Browsing by Subject "T-Lymphocytes, Regulatory"
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Item Characterizing B cell Phenotype during chronic HCV infection(2009-06-15) Ayers, Christopher Lee; Karandikar, NitinChronic hepatitis C virus (HCV) infection is characterized by an attenuation of virus-specific T cell responses. The mechanisms leading to T cell attenuation are still not well understood, and likely involve several integrating correlates. We hypothesized that dysfunctions of antigen presenting cells (APC) may contribute to the immunosuppresed phenotype. We also reasoned that direct viral interactions of HCV with immune cells may be responsible for such dysfunction. We employed a strand-specific real time RT-PCR assay and found that virus is frequently associated with B cells (predominantly positive strand was detected). Interestingly, we also found that ex-vivo derived B cells from chronic HCV individuals were better inducers of allogeneic T cell proliferation and this ability correlated with the presence of HCV RNA in those B cells. During such enhanced allostimulation, we also found an increase in the proportion of CD4+CD25+FoxP3+ T cells, which correlated with an increased suppressive capacity thereby demonstrating a paradoxical link between hyperactive B cells and the generation of suppressive T cells. Furthermore, ex-vivo derived chronic HCV B cells had an attenuated response to mitogenic stimulation with associated apoptosis. In an effort to determine direct HCV involvement in immune cell dysfunction, we evaluated the possibility of culture adapted JFH-1 virus to infect PBMC populations. While we found no evidence of viral replication in PBMC, exposure to JFH-1 resulted in vigorous activation of B cells. Interestingly, the B cell activation did not require viable virions, but was dependent upon CD81 availability and the presence of monocytes. We also determined that upon viral exposure, these B cells replicated the hyper-activation of MLR responses found in ex-vivo derived B cells from chronic HCV individuals. In all, our results suggest a novel model wherein HCV-B cell interaction leads to B cell hyper-activation and consequent paradoxical T cell suppression.Item Peripheral Generation Of Regulatory T Cells In Health And Disease(2007-05-23) Pillai, Vinodh; Karandikar, NitinCD4+CD25+FOXP3+ regulatory T cells (Tregs) form an important arm of the immune system responsible for suppressing untoward immune responses. They play a role in autoimmunity, allergy, asthma, transplantation, tumors and infectious diseases. Tregs are increased in the peripheral blood of chronic hepatitis C virus-infected patients and their depletion in-vitro increases anti HCV responses when measured by a sensitive CFSE-based flow cytometric proliferation assay. The CFSE-based assay, developed and validated by my laboratory, has a greater ability to detect low frequency and low avidity type T cell responses in the chronic HCV patients that are difficult to measure using ex-vivo assays. Using this assay in a cross sectional study, I showed that anti-viral immune responses are attenuated in untreated chronic HCV patients and are increased after anti-viral treatment with Interferon and Ribavirin. Interestingly, increase in anti-viral immune responses after Treg depletion was not seen in patients who were successfully treated with interferon and ribavirin. These results suggest that anti-viral therapy may be acting by modulating anti-viral immune responses. Tregs can be thymically derived (natural Tregs) or peripherally induced (adaptive Tregs). FOXP3 expression and in-vitro suppressive activity are considered unique hallmarks of this dedicated and stable lineage of regulatory cells. Initial evidence indicated that it is a specific marker of natural Tregs. It was thought that FOXP3+ T cells cannot arise in the periphery from na?CD4+CD25- T cells. However, using allostimulation of CFSE stained T cells and polycolor flow cytometry, I showed that virtually all human CD4+CD25-FOXP3- T cells and CD8+CD25-FOXP3- T cells attain a transient FOXP3+CD25+ state during activation. In this state of activation, these cells possess the classic phenotype of Tregs, in that they express similar markers and inhibit in-vitro proliferation of autologous CD4+CD25- T cells. This state is characterized by suppressed IFN-gamma production and robust TNF-alpha and IL-10 production. Interestingly, the great majority of the activated cells eventually downregulate FOXP3 expression, with a concomitant drop in suppressive ability. However some of the FOXP3+ T cells continue to maintain FOXP3 expression suggesting that activation might be a mechanism of producing FOXP3+ Regulatory T cells in the periphery during viral infections like chronic HCV infection. Transient FOXP3 expression in activated T cells might also be a mechanism of fine tuning excessive immune activation. These results show that, in humans, FOXP3 expression and Treg functionality are not exclusive features of a stable or unique lineage of T cells, but may also be a transient state attained by almost all T cells. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts and suggest that attempts to induce " Tregs" may paradoxically result in induction of effector T cells, unless stability is confirmed.Item Role of Allergic Conjunctivitis in Inducing Corneal Transplant Rejection(2013-01-17) Reyes, Nancy J.; Niederkorn, Jerry, Ph.D.Corneal allografts are the most commonly transplanted solid organs in humans and have a success rate of over 90%. This low incidence of graft rejection is largely due to the unique properties of the eye that decrease the likelihood of mounting an immune response, a phenomenon called “immune privilege”. Despite this characteristic, immune rejection remains the leading cause of corneal graft failure, indicating that immune privilege can be abolished. We used a model of corneal transplantation in which C57BL/6 corneal allografts transplanted to BALB/c mice experienced a 50% survival rate. This is in sharp contrast to the 100% rejection that occurs with other organ grafts exchanged between these two mouse strains. The 50% survival rate decreased to 0% in mice with ongoing allergic conjunctivitis. We set out to delineate the mechanisms by which allergic conjunctivitis abolishes immune privilege and results in corneal allograft rejection. Anterior chamber-associated immune deviation (ACAID) is initiated when foreign antigens, such as histocompatibility antigens sloughed from the cornea, enter the anterior chamber of the eye and induce a systemic down-regulation of immune responses. Studies have shown that abolishing ACAID increases the incidence of corneal allograft rejection. The hypothesis that allergic conjunctivitis abolished the induction of ACAID was tested and results showed that allergic conjunctivitis did not. Moreover, allergic conjunctivitis did not cause a qualitative or quantitative difference in cell-mediated immune responses to the donor’s alloantigens. Regulatory T cells (Tregs) have also been shown to be important in corneal allograft survival. Our results indicated that allergic conjunctivitis altered the Tregs that support corneal allograft survival. Th2 cytokines, namely IL-4, generated during allergic conjunctivitis, render effector cells resistant to the suppressive ability of Tregs. Furthermore, allergic conjunctivitis did not exacerbate corneal allograft rejection unless the host’s effector T cells were able to respond to IL-4. Moreover, corneal immune privilege was restored in short ragweed (SRW) pollen-sensitized, allergic mice if they were isolated from SRW pollen for 14 days after receiving a corneal allograft. These results suggest that the exacerbation of corneal allografts in allergic hosts is due to the production of IL-4, which renders effector T cells resistant to Treg suppression.Item Role of Splenic B Cells and Gamma Delta T Cells in the Induction of Peripheral Tolerance Elicited Through the Anterior Chamber of the Eye(2006-12-19) Ashour, Hossam Mohamed; Niederkorn, JerryAnterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance that is induced by introducing antigens into the anterior chamber (AC) of the eye, and is maintained by antigen-specific regulatory T cells (Tregs). ACAID regulates harmful immune responses that can lead to irreparable injury to innocent bystander cells that are incapable of regeneration. ACAID is a product of complex cellular interactions that involve F4/80+ ocular antigen presenting cells (APC), NK T cells, B cells, gamma delta (gamma delta ) T cells, CD4+CD25+ Tregs, and CD8+ Tregs. Antigens injected into the AC are processed by F4/80+ antigen presenting cells (APC), which migrate to the thymus and spleen. In the spleen, ocular APC induce the development of antigen-specific B cells that act as ancillary APC and are required for ACAID induction. Results show that ocular APC elicit the generation and expansion of antigen-specific splenic B cells that induce ACAID. However, direct cell contact between ocular APC and splenic B cells is not necessary for the induction of ACAID B cells. Peripheral tolerance produced by ACAID requires the participation of ACAID B cells, which induce the generation of both CD4+ Tregs and CD8+ Tregs. Using in vitro and in vivo models of ACAID, we demonstrate that ACAID B cells must express both MHC class I and class II molecules for the generation of Tregs. This suggests that ACAID requires antigen presenting B cells that simultaneously present antigens on both MHC class I and class II molecules. Gamma delta T cells are also crucial for the generation of ACAID, but their function in ACAID is largely unknown. Several hypotheses were proposed for determining the functions of gamma delta T cells in ACAID. The results indicate that gamma delta T cells neither suppress DTH directly nor do they act as antigen presenting cells. However, gamma delta T cells were shown to secrete IL-10, thus facilitating the generation of ACAID Tregs. In addition, the contribution of gamma delta T cells in ACAID generation could be replaced by adding exogenous rmIL-10 to ACAID spleen cell cultures lacking gamma delta T cells. Throughout this dissertation, we have shed light on the roles of B cells and gamma delta T cells in ACAID and have found that B cells need to express both MHC I and MHC II in order to induce the generation of Tregs and that gamma delta T cells need to secrete IL-10 for ACAID to be generated.