1) Further understanding of the role of Treg during chronic infection has been limited by our lack of knowledge regarding the stimuli that drive Treg activation and expansion in these situations. Not only are pathogen-derived antigens continuously presented during chronic infection but tissue damage may also result in the presentation of self-antigens.Two strains of LCMV are commonly used, the parental strain Armstrong and the variant Clone 13. LCMV Armstrong and Clone 13 differ by only two amino acids, neither of which has been associated with either CD4 or CD8 T cell epitopes. LCMV Armstrong causes an acute infection in normal mice in which the animal develops few, in any, symptoms and is self-limiting 6-8 days post infection (DPI). Mice that clear LCMV Armstrong infection also go on to develop life-long protective immunity from reinfection. In contrast, Clone 13 infection results in a chronic infection in which mice remain infected virtually for life. While both infections result in a long-lived pool of virus-specific T cells, those cells generated following Armstrong infection maintain all of the properties of memory T cells. In contrast, virus-specific T cells in mice infected with LCMV Clone 13 require the persistence of antigen to be maintained and they lose the ability to perform effector function (functional exhaustion). LCMV Armstrong infection is similar to many acute viral infections including influenza, and Clone 13 infection is similar to HIV and chronic hepatitis (particularly hepatitis C) both in terms of viral persistence and the down-regulation of the virus-specific immune response following infection. We have identified a subpopulation of Treg that rapidly and specifically expand early during chronic but not acute infection. The expanded Treg are restricted to cells expressing particular TCR Vβsegments (Vβ5 in C57BL/6 mice and Vβ5 and 12 in BALB/c mice). Interestingly, the Vβ-specific T cell expansion was restricted only to Treg and not Foxp3- Teff cells expressing the same Vβsegments, suggesting that the process is Treg-specific. The expanded Tregs were derived from a pre-existing population of Treg and not derived de novo during the infection. By using congenic mice that lack all endogenous Mtv proviruses (Mtv/null), we have shown that the expansion of Vβ-specific Treg during chronic LCMV infection is secondary to Mtv-superantigen (Sag)- encoded in the mouse genome. The mechanism by which chronic LCMV infection results in Mtv-Sag-dependent Treg activation seems to depend and require presentation of the endogenous SAg by DC. Taken together, these studies define a unique mechanism of Treg activation and expansion following chronic viral infection. We have addressed whether the innate inflammatory signals required for peptide antigen-specific effector CD4+ and CD8+ T cell activation are similarly required for Sag-mediated expansion of Treg following chronic LCMV infection. Our studies demonstrate that Sag-mediated Treg expansion requires intrinsic IFN-alpha/betaR signaling similar to effector CD4+ and CD8+ T cells. Unlike the effector T cell responses, Sag-specific Treg expansion does not require intrinsic MyD88 signaling, however MyD88-dependent CD8+ T cell activation itself is required. Thus, Treg expansion in this well-characterized model of chronic viral infection in response to Sag requires two major components signaling via the IFN-alpha/betaR and activation of anti-viral specific CD8+ T cells. These findings shed further light on the similarities and differences between the specific requirements for Treg versus effector T cell activation. It remains unclear whether IFN/R signaling is important in other Treg functions. It is clear in our experiments that IFN-alpha/betaR signaling alone, even in the context of a high viral load, is not sufficient to drive the expansion of Vbeta5+ Treg, as LCMV congenital carriers have an intact innate immune response, and have high serum levels of IFN-beta, but do not manifest eSag-induced Treg expansion. The mechanisms by which anti-viral specific CD8+ T cells mediate Treg expansion in this model remain unknown. It remains possible that CD8+ T cells or their products play a critical role for the enhanced expression of Sag on DC, which are required for Treg expansion. While it has not been possible to demonstrate a role for Treg in promoting CD8+ T cells exhaustion in chronic LCMV infection, depletion of Tregs in a model of chronic Friend virus infection led to the partial recovery of the exhausted CD8+ T cell phenotype and lower viral titers. Nonetheless, there appears to be a yin-yang relationship between Tregs and anti-viral CD8+ T cells. CD8+ T cells may expand Tregs to inhibit immunopathology, but overexpansion of Tregs may directly or indirectly promote CD8+ T cell exhaustion. Lastly, while the biologic significance of Sag mediated expansion of Tregs remains to be determined, endogenous retroviral sequences make up a significant percentage of both the human and mouse genome and activation of endogenous retroviral gene expression may play multiple roles in both the development and the function of the mature immune system. 2). We have also begun to explore the potential role of Mtv infection on other aspects of immune responses and have focused our studies on NK T cells and innate CD8+ T cells. Innate-like or memory-like CD8+ T cells have been characterized based on their increased expression of the phenotypic surface makers of memory cells (CD44 and CD122), increased expression of the T-box transcription factor Eomesodermin (Eomes), and increased production of IFN-γupon stimulation. Such innate CD8+ T cells were initially identified in ITK-/- mice and proposed to develop secondary to an increased TCR signaling threshold. This innate phenotype among CD8+ thymocytes was subsequently described in a number of other knockout and mutant mouse models. These studies concluded that this phenotype resulted from a cell-extrinsic factor, the overproduction of IL-4 by promyelocytic leukemia zinc-finger (PLZF)+ thymic NKT cells. Innate CD8+ T cells were present in BALB/c, but not in C57BL/6 mice. Following an extensive comparison of the phenotype of BALB/c and BALB/Mtv-null mice, we noted only one major difference. Innate CD8+ T cells were not present in the thymus of Mtv-null mice. Thymic αβ+ iNKT cells from BALB/c mice had increased expression of PLZF in iNKT cells compared to BALB/Mtv-null mice. This increased PLZF expression in BALB/c coincided with a higher percentage of iNKT cells exhibiting a phenotype consistent with that of Stage 1 and Stage 2 iNKT cells, which are capable of producing higher levels of IL-4. In the absence of endogenous Mtv, the majority of thymic TCR αβ+ iNKT cells within BALB/Mtv-null mice exhibited a phenotype that resembled that of Stage 3 iNKT cells, exhibiting increased expression of T-bet, CD122 and IFN-γ. One potential candidate proviral gene that may influence iNKT development is Rem, an Mtv-encoded protein resulting from a doubly spliced mRNA within the env gene. Rem has been demonstrated to show functional homology to other RNA-binding nuclear export proteins of endogenous human retroviruses, including Rec (HERV-K), Rev (HIV), and Rex (HTLV). Rec physically interacts with the transcriptional repressor PLZF and PLZF-related proteins. The interaction between Rec and PLZF released the transcriptional repression of certain PLZF-repressed genes. One such gene was c-myc, which has previously been shown to dramatically and specifically influence iNKT cell development.
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