments, such an approach can permit modular assembly of multiple binding agents through linkage of different specific binding moieties with the same generic binding moiety partner. In some embodiments, binding agents are or comprise polypeptides (including, e.g., antibodies or antibody fragments). In some embodiments, binding agents are or comprise small molecules. In some embodiments, binding agents are or comprise nucleic acids. In some embodiments, binding agents are aptamers. In some embodiments, binding agents are polymers; in some embodiments, binding agents are not polymers. In some embodiments, binding agents are non-polymeric in that they lack polymeric moieties. In some embodiments, binding agents are or comprise carbohydrates. In some embodiments, binding agents are or comprise lectins. In some embodiments, binding agents are or comprise peptidomimetics. In some embodiments, binding agents are or comprise scaffold proteins. In some embodiments, binding agents are or comprise mimotopes. In some embodiments, binding agents are or comprise stapled peptides. In certain embodiments, binding agents are or comprise nucleic acids, such as DNA or R A.

[0071] Combination therapy: The term "combination therapy", as used herein, refers to those situations in which two or more different pharmaceutical agents for the treatment of disease are administered in overlapping regimens so that the subject is simultaneously exposed to at least two agents. In some embodiments, the different agents are administered simultaneously. In some embodiments, the administration of one agent overlaps the administration of at least one other agent. In some embodiments, the different agents are administered sequentially such that the agents have simultaneous biologically activity with in a subject.

[0072] Comparable: The term "comparable" is used herein to describe two (or more) sets of conditions, circumstances, individuals, or populations that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some

embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0073] Cyclic Guanosine Monophosphate (cGMP)Modulator: As described herein, agents that are cGMP modulators are known in the art. In some embodiments, a cGMP modulator alters the synthesis, level, availability, metabolism, of cGMP. In some embodiments a cGMP modulator acts upstream or downstream in a cGMP signaling pathway.

[0074] Derivative: As used herein, the term "derivative" refers to a structural analogue of a reference substance. That is, a "derivative" is a substance that shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, a derivative is a substance that can be generated from the reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.

[0075] Dosage form: As used herein, the term "dosage form" refers to a physically discrete unit of a therapeutic agent for administration to a subject. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).

[0076] Dosing regimen: A "dosing regimen" (or "therapeutic regimen"), as that term is used herein, is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some

embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.

[0077] Fragment: A "fragment" of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. The whole material or entity may in some embodiments be referred to as the "parent" of the whole.

[0078] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[0079] Infection: As used herein, the term "infection" refers to the invasion of a host organism's body by a disease-causing organism that multiplies in the host. Symptoms of an infection may result from action of toxins produced by the disease-causing organism and/or be reaction of host tissues to the organisms and/or to toxins they produce.

[0080] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%>, about 20%>, about 30%>, about 40%>, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. In some embodiments, isolation involves or requires disruption of covalent bonds (e.g., to isolate a polypeptide domain from a longer polypeptide and/or to isolate a nucleotide sequence element from a longer oligonucleotide or nucleic acid).

[0081] Modulator: The term "modulator" is used to refer to an entity whose presence or level in a system in which an activity of interest is observed correlates with a change in level and/or nature of that activity as compared with that observed under otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator is an activator, in that activity is increased in its presence as compared with that observed under otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator is an antagonist or inhibitor, in that activity is reduced in its presence as compared with otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator interacts directly with a target entity whose activity is of interest. In some embodiments, a modulator interacts indirectly (i.e., directly with an intermediate agent that interacts with the target entity) with a target entity whose activity is of interest. In some embodiments, a modulator affects level of a target entity of interest; alternatively or additionally, in some embodiments, a modulator affects activity of a target entity of interest without affecting level of the target entity. In some embodiments, a modulator affects both level and activity of a target entity of interest, so that an observed difference in activity is not entirely explained by or commensurate with an observed difference in level.

[0082] Nitric Oxide (NO) Modulator: As described herein, a large number of agents that are NO modulators are known in the art. In some embodiments an NO modulator alters the synthesis, level, availability, metabolism, or activity of NO. In some embodiments, an NO modulator acts upstream/downstream of NO in an NO signaling pathway. [0083] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals {e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some

embodiments, a patient is a human. A human includes pre and post natal forms.

[0084] Pharmaceutical composition : As used herein, the term "pharmaceutical composition" refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity;

intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0085] Pharmaceutically acceptable: The term "pharmaceutically acceptable" as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0086] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0087] Prevention : The term "prevention", as used herein, refers to a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition.

[0088] Reference: The term "reference" is often used herein to describe a standard or control agent, individual, population, sample, sequence or value against which an agent, individual, population, sample, sequence or value of interest is compared. In some

embodiments, a reference agent, individual, population, sample, sequence or value is tested and/or determined substantially simultaneously with the testing or determination of the agent, individual, population, sample, sequence or value of interest. In some embodiments, a reference agent, individual, population, sample, sequence or value is a historical reference, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference agent, individual, population, sample, sequence or value is determined or characterized under conditions comparable to those utilized to determine or characterize the agent, individual, population, sample, sequence or value of interest. [0089] Refractory: The term "refractory" as used herein, refers to any subject that does not respond with an expected clinical efficacy following the administration of provided compositions as normally observed by practicing medical personnel.

[0090] Small molecule: As used herein, the term "small molecule" means a low molecular weight organic and/or inorganic compound. In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent. In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic.

[0091] Specific: The term "specific", when used herein with reference to an agent or entity having an activity, is understood by those skilled in the art to mean that the agent or entity discriminates between potential targets or states. For example, an agent is said to bind

"specifically" to its target if it binds preferentially with that target in the presence of competing alternative targets. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target. In some embodiments, the agent or entity binds with higher on-rate, lower off-rate, increased affinity, decreased

dissociation, and/or increased stability to its target as compared with the competing alternative target(s). [0092] Subject: By "subject" is meant a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some

embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. In some embodiments, a subject is an individual to whom therapy is administered.

[0093] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0094] Suffering from: An individual who is "suffering from" a disease, disorder, or condition {e.g., influenza) has been diagnosed with and/or exhibits one or more symptoms of the disease, disorder, or condition.

[0095] Susceptible to: An individual who is "susceptible to" a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, such an individual is known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., family members of individuals suffering from allergy, etc.

[0096] Symptoms are reduced: According to the present invention, "symptoms are reduced" when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom. Many cancer patients with smaller tumors have no symptoms. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that one or more symptoms is/are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated.

[0097] Thl 7 Modulator: Those skilled in the art will appreciate that a Thl7 Modulator, as that term is used herein, is an agent or entity whose presence or level correlates with increased or decreased level or activity of Thl 7 cells. A Thl 7 Modulator whose presence or level correlates with a statistically significant increase in Thl 7 level or activity is considered to be a "Thl 7 Promoter". A Thl 7 Modulator whose presence or level correlates with a statistically significant decrease in Thl 7 level or activity is considered to be a "Thl 7 Suppressor". In some embodiments, a Thl 7 Modulator is or comprises an NO Modulator and/or a cGMP Modulator.

[0098] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.

[0099] Therapeutic regimen : A "therapeutic regimen", as that term is used herein, refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome. [0100] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some

embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be "refractory" to a "therapeutically effective amount." To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

[0101] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance (e.g., anti-receptor tyrosine kinases antibodies or receptor tyrosine kinase antagonists) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition (e.g., cancer). Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0102] Unit dose: The expression "unit dose" as used herein refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical

composition. In many embodiments, a unit dose contains a predetermined quantity of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect. A unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described infra. It will be appreciated by those skilled in the art, in many embodiments, a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0103] This invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

[0104] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described. All publications mentioned are hereby incorporated by reference.

Nitric Oxide

[0105] Nitric oxide (NO) is a mediator of inflammation and immunity, involved both in the pathogenesis of and resistance against cancer, autoimmunity and infectious diseases. We observed that, while high concentrations of exogenous NO indiscriminately suppressed the proliferation and differentiation of Thl, Th2, and Thl7 cells, the physiologic NO concentrations produced by myeloid-derived suppressor cells (MDSCs), or provided in form of synthetic NO donor, support the development of RORyt(Rorc)+IL-23R+IL-17+ Thl 7 cells. In addition to the positive impact of the exogenous (MDSC-produced) NO upon the induction of Thl 7 cells, we observed that the development of Thl 7 cells from naive-, memory-, or tumor-infiltrating CD4+ T cells, driven by IL-i p/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL- ip/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cGMP/cGK pathway of NO signaling within CD4+ T cells.

Thl 7 Modulation

[0106] Those skilled in the art will appreciate that a "Thl 7 modulator", as that term is used herein, is an agent or entity whose presence or level correlates with increased or decreased level or activity of Thl 7 cells. A Thl 7 modulator whose presence or level correlates with a statistically significant increase in Thl 7 level or activity is considered to be a "Thl 7 promoter" or "Thl 7 promoting factor". A Thl 7 modulator whose presence or level correlates with a statistically significant decrease in Thl 7 level or activity is considered to be a "Thl 7

suppressor".

[0107] In some embodiments, a Thl 7 modulator is or comprises an NO modulator and/or a cGMP modulator. [0108] In some embodiments, a Thl7 modulator is or comprises one or more cytokines.

In some embodiments, a Thl7 modulator is a Thl7 promoting factor. In some embodiments, a Thl7 promoting factor comprises IL-Ιβ, IL-6 (or other STAT3 inducer), and/or IL-23.

[0109] In some embodiments, a Thl7 modulator is a Thl7 suppressor. In some embodiments, a Thl7 suppressor comprises one or more cytokines, for example, TGF-βΙ, IL-12, or the like.

[0110] In some embodiments, a Thl7 modulator comprises an NO modulator. In some embodiments, an NO modulator promotes NO levels, activity, or signaling. In some

embodiments, an NO promoting agent is or comprises nitric oxide, a nitric oxide agonist, a precursor/substrate for NO synthesis such as nitrites, and/or an NO donor (e.g., DETA- NONOate, S-nitroso-DL-penicillamine).

[0111] Nitric oxide donors (agonists of NO signaling) are known in the art, for example, including but not limited to nitrates (such as Glyceryl trinitrate, GTN, also known as

nitroglycerin; isosorbide mononitrate; or ISMN; pentaerythrityl tetranitrate, PETN) sodium nitropruside (SNP), BiDil (isosorbide dinitrate with hydralazine), Diazeniumdiolates (also known as 'NONOates'; including diethylamine NONOate: DETA/NONOate or DEA-NONOate, and other members of this group such as SPER/NO, PROLI/NI, V-PYRRO/NO or JS-K), S- Nitrothiols (S-nitroso-glutathione, GSNO; S-nitroso-acetylpenicillamine, SNAP; or S-nitroso-N- valerylpenicillamone, SNVP), NO donor hybrid drugs (NO-NSAIDS; including NicOx compounds, NCX4016 and NCX4215; Nipradilol, or noitropravastatin; S-nitroso-captopril or S- nitroso-diclofenac), nocorandil, and NO-generating materials, including Zeolites, (for a brief review of certain NO donor compounds, see Miller MR and Megson IL, Br J Pharmacol. Jun 2007; 151(3): 305-321).

[0112] In some embodiments, an NO modulator is a nitric oxide synthase (NOS) inhibitor. In some embodiments, an NO modulator inhibits NOS1, NOS2, and/or NOS3. NOS inhibitors are known in the art, for example, NG-Amino-L-arginine hydrochloride, 2-Ethyl-2- thiopseudourea, NG-Monomethyl-L-arginine, Monoacetate (L-NMMA), and the like.

[0113] In some embodiments, an NO modulator is a selective NOS2 inhibitor. Selective

NOS2 inhibitors are known in the art, for example, 1400W, l-Amino-2-hydroxyguanidine, p- Toluenesulfonate, Curcumin, 1,3-PBITU, Dihydrobromide, MEG, 2-Iminopiperidine, AMT, ADMA, BYK 191023 dihydrochloride, and the like.

[0114] In some embodiments, a Thl7 modulator comprises one or more cGMP modulators. In some embodiments, a cGMP modulator stimulates or increases cGMP signaling. In some embodiments, comprises a cGMP analogue. cGMP analogues are known in the art, for example, 8-(4-Chlorophenylthio)-guanosine 3 ',5 '--cyclic monophosphate, 8-Br-cGMP,

Dibutyryl-cGMP, and the like.

[0115] In some embodiments, a Thl7 modulator decreases cGMP levels and/or inhibits cGMP inhibits cGMP-mediated signaling. cGMP inhibitors are known in the art, for example, LH- [l,2,4]oxadiazolo[4,3,-a]quinoxalin-l-one (ODQ)), 6-anilino-5,8-quinolinedione, 4H-8- Bromo-l,2,4-oxadiazolo(3,4-d)ben(b)(l,4)oxazin-l-one, and the like.

[0116] In some embodiments, a cGMP modulator inhibits the metabolism of cGMP. In some embodiments, a cGMP modulator comprises a phosphodiesterase (PDE) inhibitor.

Phosphodiesterase inhibitors are known in the art, for example methylated xanthines, aminophylline, IBMX, theobromine, theophylline,

[0117] In some embodiments, a PDE inhibitor is a selective PDE inhibitor. In some embodiments, a PDE inhibitor is a selective PDE5 inhibitor. Selective PDE5 inhibitors are known in the art, for example, sildenafil, tadalafil, vardenafil, udenafil and avanafil.

Thl7 mediated disorders

[0118] In some embodiments, the invention provides methods for manipulating a Thl7 response to a disease, disorder, or condition. In some embodiments, a disease, disorder, or condition is treated or ameliorated by decreasing the level or activity of Thl7 cells. It may be desirable to decrease Thl7 cells, or prevent an increase of Thl7 cells, if such cells are associated with a malignant tumor, or play a role in treatment resistance or promotion of cancer, chronic inflammation, undesirable side effects of severe acute infection (e.g., viral pneumonia or bacterial sepsis), or an autoimmune disease. Thus, it may be desirable to decrease or diminish Thl7 cell activity or number according the methods described herein. The present invention contemplates, among other things, treatment of autoimmune and/or inflammatory disease such as multiple sclerosis, psoriasis, diabetes, Crohn's disease, inflammatory bowel disease, by manipulation of Thl7 responses.

[0119] In other circumstances, Thl7 cells may promote immune responses which facilitate destruction of cancer cells or infectious agents or pathogens. In such circumstances, it is desirable to increase or augment Thl7 cell number and/or activity, for example, by administering an agent that stimulates nitric oxide or cGMP/cGK signaling according to the methods described herein.

[0120] In some embodiments, agents that promote a Thl7 response are administered to treat chronic infections, including but not limited to M. Tuberculosis, M. Leprae, and other fungal, protozoan and bacterial infections.

[0121] In some embodiments, agents that promote a Thl7 response are administered to stimulate or activate T17-mediated immune responses by MDSCs, tumor-associated

macrophages, activated neutrophils; and/or inflammatory dendritic cells.

EXAMPLES

[0122] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Materials and Methods

[0123] Media and reagents. Cells were cultured in IMDM medium (Invitrogen) with

10% FCS (Gemini). DETA-NONOate was purchased from Cayman Chemical and used at the concentration of 25 μΜ, unless otherwise specified. General NOS inhibitors L-NMMA (Sigma) and AD MA (Sigma) and the NOS2-specific inhibitor 1400W (Sigma) were used at the concentrations of, respectively, 100 μΜ, 200μΜ, and 200 nM, unless indicated otherwise. Arginase inhibitor nor-NOHA (Cayman Chemical) was used at 200 μΜ, IDO inhibitor 1- Methyl-D-tryptophan (Sigma) was used at 1 mM, neutralizing a-IL-10 mAb (R&D; clone 25209) was used at 1.0 μg/ml, COX2 inhibitor celecoxib (Biovision) was used at 20 μΜ, c-GMP analogue Br-cGMP (Sigma) was used at 100 μΜ, and cGMP inhibitor ODQ was used at 10 μΜ. Thl, Thl7, and Treg-driving cytokines were used at the following concentrations (unless stated otherwise): IL-Ιβ (20 ng/ml; Miltenyi), IL-6 (50 ng/ml; ThermoFisher

Scientific), IL-23 (10 ng/ml; R&D), TGF-βΐ (5 ng/ml; R&D), and 9-cis retinoic acid (10 nM; Sigma). CFSE (Invitrogen) labeling kit to monitor cell proliferation was used according to the manufacturer's protocol. CD3/CD28 stimulation was accomplished with anti-CD3/CD28 human or mouse T cell-activator Dynabeads (at 2μ1/ιη1; Invitrogen). Soluble (s)CD40L was used at 1 μg/ml in combination with 1 μg/ml of Enhancer for Ligands (Enzo Life Sciences). Nitrite formed by the spontaneous oxidation of NO under physiological conditions in cell culture supernatants was detected with the Griess reagent kit (Invitrogen) according to the manufacturer's protocol.

[0124] Isolation of peripheral blood naive and memory human CD4⁺ T cells and mouse splenic CD4⁺ Tcells. Human PBMCs were isolated from buffy coats provided by the Central Blood Bank of Pittsburgh, PA. T cells were isolated from PBMCs by negative selection using the CD4+ T cell enrichment cocktail (Stem Cell Tech) in combination with either anti-CD45RO or CD45RA depletion antibodies, resulting in a >95% pure CD3+ population of uniform CD4+CD45RA+/-CD45RO+/- cells. Allogeneic combinations of T cells and MDSCs were used to allow testing of the impact of tumor-derived MDSCs (or control blood-isolated CD1 lb+ cells) on the differentiation of healthy donor naive or memory blood-isolated T cells. For analysis of mouse T cells, C57BL/6 (B6) mice were purchased from The Jackson

Laboratory. Naive and memory CD4+ T cells were isolated from the spleens of B6 mice using naive and memory CD4+ T cell isolation kits (Miltenyi Biotec).

[0125] Isolation of MDSCs and OvCa-infiltrating CD4⁺ T cells (TILs). Human

OvCa ascites cells wereobtained intraoperatively from previously-untreated patients with primarily advanced stage III or IV epithelial OvCa, after obtaining written informed consent. The nature and possible consequences of the studies were explained. All specimens were provided under protocols approved by the University of Pittsburgh or Roswell Park Cancer Institute Institutional Review Boards. Human OvCa ascites obtained from the University of Pittsburgh (IRB0406147) were used in the isolation of cancer- associated CDl lb+ cells (MDSCs) and subsequent isolation of CD4+ TILs. The median age of patients was 56 years old (range 39-69 years old). Twelve patients were Caucasian and one patient was African- American. The majority of patients were FIGO Stage IIIC, one patient was Stage IIIA, and one patient was Stage II A. Tumor histology was serous in 9 cases (69.2%), clear cell in 2 cases (15.4%)), mucinous in 1 case (7.7%), and mixed histology in 1 case (7.7%). Human OvCa ascites obtained from the Roswell Park Cancer Institute (CIC02-15) were used in the isolation of bulk OvCa primary cells and their CD3/CD28-driven expansion for 7 days in culture. The median age of patients was 64 (range 50-85). Nine patients were Caucasian and one was Hispanic. The majority of patients were FIGO Stage IIIC, three patients were Stage IV. Tumor histology was serous in 7 cases (70%>), papillary serous in 2 cases (20.0%>), and mixed histology in 1 case (10%>) OvCa primary cells were harvested by

centrifugation. CD l lb+ cells (i.e. MDSCs) wereobtained after centrifugation of OvCa ascites, followed by red blood cell lysis and positive magnetic selection of

CD 1 lb+ cells (CD 1 lb EasySep Isolation kit; Stem Cell Tech). The isolated cells were>95% CD l lb+ and uniformly expressed the CD 1 lb+CD33+CD34+ MDSC phenotype (Obermajer et al., 201 1). CD4+ T cells (TILs) were obtained after positive magnetic selection of CD 1 lb+ cells followed by negative selection using the CD4+ T cell enrichment cocktail (Stem Cell Tech). Control CD l lb+cells were isolated from healthy donor buffy coats, using the same method.

[0126] Thl7 cell generation. T cells were stimulated with anti-CD3/CD28 Dynabeads

(2.0 μΐ/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD1 lb+ cells, pretreated or not with inhibitors, and/or in the presence of the Thl7-inducing cytokine cocktail: IL-Ιβ (20 ng/ml), IL-23 (10 ng/ml), IL-6 (50 ng/ml), and/or TGF-β (10 ng/ml). All experiments used lxlO⁵ T cells per well at a concentration of 5xl0⁵ cells/ml. All experiments in this study used the 1 :4 ratio of MDSC (or control CD1 lb+ cells) to T cells, determined to be optimal based on our preliminary experiments (which tested the MDSC:T cell ratios of 1 : 1 , 1 :2, 1 :4, and 1 :8). As an alternative to stimulation with anti- CD3/CD28 beads, T cells were stimulated with mature DCs (mDCs) [monocytes were isolated by positive magnetic selection using the EasySep CD 14+ isolation kit (Stem Cell Tech) and cultured for 6 days in 24- well plates (BD) in the presence of rhuGM-CSF and IL-4 (both 1000 U/ml; gifts from Schering Plough) and afterwards matured for 48 h with TNF-a], with a DC:T cell ratio of 1 : 10. On day 4- 6, expanded T cells were analyzed for the expression of Thl7-associated factors (mR A expression) and cytokine secretion (ELISA). On day 7-8, expanded T cells were assayed for intracellular NOS2 and NO, and were further re-stimulated for intracellular cytokine staining (all as further described below). The purity of anti-CD3/CD28-activatedT cell cultures increased from an initial >95% to over 99% by the time of analysis, as determined by flow cytometry. Note that, consistent with the key role of IL-Ιβ and IL-6, and the negative role of TGF-βΙ, in the induction of human Thl7 cells (Acosta- Rodriguez et al, 2007a; Wilson et al, 2007), and a similar recently-reported synergy between IL-Ιβ and other Stat3 activators (Guo et al., 2009), the combination of IL-Ιβ with IL-6 and/or IL-23 was sufficient for the optimal induction of IL-17A production, with TGF-βΐ having a negative effect (Fig. 3G).

[0127] ELISA. ELISA analysis was performed for IL-17A (R&D) and IFN-γ secretion by day 5 expanded T cells in culture [or by day 7-8 expanded T cells washed and re-plated at 1x106 cells/ml and re- stimulated with anti-CD3/CD28 beads for 24-48 h]. ELISA analysis of IL-23 production by OvCa ascites-isolated MDSCs or control blood-isolated CDl lb+ cells was performed after 24 h stimulation with CD40L-expressing J558 cells [or sCD40L (Enzo Life Sciences)] or CD4+ T cells. 24 h- conditioned medium from OvCa ascites-isolated MDSCs or control blood-isolated CDl lb+ cells were analyzed for IL-6, IL-10, IL-23, IL-Ιβ and TGF-βΙ by sandwich ELISA (R&D).

[0128] Flow cytometry. Two- and three-color cell surface and intracellular immunostaining analyses were performed using an Accuri C6 flow cytometer. OvCa ascites- isolated cells were stained with the antibodies CDl lb-FITC, CD33-APC, CD34-PE/Cy7, HLA-DR-FITC, CD14-PE, CD80-FITC, CD83- PE, CD15-PE, and CD8-FITC (BD and eBioscience) (see (Obermajer et al, 2011) for full phenotype). IL-23R was detected with IL- 23R-FITC mAb (R&D). Rat IgG2a-PE, IgGl-FITC, IgGl-APC, and IgGl- PE/Cy7 isotype controls, and the rat IgG2a-FITC isotype control, were from BD PharMingen.

[0129] Intracellular staining. Cells were harvested, fixed, and permeabilized using the Foxp3 Fix/Perm Buffer Set solution (eBioscience). For intracellular cytokine production only, T cells were stimulated with PMA (50 ng/ml; Sigma- Aldrich) and ionomycin (1 μg/ml; Sigma- Aldrich), and after 4 h, brefeldin A (10 μg/ml) was added for an additional 4- 10 h prior to staining. The following antibodies were used: IFN-y-FITC and IL-17A-PE (eBio64DEC17; eBioscience), Foxp3-Alexa Fluor 488 (BioLegend), CD3 [unlabeled monoclonal mouse anti-human CD3 (eBioscience) followed by secondary goat anti-mouse IgG F(ab')2-Alexa Fluor 647 (Cell Signaling Technology)], and NOS2 [unlabeled polyclonal rabbit anti-human NOS2 (Millipore) followed by secondary goat anti-rabbit IgG F(ab')2-Alexa Fluor 488 (Cell Signaling Technology)]. Stainings (including for both primary and secondary antibodies, where appropriate) were performed at room temperature for 30 min, then washed and resuspended in FACS buffer prior to analysis. For intracellular NO detection, DAF-FM diacetate (Molecular Probes) was used, which passively diffuses across cell membranes and is deacetylated by intracellular esterases to membrane-impermeant DAF-FM, which then reacts with NO to form a fluorescent benzotriazole with excitation/emission maxima of 495/515 nm (Nagy et al.,2003). DAF-FM diacetate was loaded at 10 μΜ for 2 h at 37°C.

[0130] Confocal microscopy. T cells were harvested and directly centrifuged onto 12 mm diameter circular glass coverslips (Propper) coated for 1 h at 37°C with 0.005% human fibronectin (Sigma) in PBS in 24 well plates. The coverslips were then incubated in 4% paraformaldehyde for 15 min, washed with PBS, and incubated for 1 h at room temperature in staining buffer containing 0.3% Triton X-100 (Sigma), 5% goat serum (Life Technologies), and 1% BSA (Fisher Scientific) in PBS. The slides were then incubated for 3 h at room temperature with staining buffer containing unlabeled primary antibodies for NOS2 (Millipore) and CD3 (eBioscience), washed with PBS, and incubated for 30 min at room temperature with staining buffer containing the secondary antibodies anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 647 (Cell Signaling Technology). Coverslips were washed with PBS and mounted on SuperFrost Plus Slides (Thermo Scientific) using ProLong Gold antifade reagent

(Invitrogen). Confocal analyses were conducted using a LEICA TCS SL DMRE Microsystem.

[0131] Taqman analysis of mRNA expression. m NA expression was analyzed in day 7-

8 anti-CD3/CD28- expanded OvCa primary cell cultures, and in day 4-6 expanded CD4+TILs and naive and memory CD4+ T cells. Taqman analysis was performed on the StepOne Plus System (Applied Biosystems) using Taqman-recommended inventoried or made-to-order assays (Gene IDs: ill7a:Hs00174383, ill 7f:HsO 1028648, il2ra:Hs00907779, il23r:Hs00332759, nos2:HsO 1075527, rare :HsO 1076112, tbet:Hs00203436, gata3 :NM_002051 , foxp3:Hs00203958). The expression of each gene was normalized to HPRT1 and expressed as relative expression, i.e. fold increase (2- CT), where CT=CT(Target gene)-CT(HPRTl).

[0132] Statistical analysis. The figures demonstrating key phenomena and critical mechanisms involve aggregate data from multiple patients and healthy donors (expressed as means ± SD; the donor numbers are provided in the individual figure panels and the corresponding legends). Data from representative experiments (typically used in the studies comparing different reagents or different concentrations) were obtained from replicate cultures (means ± SD; numbers of replicates provided in figure legends) with each experiment confirmed in additional independent experiments with cells from different donors, as indicated in the figure legends. All data were evaluated using GraphPad Prism Version 5 software and analyzed using Student's t test (two-tailed) and 1-way and 2-way analysis of variance, where appropriate, with P<0.05 considered as significant (P<0.05 marked *; P<0.01 marked **;

P<0.001 marked ***). A linear correlation between two continuous variables was tested with the r coefficient of determination.

[0133] Table 1. Demographic and tumor characteristics of the 13 UPCI patients from which ascites samples were obtained. Median age at diagnosis was 56 years (range 39-69 years).

Lab SO/ Can er Age (at Race Tumor Stage

Patieirt diagnosis) f rfiBaif/fBetastaxis!

2 Ovarian 62 W i e mc Serous

2 Ovarian 39 W¾^" e mc Sero-us

3 Gvsrssrs 4S White mc Serous

4 Ovasian 63 W i e mc Serous

5 Ovarian 41 White U\€ mxed

5 Ovarian m White mc Serous

7 Gvsnarc m Whits mc Serous

& Ovarian 60 White U\€ Serous

3 Ovarian 5s White U\€ Serous

10 Gvsnarc SO ^'Whits IUA Clear∞&\

11 Ovarian 44 AA UA CSear TransitionaS eel

12 Ovarian 53 White U\€ Serous

13 Gvsdarc 53 White mc Serous (iow grade)

Table 2. Demographic and tumor characteristics of the 10 RPCI patients from ascites samples were obtained. Median age at diagnosis was 64 years (range 50-85 years).

Example 2. MDSC-associated NOS2 and MDSC-produced exogenous NO promote the Thl7 phenotype in ovarian cancer patient TILs and naive and memory CD4+ T cells

[0135] Thl7 cells are observed in high frequency in ovarian cancer (OvCa; (Kryczek et al., 2009; Miyahara et al., 2008)). We have observed that the OvCa environment is a potent inducer of Thl7 responses (Miyahara et al, 2008). We tested the role of cancer-associated MDSCs and MDSC-produced factors in the development of human Thl7 cells.

[0136] As shown in Fig. 1, OvCa patient-isolated MDSCs, expressing the typical

CDl lb+CD33+HLADR-/lowCD80- phenotype (Gabrilovich and Nagaraj, 2009; Nagaraj and Gabrilovich, 2010) (see (Obermajer et al, 2011) for complete MDSC phenotype), selectively enhanced the expression of Rorc (encoding RORyt) and IL-17A (Fig. 1 A) and the production of IL-17A protein (Fig. 1, B and C) by CD4+ T cells, activated by anti-CD3/CD28 antibodies or allogeneic DCs (Fig. 1 C), showing no impact on the expression of the Thl marker, T-bet, and an inhibitory impact on the Treg marker, FoxP3 (Fig. 1 A). Unexpectedly, while inhibition of other MDSC products, including IL-10, IDO, and arginase, did not impact the induction of Thl 7 cells (Fig. 1, D and E), the inhibition of NOS, a key enzyme in the synthesis of the nominally- suppressive MDSC product NO (Huang et al, 2006; Movahedi et al, 2008), dose-dependently inhibited the ability of MDSCs to enhance IL-17A production (Fig. 1, D-F) and Rorc expression (Fig. 1 G) in activated CD4+ T cells. In addition, the Thl7-promoting effect of MDSCs was also reduced by the inhibition of COX2 (Fig. 1, D-F), the factor needed for the optimal MDSC expression of NOS2 (Obermajer et al, 2011).

[0137] In accordance with a role of MDSC-associated NOS2 in the induction of Thl 7 responses in cancer patients in vivo, we observed that the levels of NOS2 expression in the ascites cells from OvCa patients positively correlated with the ability of these cells to produce IL-17A after short-term ex vivo stimulation (Fig. 1 H). Moreover, we observed that the Thl 7- promoting effects of MDSCs could be prevented both by the selective inhibition of NOS2 activity as well as the inhibition of cGMP function (Fig. 1 I), further demonstrating that human Thl 7 responses depend on NOS2 and the canonical cGMP/cGK-mediated signaling pathway associated with the physiologic NO concentrations produced by human cancer-isolated MDSCs (Fig. 2 A). [0138] While MDSCs produced all the previously-identified Thl7-driving cytokines

(Fig. 2 B) either spontaneously (IL-Ιβ, IL-6, and TGF-βι) or after their stimulation (IL-23) with CD40L (the CD4+ T cell-expressed APC-activating factor (Lane et al., 1992)), the advantage of MDSCs in driving the Thl7 phenotype was particularly pronounced in the case of naive, as compared to memory, Th cells (Fig. 1 C). Human naive Th cells, compared to their memory counterparts, were previously shown to be less sensitive to the induction of the Thl7 phenotype by recombinant cytokines (van Beelen et al., 2007), indicating that such an additional signal, which can be promoted by MDSCs, is essential for the de novo induction of Thl7 cells.

[0139] Whereas, consistent with previous reports (Mahidhara et al, 2003; Niedbala et al,

2011), high doses of exogenous NO (higher than 100 μΜ; known to have cytostatic function (Bogdan, 2001)) nonselectively blocked CD4+ T cell differentiation (Fig. 2 C) and their proliferation (Fig. 2 D) in all conditions tested (Thl7-, Thl-, or Treg-driving conditions), the application of lower, standard cell signaling doses (Karpuzoglu and Ahmed, 2006; Kolb and Kolb-Bachofen, 1998) of NO donor (10-25 μΜ; comparable to the MDSC-produced NO levels; see Fig. 2 A) did not affect CD4+ T cell proliferation. Instead, these lower doses of NO selectively enhanced IL-17A production (Fig. 2 C) without affecting IFN-γ production (Fig. 2 C), further confirming the ability of physiologic NO concentrations to selectively support human Thl 7 development.

[0140] Exogenous NO strongly enhanced the induction of Thl 7 cells driven by recombinant cytokines (Fig. 2, C and E-F), indicating its direct impact on T cells rather than its modulation of MDSC functions. NO induced IL-17A expression (Fig. 2 E) and secretion (Fig. 2 F) by both naive and memory CD4+ T cells and enhanced their expression of Rorc (but not GATA3, FoxP3, or T-bet; Fig. 2 E). The distinct Thl7-promoting effect of exogenous NO was evident even in the absence of Thl7-driving cytokines (IL-Ιβ, IL-6, and IL-23; Fig. 2, C and E- F), indicating that NO is a direct inducer of Thl 7 differentiation, rather than a mere enhancer of the effects of Thl7-inducing cytokines. Example 3. Cytokine- or MDSC-driven Thl7 differentiation depends on the induction of endogenous NOS2 in naive CD4+ T cells and elevation of endogenous NOS2 in memory CD4+ T cells.

[0141] Unexpectedly, we observed that Thl7-driving cytokines, as well as exogenous

NO, all induce the expression of endogenous NOS2 in the expanding population of naive CD4+ T cells (>99% pure at the day of analysis) and further enhance its expression in memory Th cells (Fig. 3, A-C). A similar induction of endogenous NOS2 was also observed in blood-isolated naive and memory CD4+ T cells, as well as tumor-isolated TILs, differentiated in the presence of cancer-isolated MDSCs.

[0142] To eliminate the possibility that such enhanced NOS2 levels in expanding T cell cultures result from their contamination with rare non-T cells expressing very high levels of NOS2, we evaluated the presence of intracellular NOS2 protein in individual CD4+ T cells. As shown in Fig. 3 D, the addition of Thl7-driving cytokines uniformly induced distinct elevation of NOS2 in the individual differentiating T cells, with further enhancement of intracellular NOS2 protein observed in the T cells that developed in the added presence of NO donor.

[0143] In accordance with the enzymatic activity of the CD4+ T cell-expressed NOS2, we observed intracellular accumulation of NO in the individual Thl7-differentiated cells by flow cytometry (Fig. 3 E). The intracellular NO accumulation resulted from endogenous NOS2 activity, as it was completely blocked using two different small molecule inhibitors of NOS2 activity (pan-NOS-inhibitor and NOS2-selective inhibitor; Fig. 3 E). In line with the requirement for endogenous NOS2/NO-signaling in the effective induction of human Thl7 cells, blockade of the endogenous NOS2 in CD4+ T cells differentiating in the presence of Thl7-driving cytokines suppressed their ability to secrete IL-17A (Fig. 3 F).

[0144] The induction of NOS2 in CD4+ T cells was closely correlated with the activity of the individual Thl7-driving factors (and their combinations) in inducing Thl7 differentiation (Fig. 3 G). IL-Ιβ recently shown to be the critical component of the Th 17 -promoting cytokine cocktail (Chung et al, 2009; Guo et al, 2009), was sufficient to induce significant expression of NOS2 in CD4+ T cells, but its combination with the additional Thl7-driving cytokines (IL-6 and IL-23) was needed for the optimal induction of NOS2 (Fig. 3 G). This effect was further amplified by exogenous NO (Fig. 3, B, D and E). In contrast to ILip, IL-6, and IL-23, which together promoted the elevation of endogenous NOS2 in human CD4+ T cells (and associated IL- 17A production), human TGF-βΙ proved to be a suppressor of both IL-17A and NOS2 (Fig. 3 G). Unlike the potent induction of endogenous NOS2 in CD4+ T cells driven toward Thl7 development, neither NOS1 (undetectable) nor NOS3 expression were induced during this process.

[0145] The induced levels of endogenous NOS2 were much higher in human memory than in naive CD4+ T cells (Fig. 3 A), consistent with the observed differences in the

effectiveness of IL-17 induction in these two populations (see ref. (van Beelen et al, 2007) and current Fig. 1, C and I, Fig. 2 F, and Fig. 3 A). However, very high differences in baseline NOS2 levels between human memory and naive CD4+ T cells (mean ± SD: naive: 0.00003±0.00002 vs. memory: 0.0006±0.0002, p=0.0053) could not be seen in their murine counterparts, where both memory and naive CD4+ T cells expressed similarly high baseline levels of NOS2 (naive:

0.0034±0.001 vs. memory: 0.0032±0.001, not significant), and were not significantly modulated in the presence of Thl7-promoting cytokines (mouse naive: 0.0048±0.001 vs. memory:

0.005±0.001, not significant). These observations are consistent with the previously-reported differences in the regulation of mouse and human immune system by NO (Fang, 2004;

Schneemann and Schoeden, 2007; Schneemann and Schoedon, 2002), different susceptibility of human and mouse naive CD4+ T cells to Thl7-inducing factors (Acosta- Rodriguez et al., 2007a; Acosta-Rodriguez et al, 2007b; van Beelen et al, 2007), and the recently reported lack of positive impact of exogenous NO on mouse CD4+ T cells at any concentrations of NO donor (ref. (Niedbala et al., 2011).

Example 4. Persistent expression of endogenous NOS2 and persistent cGMP-signaling are required for the functional stability of Thl7 cells: Reversal of established Thl7 cells from cancer patients by NOS- and cGMP-inhibitors

[0146] NOS2 blockade in CD4+ T cell cultures activated in the presence of Thl7-driving cytokines revealed that endogenous NO is not only required for the induction of IL-17A and IL- 17F expression, but also for the optimal expression of IL-23 receptor (Fig. 4, A and B), important for the maintenance of Thl7 function (Stritesky et al., 2008). NO induced IL-23R expression on naive CD4+ T cells, which express significantly less IL-23R than memory CD4+ T cells (Fig. 4 B, left). In contrast to IL-23R, no impact of NO on IL-2R expression was observed (Fig. 4 B, right). Furthermore, NOS inhibition reduced IL-23R expression by memory CD4+ T cells (Fig. 4 B), suggesting the requirement for NO in the optimal delivery of IL-23 -mediated signals, which may contribute to the persistence of the Thl7 phenotype.

[0147] NO has been shown to signal predominantly via the cGMP/cGK signaling cascade (Fischer et al., 2001), while high concentrations of NO involve additional NO-dependent modification of a wider spectrum of endogenous proteins and nonspecific cytotoxic effects (de Vera et al., 1996; Thomas et al, 2004). In order to define the pathway of NO signaling relevant to Thl7 induction, and to identify new targets for therapeutic modulation of Thl7 responses and Thl7-dependent pathologies, we evaluated the role of cGMP/cGK signaling in these phenomena. Our data show that the cGMP-specific inhibitor ODQ blocks IL-17A production in naive CD4+ T cells activated in the presence of Thl7-driving cytokines, while the addition of a membrane- permeable cGMP analogue alone can induce IL-17A production and further increase the production of IL-17A induced by Thl7-driving cytokines (Fig. 4 C).

[0148] Both NO and the cGMP signaling cascade proved to be required for the stability of established Thl7 cells that developed in cancer patients (OvCa TILs) in vivo or were generated from healthy donors in vitro, since inhibition of either NOS or cGMP selectively suppressed IL-17A but not IFN-γ production by these cells (Fig. 4, D and E). These data indicate that targeting of NO-activated cGMP/cGK signaling can be evaluated for therapeutic

intervention in Thl7-mediated disorders.

Example 5. Discussion

[0149] Local expression of IL-17A and NOS2 is observed in the tumor environment of patients with ovarian cancer. We tested the role of the NOS2/NO/cGMP/cGK pathway in the development of Thl7 cells from human naive, memory, and tumor-infiltrating CD4+ T cells. We observed that endogenous NOS2 activity and intracellular NO production induced in CD4+ T cells by previously-identified Thl7 inducers (IL-Ιβ, IL-6, and IL-23) or by cancer-infiltrating MDSCs is critically required for the de novo induction of Thl7 cells in vitro and for the stability of in vivo arising Thl7 cells from cancer patients. While, in accordance with previous reports (Bogdan, 2001; Kolb and Kolb-Bachofen, 1998; Mahidhara et al, 2003; Niedbala et al, 2011), high concentrations of exogenous NO non-selectively inhibited immune activation, including the proliferation and differentiation of Thl7 cell precursors, the levels of NO produced by myeloid cells and, endogenously, by CD4+ T cells supported the induction of Thl7 responses and were essential for the functional stability of Thl7 cells. These results help to explain the

heterogeneous and often paradoxical effects of NO and Thl7 cells in the regulation of inflammation, autoimmunity, and cancer (Bogdan, 2001).

[0150] The current data and the previously-reported importance of COX2 and PGE2 in the induction and stability of NOS2 production by MDSCs (Obermajer et al., 2011) and the ability of NO to enhance COX2 activation (Kim et al., 2005) indicate a close interplay between these two inflammatory systems, which may provide new insights to the mechanism of the COX2/PGE2-deiven development of Thl7 responses to different pathogens (Boniface et al, 2009; Chizzolini et al., 2008; Gopal et al, 2012) and potential new therapeutic targets.

[0151] While MDSCs suppress naive CD8+ T cell proliferation and acquisition of cytolytic functions (Gabrilovich and Nagaraj, 2009; Kusmartsev et al., 2004; Nagaraj and Gabrilovich, 2010; Obermajer et al., 2011), they do not impair naive CD40L-expressing CD4+ T cell proliferation, but instead promote the de novo induction of Thl7 cells, an effect that may explain the paradoxic generation and presence of inflammatory Thl7 cells in the

immunosuppressive cancer-associated environment (Coussens and Werb, 2002; Kryczek et al, 2009; Mantovani et al., 2008; Martin-Orozco and Dong, 2009). Our data also indicate that in analogy to the positive feedback loop between Thl cells and IFN-y-producing NK and CD8+ T cells (Kalinski and Moser, 2005), Thl 7 cells not only promote NO-dependent effector mechanisms of immunity (Miljkovic and Trajkovic, 2004), but also benefit from interaction with NO-producing cells (neutrophils, macrophages, MDSCs), leading to the establishment of a positive feedback between such NO- and IL-17-producing 'effector' and 'helper' cells.

[0152] Our observations demonstrating that the stability of Thl 7 function requires endogenous NOS2 and that the induction of endogenous NOS2 in CD4+ T cells benefits from the synergy between the previously-identified Thl7-driving cytokines (IL-Ιβ, IL-6, and IL-23; Fig. 3 G) help to explain the paradox that while the synergy between IL-Ιβ and IL-6 (or other Stat3 inducers) is sufficient for the effective induction of Thl 7 cells (Bettelli et al, 2007; Guo et al., 2009), IL-23 signaling is an important component of the development of the Thl7 phenotype and Thl7 functions in most models (Acosta-Rodriguez et al, 2007a; Acosta-Rodriguez et al, 2007b; Bettelli et al, 2006; Ivanov et al, 2006; van Beelen et al, 2007; Veldhoen et al, 2006; Wilson et al, 2007; Zhou et al, 2007). Similar to previous reports (Acosta-Rodriguez et al, 2007a; Wilson et al, 2007), we observed that TGF-βΙ suppresses the development of human Thl7 cells (Fig. 3 G), an effect that is mirrored by the ability of TGF- βΐ to suppress endogenous NOS2 in CD4+ T cells. These data suggest that the differences in the relative importance of TGF- βΐ in the development of mouse Thl7 cells in different models (Bettelli et al., 2006;

Ghoreschi et al., 2010; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007) may need to be evaluated in the context of its impact on the production of endogenous (T cell-derived) and exogenous (produced by MDSCs and other myeloid or stromal cells) NO, which may potentially differ in different settings. Likewise, fundamental differences between mice and humans regarding NOS activity, NO production, and NO regulation ((Fang, 2004; Schneemann and Schoeden, 2007; Schneemann and Schoedon, 2002) and current data) may explain different requirements for TGF-βΙ in the development of Thl7 cells in mice versus humans.

[0153] Similarly, it remains to be seen if such TLR ligands as LPS or peptidoglycan, shown to be particularly effective in promoting the DC-mediated induction of human Thl7 cells (Acosta-Rodriguez et al., 2007a; van Beelen et al., 2007), are particularly effective in inducing DC production of NO or alternative activators of the cGMP/cGK-mediated signaling pathway. Whether potential differences in this regard may contribute to the different efficiency of induction of Thl7 cells from naive versus memory and effector precursors ((van Beelen et al., 2007) and the current data) deserves further exploration.

[0154] Our data suggest that the previously-observed differences in the ability of human memory and naive CD4+ T cells to develop into Thl7 cells (see ref. (van Beelen et al., 2007) and current Fig. 1 C and Fig. 2 F) may, at least partially, result from much higher baseline levels of endogenous NOS2 in human memory CD4+ T cells, compared to naive CD4+ T cells (Fig. 3 A). Our preliminary data indicate that a similar difference does not exist in the mouse system, where both naive and memory cells express very high baseline levels of NOS2, and do not further elevate its levels during Thl7 differentiation (unpublished data). These differences in the baseline levels of NOS2 between human and mouse cells and the requirement for inflammatory factors in the expression of NOS2 by human cells are consistent with the significant delay in the demonstration of the presence of NOS2 in human cells (Geller et al, 1993; Hibbs et al, 1992; Ochoa et al, 1992) and its cloning (Geller et al, 1993). [0155] The present invention encompasses the discovery that NO, NOS/NOS2, and the cGMP/cGK-signaling pathway are critical requirements for the induction and stability of human Thl7 cells, including, for example, Thl7 cells arising in vivo in cancer bearing patients and induced in vitro from naive and memory precursor cells from healthy donors. Manipulation of NO, NOS/NOS2, and/or the cGMP/cGK- signaling pathway may be employed in several therapeutic strategies, for example, inhibition of NO production or signaling in Thl7-dependent malignant tumors or in Thl7-mediated inflammatory/autoimmune processes, or the activation of these pathways to boost desirable Thl7 immunity in Thl7-susceptible tumors or chronic infections, such as M. tuberculosis. NO donors and inhibitors are available which may be useful for the treatment of autoimmune and inflammatory diseases, as well as for vasodilation in hypertensive coronary disease, erectile dysfunction, and pulmonary hypertension.

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[0156] While the described invention has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the described invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Claims What is claimed is:

1. A method of manipulating a Thl7 response to a disease, disorder, or condition, the method comprising a step of: administering an agent that modulates Thl7 cell number or activity, wherein the agent comprises nitric oxide or a mediator of nitric oxide synthesis, levels, or signaling.

2. A method of manipulating a Thl7 response to a disease, disorder, or condition, the method comprising a step of: administering an agent that modulates cyclic guanosine monophosphate (cGMP)/cyclic GMP dependent protein kinase (cGK) signaling.

3. The method of any of claims 1 or 2 wherein the disease, disorder, or condition is selected from cancer, autoimmune disease, inflammatory disease, and infectious disease.

4. The method of any of claims 1 to 3 further comprising administering one or more cytokines.

5. The method of claim 4 wherein the one or more cytokines are selected from IL-Ιβ, IL-6 or other STAT3 inducer, IL-23, TGF-βΙ, and IL-21.

6. The method of any of claims 1-5 wherein Thl7 cell number increases by developing from naive, effector, and/or memory CD4+ T cell precursors.

7. The method of any of claims 1-5 wherein the Thl7 cell activity comprises an increase in IL- 17A production , level, or secretion.

8. The method of claim 7, wherein the agent does not significantly alter detectable IFN-γ production or levels.

9. The method of any of claims 1-5 wherein the agent increases Thl7 cell number or activity.

10. The method of claim 9 wherein the agent comprises nitric oxide, a nitric oxide agonist, a nitric oxide donor, a nitric oxide stabilizer, an inhibitor of nitric oxide catabolism, a nitric oxide synthase stimulator, or substrate for nitric oxide production.

11. The method of any of claims 1-5 wherein the agent decreases Thl7 cell number or activity.

12. The method of claim 11 wherein the agent comprises a nitric oxide synthase inhibitor.

13. The method of claim 11 wherein the agent is a selective nitric oxide synthase 2 (NOS2) inhibitor.

14. The method of claim 11 wherein the agent decreases the number of myeloid-derived suppressor cells (MDSCs) and/or decreases the production of nitric oxide from MDSCs.

15. A method comprising a step of: administering to a subject suffering from a Thl7-associated disease, disorder, or condition a therapeutic regimen that inhibits NO production or signaling.

16. The method of claim 15, wherein the Thl7-associated disease, disorder or condition is or comprises Thl7-malignant tumors.

17. The method of claim 15, wherein the Thl7-associated disease, disorder or condition is or comprises a Thl7-mediated inflammatory process.

18. The method of claim 15, wherein the Thl7-associated disease, disorder or condition is or comprises a Thl7-mediated autoimmune process.

19. The method of claim 15, wherein the therapeutic regimen comprises one or more

NOS inhibitors; cGMP/cGk inhibitors; and combinations thereof.

20. A method comprising a step of: administering to a subject suffering from a Thl7-susceptible disease, disorder, or condition a therapeutic regimen that stimulates or supports NO production or signaling.

21. The method of claim 20, wherein the Thl7-susceptible disease, disorder or condition is or comprises a TH17-susceptible tumor or infection.

22. The method of claim 20, wherein the Thl7-susceptible disease, disorder or condition is or comprises a chronic infection.

23. A method of identifying and/or characterizing therapeutic agents, the method comprising steps of: contacting a system in which presence or level of Thl7 activity is detectable with a test therapeutic agent to be characterized; and detecting a difference in the presence or level of Thl7 activity that correlates with presence or level of the test therapeutic agent as compared with a comparable reference condition, wherein the test therapeutic agent is identified and/or characterized as a Thl7 inhibitory agent if the detected difference is or comprises a decrease in the presence or level of Thl7 activity, and agent is identified and/or characterized as a Thl7 stimulatory agent if the detect difference is or comprises an increase in the presence or level of Thl7 activity.

24. The method of claim 23, wherein the comparable reference condition lacks the test therapeutic agent.

25. The method of claim 23, wherein the comparable reference condition includes a reference agent with known effect on presence or level of Thl7 activity.

26. A method comprising steps of: providing results analyzing presence or level of Thl7 activity in a sample from a patient suffering from or susceptible to a proliferative condition selected from the group consisting of tumors, inflammatory disorders, autoimmune disorders, and infections, which results indicate elevated presence or level of Thl7 activity relative to a reference; and administering a therapeutic regimen that modulates NO production or signaling.

27. The method of claim 26, wherein the step of administering comprises administering an agent selected from the group consisting of:

NOS inhibitors; cGMP-specific inhibitors; and combinations thereof.