Subhasis Chattopadhyay

Regulatory T cells and tumor immunity.

Central deletion of “self-reactive” T cells has been the textbook paradigm for inducing “self-tolerance” in the periphery and the concept of a role of T cell-mediated suppression in this process has long been controversial. A decisive shift in the opinion on suppressor T cells has lately occurred with the observations of Sakaguchi’s group that linked a class of CD4+CD25+ T cells to the prevention of autoimmunity from neonatal thymectomy in mice. These CD4+CD25+ T cells have been named T regulatory (Treg) cells. They are believed to be selected in the thymus as an anti-self repertoire. Hence they were referred to as natural T regulatory (nTreg) cells. Presently, in addition to their role in autoimmunity, they are believed to exert regulatory function in infection, in transplantation immunity as well as in tumor immunity. In contrast to these nTreg cells, another class of CD4+ Treg cells also exercises regulatory function in the periphery. These Treg cells are also CD4+ T cells and after activation they also become phenotypically CD4+CD25+. They are, however induced in the periphery as Treg cells. Hence, they are termed as induced Treg (iTreg) cells. There are major differences in the biology of these two types of Treg cells. They differ in their requirements for activation and in their mode of action. Nonetheless, evidence indicates that both nTreg cells and iTreg cells are involved in the control of tumor immunity. The question of how to circumvent their regulatory constraints, therefore, has become a major challenge for tumor immunologists.

Human Neutrophil-Expressed CD28 Interacts with Macrophage B7 to Induce Phosphatidylinositol 3-Kinase-Dependent IFN-γ Secretion and Restriction of Leishmania Growth

We previously showed that CD28 is expressed on human peripheral blood neutrophils and plays an important role in CXCR-1 expression and IL-8-induced neutrophil migration. In this work we demonstrate that Leishmania major infection of macrophages results in parasite dose-dependent IL-8 secretion in vitro and in IL-8-directed neutrophil migration, as blocked by both anti-IL-8 and anti-IL-8R Abs, toward the L. major-infected macrophages. In the neutrophil-macrophage cocultures, both CTLA4-Ig, a fusion protein that blocks CD28-CD80/CD86 interaction, and a neutralizing anti-IFN-γ Ab inhibit the anti-leishmanial function of neutrophils, suggesting that the neutrophil-macrophage interaction via CD28-CD80/CD86 plays an important role in the IFN-γ-dependent restriction of the parasite growth. Cross-linking of neutrophil-expressed CD28 by monoclonal anti-CD28 Ab or B7.1-Ig or B7.2-Ig results in phosphatidylinositol 3-kinase association with CD28 and in wortmannin-sensitive but cyclosporin A-resistant induction and secretion of IFN-γ. Whereas the neutrophils secrete IFN-γ with CD28 signal alone, the T cells do not secrete the cytokine in detectable amounts with the same signal. Thus, neutrophil-expressed CD28 modulates not only the granulocyte migration but also induction and secretion of IFN-γ at the site of infection where it migrates from the circulation.

Effect of CD4+CD25+ and CD4+CD25− T Regulatory Cells on the Generation of Cytolytic T Cell Response to a Self but Human Tumor-Associated Epitope In Vitro

CD4+ T cells naturally expressing CD25 molecules (natural T regulatory cells (Tregs)) have a role in maintaining self tolerance and in regulating responses to infectious agents, transplantation Ags, and tumor Ags. CD4+ Tregs induced from CD4+CD25− precursors (induced Tregs) also regulate immune responses in the periphery. However, which of these Tregs is a major impediment in generating antitumor CTL responses is not clear. We show that although the CD4+CD25+ subsets isolated from peripheral blood-derived lymphocytes do suppress the proliferation of CD4+CD25− effector T cells, they do not suppress the activation and expansion of the self but melanoma-associated, melanoma Ag-reactive T cell 1 (MART-1)27–35-specific CD8+ T cells stimulated by the respective peptide-loaded matured dendritic cells in vitro. The CD4+CD25− counterparts, in contrast, lead to the generation of CD25+ glucocorticoid-inducible TNFR+-Forkhead/winged helix transcription factor+ populations and efficiently suppress the activation and expansion of the MART-127–35 epitope-specific CTLs. Our data suggest that when CTL precursors are optimally stimulated, natural Tregs are not a formidable constraint toward generating a robust antitumor CTL response, but induced Tregs could be.

Rescuing Melanoma Epitope-Specific Cytolytic T Lymphocytes from Activation-Induced Cell Death, by SP600125, an Inhibitor of JNK: Implications in Cancer Immunotherapy

Activation-induced cell death (AICD) as well as programmed cell death (PCD) serve to control the expansion of activated T cells to limit untoward side effects of continued effector responses by T cells and to maintain homeostasis. AICD of T cells in tumor immunotherapy can be counterproductive particularly if the activated T cells undergo apoptotic death after the very first secondary encounter of the specific epitope. We examined the extent to which tumor epitope-specific CTLs that are activated and expanded in an in vitro-matured dendritic cell-based primary stimulation protocol undergo AICD following their first secondary encounter of the cognate epitope. Using the MART-127–35 epitope as a prototype vaccine epitope, we also examined whether these CTLs could be rescued from AICD. Our results demonstrate that a substantial fraction of MART-127–35 epitope-specific primary CTLs undergo AICD upon the very first secondary encounter of the cognate epitope. The AICD in these CTLs is neither caspase dependent nor is it triggered by the extrinsic death signaling pathways (Fas, TNFR, etc.). These CTLs, interestingly, could be rescued from AICD by the JNK inhibitor, SP600125. We also found that SP600125 interferes with their IFN-γ response but does not block their cytolytic function. The rescued CTLs, however, regain their capacity to synthesize IFN-γ if continued in culture without the inhibitor. These observations have implications in tumor immunotherapy and in further studies for regulation of AICD in CTLs.

Heat Shock Protein 90 Positively Regulates Chikungunya Virus Replication by Stabilizing Viral Non-Structural Protein nsP2 during Infection

Background The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV) emphasize the need to understand the biology of the virus for developing effective antiviral therapies. Methods and Findings In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90) mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27) and Indian outbreak strain of 2006 (DRDE-06). Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA) can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production. Conclusion Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06, which will be informative for developing effective anti-CHIKV therapies in future.

High rates of co-infection of Dengue and Chikungunya virus in Odisha and Maharashtra, India during 2013.

Dengue viral (DENV) infection is endemic in different parts of India and because of similar primary signs and symptoms, Chikungunya virus (CHIKV) is mostly undiagnosed. Hence, we investigated 204 suspected Dengue cases in a hospital based cross-sectional study in Odisha, India in 2013. It was observed that 50 samples were positive for DENV only, 28 were positive for CHIKV only and interestingly, 28 patients were co-infected with both DENV and CHIKV. Additionally, a total of 18 confirmed Dengue samples from Maharashtra, India were screened for CHIKV and out of those, 15 were co-infected. All CHIKV strains were of East Central South African (ECSA) type and serotype 2 (genotype IV) was predominant in the DENV samples. Additionally, Dengue serotype 1 and 3 were also detected during this time. Further, sequence analysis of E1 gene of CHIKV strains revealed that two substitution mutations (M269V and D284E) were observed in almost 50% strains and they were from co-infected patients. Similarly, sequence analysis of C-prM gene showed the presence of five substitution mutations, (G70S, L72F, N90S, S93N and I150L) in all serotype 1 and two consistent mutations (A101V and V112A) in serotype 2 Dengue samples. Together, it appears that a significantly high number of dengue patients (43, 44.8%) were co-infected with DENV and CHIKV during this study. This emphasizes the need of a routine diagnosis of CHIKV along with DENV for febrile patients. This will be useful in early and proper recognition of infecting pathogen to study the correlation of clinical symptoms with single or co-infection which will ultimately help to implement proper patient care in future.

Implication for the CD94/NKG2A-Qa-1 system in the generation and function of ocular-induced splenic CD8+ regulatory T cells

The injection of antigen into the anterior chamber (AC) induces the production of antigen-specific splenic CD8+ regulatory T cells (Tregs) /suppressor T cells that perform the local suppression of delayed-type hypersensitivity (DTH) responses. Because CD94/NKG2A-Qa-1-dependent interactions have been implicated in CD8+ Treg-mediated immune suppression and DBA/2J mice are deficient in CD94/NKG2R, we have utilized these mice to test the hypothesis that the CD94/NKG2A-Qa-1 system is essential to the induction and immunosuppressive function of CD8+ Tregs in anterior chamber-associated immune deviation (ACAID). We show that: (i) neither ACAID-mediated suppression of DTH to ovalbumin nor splenic Tregs/suppressor T cells was induced in DBA/2J mice that received an injection of antigen into the AC; (ii) splenic CD8+ Tregs from ACAID-induced DBA/2NCr mice suppressed the initiation of DTH when transferred to DBA/2J mice; (iii) following injection of antigen into the AC, intravenous administration of splenocytes or Peripheral Blood Mononuclear Cells (PBMC) isolated from DBA/2NCr but not from DBA/2J mice transferred suppression of DTH to DBA/2NCr mice; (iv) antibodies to CD94/NKG2A reduced the ACAID CD8+ T cell-mediated suppression of DTH and (v) The deficiency of such immune regulation in DBA/2J mice also correlated with a decreased number of Qa-1(b+) B cells, F4/80+ cells, a deficient number of CD94/NKG2AR and Qa-1 tetramer binding by CD8+ T cells. These results demonstrate that defective ACAID in DBA/2J mice involves multiple regulatory lesions resulting in a lack of induction of a CD8+ Treg response and possibly defective CD94/NKG2A-dependent suppression of peripheral cell-mediated immunity.

Functional expression of TRPV channels in T cells and their implications in immune regulation.

The importance of Ca2+ signalling and temperature in the context of T cell activation is well known. However, the molecular identities of key players involved in such critical regulations are still unknown. In this work we explored the endogenous expression of transient receptor potential vanilloid (TRPV) channels, a group of thermosensitive and non‐selective cation channels, in T cells. Using flow cytometry and confocal microscopy, we demonstrate that members belonging to the TRPV subfamily are expressed endogenously in the human T cell line Jurkat, in primary human T cells and in primary murine splenic T cells. We also demonstrate that TRPV1‐ and TRPV4‐specific agonists, namely resiniferatoxin and 4α‐phorbol‐12,13‐didecanoate, can cause Ca2+ influx in T cells. Moreover, our results show that expression of these channels can be upregulated in T cells during concanavalin A‐driven mitogenic and anti‐CD3/CD28 stimulated TCR activation of T cells. By specific blocking of TRPV1 and TRPV4 channels, we found that these TRPV inhibitors may regulate mitogenic and T cell receptor mediated T cell activation and effector cytokine(s) production by suppressing tumour necrosis factor, interleukin‐2 and interferon‐γ release. These results may have broad implications in the context of cell‐mediated immunity, especially T cell responses and their regulations, neuro‐immune interactions and molecular understanding of channelopathies.

Presence of Low Dose of Fludarabine in Cultures Blocks Regulatory T Cell Expansion and Maintains Tumor-Specific Cytotoxic T Lymphocyte Activity Generated with Peripheral Blood Lymphocytes

Background: For tumor vaccine-based immunotherapy of cancer, the expansion of tumor antigen-specific cytotoxic T lymphocytes (CTL) in the patients by blocking induced regulatory T (Treg) cells is the most important objective now. Fludarabine (FLU), a known anticancer drug, has been shown to downregulate Treg cells in vivo in chronic leukemia patients. Melanoma tumor antigen Mart-127–35-specific CD8+ CTLs generated in vitrowith total peripheral blood lymphocytes (PBL) lose their activity within 14–21 days with concomitant expansion of Treg cells. When CD4+ cells are removed from PBL and CTL are generated with purified CD8+ cells, the CTL survive and maintain their activity for a significantly longer period. Methods: We used a low dose of FLU in the cultures in Mart-1-specific CTL generation assays with total PBL. Blood samples were taken from HLA-A2-positive melanoma patients and normal donors. Autologous matured dendritic cells pulsed with Mart-127–35 peptide were used to generate CTL responses using purified CD8+ cells or total PBL. Results: The presence of FLU in the cultures with PBL helped to generate a significantly higher number of antigen-specific CTLs as detected by Mart-1 HLA-A2 tetramer staining. Specificity of such CTLs was determined by IFN-γ secretion or by cytotoxicity against the target cells bearing the specific antigen. The presence of FLU stopped the expansion of IL-10-producing CD4+ Treg cells in the cultures with PBL. Analyses of expanded CD4+ cells isolated from PBL in vitro cocultures with FLU showed a Th1 type of function. Those cells secreted higher amounts of IFN-γ and very low levels of IL-10, or no IL-10 at all, upon restimulation. Conclusion: The observations of the study are as important for adaptive immunotherapy of cancer as they are for vaccine-based approaches.

CD28 signaling in neutrophil induces T-cell chemotactic factor(s) modulating T-cell response.

We previously reported that human peripheral blood neutrophils express CD28 and interact with macrophage B7 to generate CD28 signaling through PI-3 kinase. Here, we demonstrate that crosslinking of CD28 on neutrophils results in the release of IFN-gamma, which restricts amastigote growth and modulates CD4+ T cells cytokine secretion. CD28 crosslinking also induces a T-cell chemotactic factor (TCF) that induces chemotactic migration of CD4+ T cells. Based on our previous and the current set of data, we propose an operational model explaining how neutrophils are involved in Leishmania infection and how the reported effect of neutrophils on the control of infection is mediated by alteration of T-cell function.