It is known that a subpopulation of T cells expresses two T cell receptor (TCR) clonotypes, though the extent and functional significance of this is not established. To definitively evaluate dual TCRα cells, we generated mice with green fluorescent protein and red fluorescent protein reporters linked to TCRα, revealing that ∼16% of T cells express dual TCRs, notably higher than prior estimates. Importantly, dual TCR expression has functional consequences, as dual TCR cells predominated response to lymphocytic choriomeningitis virus infection, comprising up to 60% of virus-specific CD4+and CD8+T cells during acute responses. Dual receptor expression selectively influenced immune memory, as postinfection memory CD4+populations contained significantly increased frequencies of dual TCR cells. These data reveal a previously unappreciated contribution of dual TCR cells to the immune repertoire and highlight their potential effects on immune responses.

Approximately 10% of peripheral T cells express 2 functional TCR αβ heterodimers. Receptor co-expression changes the repertoire of TCRs produced during thymic development, enabling generation of T cells bearing TCRs not capable of mediating positive selection or that would normally be negatively selected. The effect of receptor co-expression on the composition and functionality of the peripheral TCR repertoire is not well defined, though evidence demonstrates dual TCR cells pose an increased risk for unwanted immune responses such as autoimmunity and alloreactivity. Based on our previous finding that dual TCR expression promotes positive selection, we hypothesized that dual TCR expression may enhance T cell homeostasis via increased reactivity against self-peptide:MHC (pMHC) ligands. To examine the effect of dual TCR expression on T cell homeostasis, we performed cotransfer experiments comparing T cells genetically deficient for dual TCR expression (TCRα ) with wild-type T cells in models of acute and chronic lymphopenia-induced proliferation (LIP). Lack of dual TCR expression resulted in reduced LIP. The effect of dual TCR expression on LIP was most pronounced in acute lymphopenia, which is driven by recognition of low-affinity self-pMHC ligands. Differences in homeostatic proliferation were not attributable to differences in total TCR expression or signaling, but were dependent on interaction with MHC and associated with increased affinity for positively selecting self-pMHC as evidenced by higher expression of CD5 by dual TCR cells from wild-type mice. These results represent an unappreciated novel mechanism driving homeostasis and shaping the T cell repertoire, potentially promoting autoreactive or heterologous immune responses.

Background Hematopoietic stem cell transplant is a crucial intervention to definitively treat many hematopoietic malignancies, but it carries great risks of morbidity and mortality often associated with graft-versus-host disease (GVHD). Acute and chronic GVHD are distinct entities, defined by a combination of historical, clinical, and pathologic data, but both are generally thought to stem from self-propagating aberrantly activated immune cells inflicting end organ damage, with the potential to cause significant illness or even death. Event-free survival rates after hematopoietic stem cell transplant continue to improve each year, but GVHD remains a major hurdle in improving the efficacy and safety of transplant. Objective Recent studies demonstrating tissue-specific immune effector phenotypes underscore the need for a deeper understanding of the cellular and molecular pathways driving the destruction of target tissues in patients with acute GVHD. Methods Samples were collected from lesional and unaffected skin in five patients with acute cutaneous GHVD. Fresh tissue was processed for fluorescence-activated cell sorting and analysis of macrophages and lymphocytes. Results The percentage of lymphocytes and macrophages as a representation of total cells varied among patients and was not always consistent between lesional and unaffected sites. The heterogeneity in immune cell profiling observed in patients in this study could reflect the diverse demographics, conditioning, and transplant conditions of each individual. Conclusion This study provides initial insight into the underlying molecular mechanisms of cutaneous GVHD progression and paves the way for additional studies to examine the cellular and molecular landscape in greater detail.

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphoproliferative disorders derived from skin-homing memory T cells. Mycosis fungoides (MF) and Sezary syndrome (SS) are the most common subtypes. Aberrant cytokine expression in the MF/SS tumor microenvironment (TME) is a major factor in disease pathogenesis and progression. We have previously shown that MF/SS malignant lymphocytes produce high levels of IL-13, which acts as an autocrine factor for tumor cells and suppresses tumor-cell immunosurveillance. Furthermore, our studies indicate that IL-13 synergizes with IL-4 in inducing SS cell growth and implicate IL-13 signaling via the Signal Transducer and Activator of Transcription-6 (STAT-6), an upstream mediator common to both IL-4 and IL-13 signaling. Significantly, we found high numbers of activated STAT-6 + cells in the affected skin of MF patients, particularly in advanced stages, implicating STAT-6 as a critical signaling mediator in MF/SS lymphocytes. To investigate the underlying molecular mechanism, we combined genome-wide transcriptional profiling with STAT-6 inhibition to identify the STAT-6-regulated genes in advanced-stage MF/ SS tumors. In malignant lymphocytes, we found that STAT-6 regulates the expression of genes associated with control of cell cycle progression and genomic stability, and its inhibition decreased proliferation. Furthermore, we showed that STAT-6 enhances expression of Th2 cytokines in malignant and reactive T cells by up-regulating the expression of GATA-3. Finally, we demonstrated that STAT-6 contributes to the pro-tumoral M2-like phenotype of macrophages in the TME of advanced-stage MF by up-regulating the expression of genes associated with immunosuppression, chemotaxis, and tumor matrix remodeling. Thus, STAT-6 contributes to MF/SS malignancy by several mechanisms including enhancing proliferation of tumor lymphocytes and promoting an immunosuppressive and pro-tumoral microenvironment that favors tumor growth and invasion.

Generation of CD4+FoxP3+ regulatory T cells (Treg) in the thymus is essential for maintenance of self-tolerance. Treg lineage commitment occurs via agonist selection, via TCR high-affinity interaction with self-peptide-MHC (pMHC) ligand. We have previously demonstrated that natural co-expression of dual TCRs, occurring in ~10% of T cells, results in increased ability to recognize self-pMHC ligands and promote positive selection. We hypothesized that dual TCR expression could similarly promote agonist Treg selection. Examination of Treg development in B6.TCRa+/− mice, heterozygous for disruptive mutation of TCRAC and unable to express dual TCRs, demonstrated decreased frequency of Tregs in the thymus (2.76+0.08% of CD4 single-positive thymocytes) compared to age-matched wild-type B6 mice (3.26+0.09%, P < 0.001). To more precisely compare Treg agonist selection, we co-transferred 1:1 ratios of T cell-depleted B6.TCRa+/−.Thy1.1 and B6.Ly5.1 bone marrow to lethally-irradiated B6 recipients and monitored thymic selection. TCRa+/− cells were deficient in Treg selection compared to co-transferred wild-type cells; TCRa+/− cells produced 37.57+4.16% of donor-derived thymic Tregs (P = 0.003). This defect was specific for Treg selection, as TCRa+/− cells comprised 44.68+6.33% of double-positive thymocytes and 45.50+5.77% of CD4 single-positive thymocytes. Tregs generated from TCRa+/− or wild-type cells were phenotypically similar, with no differences in expression of FoxP3 or CD5. We propose that the difference in agonist selection of dual TCR and single TCR Tregs could have important consequences in the TCR repertoire and antigenic specificity of peripheral Tregs, impacting immunologic tolerance.