Blockade of CD127 Exerts a Dichotomous Clinical Effect in Marmoset Experimental Autoimmune Encephalomyelitis.

Non-human primate models of human disease have an important role in the translation of a new scientific finding in lower species into an effective treatment. In this study, we tested a new therapeutic antibody against the IL-7 receptor α chain (CD127), which in a C57BL/6 mouse model of experimental autoimmune encephalomyelitis (EAE) ameliorates disease, demonstrating an important pathogenic function of IL-7. We observed that while the treatment was effective in 100 % of the mice, it was only partially effective in the EAE model in common marmosets (Callithrix jacchus), a small-bodied Neotropical primate. EAE was induced in seven female marmoset twins and treatment with the anti-CD127 mAb or PBS as control was started 21 days after immunization followed by weekly intravenous administration. The anti-CD127 mAb caused functional blockade of IL-7 signaling through its receptor as shown by reduced phosphorylation of STAT5 in lymphocytes upon stimulation with IL-7. Group-wise analysis showed no significant effects on the clinical course and neuropathology. However, paired twin analysis revealed a delayed disease onset in three twins, which were high responders to the immunization. In addition, we observed markedly opposite effects of the antibody on pathological changes in the spinal cord in high versus low responder twins. In conclusion, promising clinical effect of CD127 blockade observed in a standard inbred/SPF mouse EAE model could only be partially replicated in an outbred/non-SPF non-human primate EAE model. Only in high responders to the immunization we found a positive response to the treatment. The mechanism underpinning this dichotomous response will be discussed.

Receptor occupancy and blocking of STAT5 signaling by an anti-IL-7 receptor α antibody in cynomolgus monkeys.

BACKGROUND: Interleukin-7 receptor α (IL-7Rα) is associated with autoimmune disease. Blocking its activation by interleukin-7 (IL-7) with a therapeutic monoclonal antibody may reduce pathogenic T cells and effectively control the autoimmune response in these disorders. METHODS: Two flow cytometry-based assays were developed and implemented to evaluate the interaction between cell surface IL-7Rα and an anti-IL-7Rα monoclonal antibody (Ab1). The receptor occupancy assay utilized competing and noncompeting commercial detection antibodies for "free" and "total" IL-7Rα, respectively. STAT5 phosphorylation (pSTAT5) was measured as a proximal biomarker of IL-7Rα inhibition by Ab1. RESULTS: Monkeys administered Ab1 had no free IL-7Rα detectable on the CD3+ T cell surface at 0.25 hours postdose through day 4, in all treatment groups. Ab1 treatment resulted in a significant reduction in total IL-7Rα, dropping to 53%, 44%, and 55% on day 4 at 0.3, 3, and 30 mg/kg, respectively, compared to predose levels. There were treatment-related decreases in the ability of IL-7 to induce STAT5 phosphorylation in both CD4+ and CD8+ T cells in monkey blood samples from all treated animals from 0.25 hours through Day 4 postdose. CONCLUSIONS: The nonclinical receptor occupancy assay was developed and applied to detect free and total IL-7Rα on the surface of CD3+ T cells in cynomolgus monkeys treated with Ab1. The results showed good correlation with the phosphorylation of STAT5 and serum concentration of Ab1. The approach for IL-7Rα occupancy and pSTAT5 measurements established in monkeys can be utilized in clinical trials for pharmacokinetic/pharmacodynamic evaluation of Ab1 effect in humans.

Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 T cells in a poorly immunogenic tumor model.

Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti-4-1BB/anti-PD-1 combination with that of the anti-PD-1/anti-LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti-PD-1 and anti-4-1BB concomitantly, while combining anti-PD-1 with anti-LAG-3 led to a modest degree of tumor suppression. The activity of the anti-4-1BB/anti-PD-1 combination was dependent on IFNγ and CD8(+) T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8(+) T cells by anti-4-1BB/anti-PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8(+)/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8(+) CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti-4-1BB/anti-PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients.

Anti-IL-7 receptor-α reverses established type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function.

Genetic variation in the IL-7 receptor-α (IL-7R) gene is associated with susceptibility to human type 1 diabetes (T1D). Here we investigate the therapeutic efficacy and mechanism of IL-7Rα antibody in a mouse model of T1D. IL-7Rα antibody induces durable, complete remission in newly onset diabetic mice after only two to three injections. IL-7 increases, whereas IL-7Rα antibody therapy reduces, the IFN-γ-producing CD4(+) (T(H)1) and IFN-γ-producing CD8(+) T cells. Conversely, IL-7 decreases and IL-7Rα antibody enhances the inhibitory receptor Programmed Death 1 (PD-1) expression in the effector T cells. Programmed Death 1 blockade reversed the immune tolerance mediated by the IL-7Rα antibody therapy. Furthermore, IL-7Rα antibody therapy increases the frequency of regulatory T cells without affecting their suppressor activity. The durable efficacy and the multipronged tolerogenic mechanisms of IL-7Rα antibody therapy suggest a unique disease-modifying approach to T1D.

IL-7 promotes T(H)1 development and serum IL-7 predicts clinical response to interferon-ß in multiple sclerosis.

The interleukin-7 receptor α chain (IL-7Rα) gene was identified as a top non-major histocompatibility complex-linked risk locus for multiple sclerosis (MS). Recently, we showed that a T helper 1 (T(H)1)-driven, but not a T(H)17-driven, form of MS exhibited a good clinical response to interferon-ß (IFN-ß) therapy. We now demonstrate that high serum levels of IL-7, particularly when paired with low levels of IL-17F, predict responsiveness to IFN-ß and hence a T(H)1-driven subtype of MS. We also show that although IL-7 signaling is neither necessary nor sufficient for the induction or expansion of T(H)17 cells, IL-7 can greatly enhance both human and mouse T(H)1 cell differentiation. IL-7 alone is sufficient to induce human T(H)1 differentiation in the absence of IL-12 or other cytokines. Furthermore, targeting IL-7/IL-7Rα is beneficial in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Mice treated with IL-7Rα-blocking antibodies before or after onset of paralysis exhibited reduced clinical signs of EAE, with reduction in peripheral naïve and activated T cells, whereas central memory T, regulatory T, B, and natural killer cell populations were largely spared. IL-7Rα antibody treatment markedly reduced lymphocyte infiltration into the central nervous system in mice with EAE. Thus, a serum profile of high IL-7 may signify a T(H)1-driven form of MS and may predict outcome in MS patients undergoing IFN-ß therapy. Blockade of IL-7 and the IL-7Rα pathway may have therapeutic potential in MS and other autoimmune diseases.

Genomic expression profiling of TNF-alpha-treated BDC2.5 diabetogenic CD4+ T cells.

TNF-alpha plays an important role in immune regulation, inflammation, and autoimmunity. Chronic TNF exposure has been shown to down-modulate T cell responses. In a mouse T cell hybridoma model, TNF attenuated T cell receptor (TCR) signaling. We have confirmed that chronic TNF and anti-TNF exposure suppressed and increased T cell responses, respectively. In adult TCR (BDC2.5) transgenic nonobese diabetic mice, DNA microarray analysis of global gene expression in BDC2.5 CD4(+) T cells in response to chronic TNF or anti-TNF exposure showed that genes involved in functional categories including T cell signaling, cell cycle, proliferation, ubiquitination, cytokine synthesis, calcium signaling, and apoptosis were modulated. Genes such as ubiquitin family genes, cytokine inducible Src homology 2-containing genes, cyclin-dependent kinase inhibitors p21, p57, calmodulin family genes (calmodulin-1, -2, and -3) and calcium channel voltage-dependent, N type alpha1B subunit (CaV2.2) were induced by TNF, whereas Vav2, Rho GTPase-activating protein, calcium channel voltage-dependent, L type alpha1C subunit (CaV1.2), IL-1 receptor-associated kinase-1 and -2, and IL enhancer binding factor 3 were reduced by TNF. Genes such as CaV1.2 and proliferating cell nuclear antigen, repressed by TNF, were induced by anti-TNF treatment. Further, we showed that chronic TNF exposure impaired NF-kappaB and adaptor protein 1 transactivation activity, leading to T cell unresponsiveness. Thus, our results present a detailed picture of transcriptional programs affected by chronic TNF exposure and provide candidate target genes that may function to mediate TNF-induced T cell unresponsiveness.

The role of TNF-alpha in the pathogenesis of type 1 diabetes in the nonobese diabetic mouse: analysis of dendritic cell maturation.

TNF-alpha has been linked to the development of type 1 diabetes (T1D). We previously reported that neonatal treatment of nonobese diabetic (NOD) mice with TNF-alpha accelerated the onset of T1D, whereas TNF-alpha blockade in the same time period resulted in a complete absence of diabetes. The mechanisms by which TNF-alpha modulates development of T1D in NOD mice remain unclear. Here we tested the effects of TNF-alpha on the maturation of dendritic cells (DCs) in the NOD mouse. We found that neonatal treatment with TNF-alpha caused an increase in expression of maturation markers on CD11c(+)CD11b(+) DC subpopulations, whereas treatment with anti-TNF-alpha resulted in a decrease in expression of maturation markers in the CD11c(+)CD11b(+) subset. Moreover, neonatal treatment with TNF-alpha resulted in skewed development of a CD8alpha(+)CD11b(-)CD11c(+) DC subset such that TNF-alpha decreases the CD8alpha(+)CD11c(+) DC subset, increases the CD11c(+)CD11b(+) subset, and causes an increase in the expression of CD40 and CD54 on mature DCs capable of inducing immunity. Anti-TNF-alpha-treated mice had an increase in the CD8alpha(+)CD11c(+) DCs. Notably, adoptively transferred naïve CD4(+) T cells from BDC2.5 T cell receptor transgenic mice proliferated in the pancreatic lymph nodes in TNF-alpha-treated NOD mice but not in anti-TNF-alpha-treated mice. Finally, we show that anti-TNF-alpha-treated mice showed immunological tolerance to islet cell proteins. We conclude that TNF-alpha plays an important role in the initiation of T1D in the NOD mouse by regulating the maturation of DCs and, thus, the activation of islet-specific pancreatic lymph node T cells.

Interleukin-8 confers androgen-independent growth and migration of LNCaP: differential effects of tyrosine kinases Src and FAK.

Interleukin-8 (IL-8), a chemokine implicated in the metastasis and angiogenesis of a variety of cancers, has been reported to be overexpressed in prostate cancer. In this study, we ascribe a new role for IL-8 in prostate cancer progression using LNCaP cells. We demonstrate that IL-8 activates the androgen receptor and confers androgen-independent growth, while serving as a potent chemotactic factor. Our evaluation of the possible signal pathways involved in androgen-independence and cell migration shows that the tyrosine kinases Src and FAK (focal adhesion kinase) are involved in IL-8-induced signaling. Pharmacological and genetic inhibitors of Src and FAK interfere with IL-8-induced cell migration, while only the Src inhibitor was able to repress androgen-independent growth. This suggests that both growth and migration depend on the activity of Src, whereas cell migration also requires the activation of FAK. Our evidence that IL-8-induced androgen-independent growth is, at least in part, due to androgen receptor activation includes (1) an inhibitor of androgen receptor activity diminishes cell growth; (2) androgen receptor transactivation potential is augmented by IL-8 and (3) androgen receptor is recruited to the promoter of prostate specific antigen (PSA) upon IL-8 treatment, based on chromatin immunoprecipitation experiments. Taken together, our data suggest that in addition to its role in metastasis and angiogenesis, IL-8 may also serve as a facilitator for androgen-independent transition of prostate cancers. To our knowledge, this is the first report about the tyrosine kinase signals and androgen receptor activation induced by IL-8 in prostate cancer cells. The observation that IL-8 mediates its growth and chemotactic effects via Src and FAK suggests the potential use for tyrosine kinase inhibitors at early stage of prostate cancer development.

Characterization of a novel androgen receptor mutation in a relapsed CWR22 prostate cancer xenograft and cell line.

CWR22 has been a valuable xenograft model for the study of prostate cancer progression from an androgen-dependent tumor to one that grows in castrated animals. Herein, we report the identification and characterization of a novel androgen receptor (AR) mutation occurring in a relapsed tumor (CWR22R-2152) and in the CWR22Rv1 cell line established from it. The mutation was not detected in the original, hormone-dependent CWR22 xenograft, indicating that this change occurred during the progression to androgen independence. It is characterized by an in-frame tandem duplication of exon 3 that encodes the second zinc finger of the AR DNA-binding domain. Accordingly, immunoblot analyses demonstrated the expression of an AR species having an approximately 5-kDa increase in size relative to the LNCaP AR. This was accompanied by a COOH-terminally truncated AR species migrating with a relative mass of 75-80 kDa, referred to as ARDeltaLBD because it lacks the ligand-binding domain. By recreating the exon 3 duplication mutation in a wild-type AR expression construct, the generation of ARDeltaLBD could be recapitulated. Whereas ARDeltaLBD exhibited constitutive nuclear localization and DNA binding, these functions in the full-length AR remained androgen dependent. The CWR22Rv1 AR repertoire displayed dose-dependent, androgen-responsive transcriptional transactivation in reporter assays, albeit to a lesser extent in comparison with LNCaP. This cell line also expressed low levels of prostate-specific antigen mRNA and did not express or secrete detectable levels of prostate-specific antigen protein in androgen-depleted medium or in response to physiological androgenic stimulation. In summary, the CWR22Rv1 cell line displays both androgen-responsive and androgen-insensitive features due, at least in part, to a novel insertional mutation of the AR.