The ability to rearrange dual TCRs enhances positive selection, leading to increased Allo- and Autoreactive T cell repertoires.

Thymic selection is designed to ensure TCR reactivity to foreign Ags presented by self-MHC while minimizing reactivity to self-Ags. 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 two TCRs, one 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 peptide-MHC 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 with 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.

Alloreactivity is limited by the endogenous peptide repertoire.

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.

Listeria monocytogenes 6-Phosphogluconolactonase mutants induce increased activation of a host cytosolic surveillance pathway.

Infection with wild-type Listeria monocytogenes activates a host cytosolic surveillance response characterized by the expression of beta interferon (IFN-beta). We performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced altered levels of host IFN-beta expression. One mutant from this screen induced elevated levels of IFN-beta 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 beta-glucosidases, consistent with known inhibition of beta-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-beta expression.

Both positive and negative effects on immune responses by expression of a second class II MHC molecule.

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-E(b), due to a monomorphic Eα(k) transgene that pairs with the endogenous I-Eß(b) chain. First, the naive T cell Vß 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.