Thymic selection is designed to ensure T cell receptor (TCR) reactivity to foreign antigens presented by self-MHC while minimizing reactivity to self-antigens. We hypothesized that the repertoire of T cells with unwanted specificities such as alloreactivity or autoreactivity are a consequence of simultaneous rearrangement of both TCRα loci. We hypothesized that this process helps maximize production of thymocytes capable of successfully completing thymic selection, but results in secondary TCRs that escape stringent selection. In T cells expressing 2 TCRs, 1 TCR can mediate positive selection and mask secondary TCR from negative selection. Examination of mice heterozygous for TRAC (TCRα+/−), capable of only one functional TCRα rearrangement, demonstrated a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of TCRα repertoires of dual TCR T cells and TCRα+/− T cells demonstrated unique TCRs in the presence of secondary rearrangements. The functional impact of secondary TCRs on the naive peripheral repertoire was evidenced by reduced frequencies of T cells responding to autoantigen and alloantigen pMHC tetramers in TCRα+/− mice. T cell populations with secondary TCRs had significantly increased ability to respond to altered peptide ligands related to their allogeneic ligand as compared to TCRα+/− cells, suggesting increased breadth in peptide recognition may be a mechanism for their reactivity. Our results imply that the role of secondary TCRs in forming the T cell repertoire is perhaps more significant than what has been assumed.

A significant portion of the naive T-cell repertoire is capable of responding to allogeneic MHC, violating the paradigm of self-MHC restriction. Recent studies have demonstrated convincing evidence for germ-line affinity of T-cell receptors (TCR) for MHC, providing explanation for recognition of MHC not encountered during thymic development. However, although germ-line affinity proposes all TCR have inherent affinity for MHC, most T cells are not alloreactive to a given MHC. We propose that specific recognition of endogenous presented peptides, rather than inability to interact with allogeneic MHC molecules, is the primary determinant of alloreactivity. Here, we demonstrate that alloreactive and nonalloreactive TCR differ specifically in the CDR3 sequences responsible primarily for the peptide specificity of T-cell recognition. Limitations on alloreactivity imposed by a requirement for recognition of presented peptides are directly demonstrated by expansion of the alloreactive T-cell repertoire through the addition of peptide mimotopes enabling response to two distinct allogeneic MHC by otherwise nonalloreactive T cells. Responses to peptide mimotopes were specific and depended on TCR interaction with MHC. These results demonstrate that recognition of presented endogenous peptides, and not the inability to interact with allogeneic MHC, is the primary limiter on alloreactivity. This observation reconciles the concept of an inherently MHC-reactive TCR repertoire with observed frequencies of T cells responding to allogeneic stimulation and underscores the fundamental nature of TCR recognition of ligands, where both MHC and presented peptides contribute critically to T-cell recognition.A lthough the diverse T-cell receptor (TCR) repertoire generated by germ-line V(D)J recombination is optimized during thymic selection for recognition of foreign antigens presented by self-MHC (1), it is hypothesized that response to MHC not encountered during thymic development is driven by inherent TCR affinity for MHC (2, 3). Recent studies have provided convincing evidence of germ-line affinity (4-8). However, it is not clear that response to allogeneic stimulation is determined primarily by the ability to make sufficient interactions between germ-line TCR elements and allogeneic MHC. Assessments of polyclonal alloreactive responses have demonstrated broad use of germ-line TCR segments (9, 10), suggesting that differential MHC affinity among germ-line elements does not account for differential alloreactivity among T cells. Conversely, analyses of non-germ-line-encoded CDR3 regions have demonstrated skewing in alloreactive responses. In a MHC-centric view of alloreactivity, CDR3 could determine alloreactivity through direct interaction with the MHC (11) or by imposing limitations on critical germ-line-encoded contacts (12).However, placing the entirety of determination for alloreactive potential on interaction with MHC does not address clear evidence for recognition of presented peptides in alloreactivity. Alterations to peptide pr...

Infection with wild-type Listeria monocytogenes activates a host cytosolic surveillance response characterized by the expression of beta interferon (IFN-␤). We performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced altered levels of host IFN-␤ expression. One mutant from this screen induced elevated levels of IFN-␤ and harbored a Tn917 insertion upstream of lmo0558. This study identified lmo0558 as the 6-phosphogluconolactonase gene (pgl), which encodes the second enzyme in the pentose phosphate pathway. pgl mutant L. monocytogenes accumulated and secreted large amounts of gluconate, likely derived from labile 6-phosphogluconolactone, the substrate of Pgl. The pgl deletion mutant had decreased growth in glucose-limiting minimal medium but grew normally when excess glucose was added. Microarray analysis revealed that the pgl deletion mutant had increased expression of several ␤-glucosidases, consistent with known inhibition of ␤-glucosidases by 6-phosphogluconolactone. While growth in macrophages was indistinguishable from that of wild-type bacteria, pgl mutant L. monocytogenes exhibited a 15-to 30-fold defect in growth in vivo. In addition, L. monocytogenes harboring an in-frame deletion of pgl was more sensitive to oxidative stress. This study identified L. monocytogenes pgl and provided the first link between the bacterial pentose phosphate pathway and activation of host IFN-␤ expression.Listeria monocytogenes is a gram-positive, food-borne facultative intracellular pathogen that causes invasive, life-threatening infections, mainly in pregnant women, newborns, the elderly, and the immunosuppressed (39). In addition, L. monocytogenes has been studied for decades as a model pathogen, illuminating many aspects of host-pathogen interaction. The cell biology of its intracellular life cycle has been particularly well characterized. After phagocytosis by a macrophage, L. monocytogenes rapidly escapes from the phagosome into the cytosol, an event mediated by the pore-forming cytolysin listeriolysin O (29). L. monocytogenes is well adapted to its cytosolic niche, possessing virulence factors that allow utilization of cytosolic sugar sources and polymerization of host actin to move from cell to cell (4, 25). It is evident that L. monocytogenes has evolved specific mechanisms to grow in the host cytosol; however, the presence of cytosolic L. monocytogenes triggers a host innate immune response. Upon entry of L. monocytogenes into cells, a host cell cytosolic surveillance pathway is activated, including a transcriptional program that leads to the robust expression of beta interferon (IFN-␤) (19,22,23,34). However, the host and bacterial components responsible for the activation of the cytosolic surveillance pathway remain largely unknown.Our lab previously performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced enhanced or diminished activation of the host cytosolic surveillance system (6). We found that bacterial multidrug resistance tra...

It is perplexing why vertebrates express a limited number of Major Histocompatibility Complex (MHC) molecules when theoretically, having a greater repertoire of MHC molecules would increase the number of epitopes presented, thereby enhancing thymic selection and T cell response to pathogens. It is possible that any positive effects would either be neutralized or outweighed by negative selection restricting the T cell repertoire. We hypothesize that the limit on MHC number is due to negative consequences arising from expressing additional MHC. We compared T cell responses between B6 mice (I-A+) and B6.E+ mice (I-A+, I-E+), the latter expressing a second class II MHC molecule, I-Eb, due to a monomorphic Eαk transgene that pairs with the endogenous I-Eβb chain. First, the naive T cell Vβ repertoire was altered in B6.E+ thymi and spleens, potentially mediating different outcomes in T cell reactivity. Although the B6 and B6.E+ responses to hen egg-white lysozyme (HEL) protein immunization remained similar, other immune models yielded differences. For viral infection, the quality of the T cell response was subtly altered, with diminished production of certain cytokines by B6.E+ CD4+ T cells. In alloreactivity, the B6.E+ T cell response was significantly dampened. Finally, we observed markedly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE) in B6.E+ mice. This correlated with decreased percentages of nTreg cells, supporting the concept of Tregs exhibiting differential susceptibility to negative selection. Altogether, our data suggest that expressing an additional class II MHC can produce diverse effects, with more severe autoimmunity providing a compelling explanation for limiting the expression of MHC molecules.

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