Peripheral CD4+CD8+ T cells arise from tissue specific CD4+ T cells and regulate CD8+ single-positive T cell function in the skin

T cells are key players in the establishment of the adaptive immune response. T cells are classified into two lineages, CD4+ or CD8+ T cells, based on the expression of either CD4 or CD8 co-receptors. Interestingly, accumulating evidence supports the existence of a third T cell lineage; mature CD4+CD8+ double-positive T cells (DPs). Mature peripheral DPs are detectable in a variety of lymphoid and non-lymphoid tissues (spleen, lymph nodes, blood, gut), species (human, mouse, pig, rat, monkey), and pathological scenarios such as cancer (melanoma, T cell lymphoma), suggesting that DPs might affect the outcome of autoimmune and malignant skin disorders. Our group is interested in gaining fundamental insight into peripheral DPs biology in order to identify new therapeutic targets. However, the study of this cell-subset presents several difficulties: peripheral DP are a minority within the large T cell compartment, and 98% of un-sorted cells that appear to be DPs are in fact CD4+-CD8+ T cell aggregates. We have overcome these issues using an isolation/enrichment strategy which allows us to analyze the profile of individual cells in a >95% pure DPs suspension. To determine whether naïve peripheral DPs arise from CD4+ T cells or CD8+ T cells, we analyzed the T cell receptor (TCR) diversity by flow cytometry using monoclonal antibodies that recognize the variant region of the  chain (TCRVB). Interestingly, principal component analysis (PCA) of the surface expression of 14 TCRVB chains in individual cells demonstrated that DPs from the spleen clustered with CD4+ T cells from the spleen and not with CD4+ T cells from lymph nodes. Moreover, the high proportion of V7 and V6-expression by DP cells in the lymph-nodes was sufficient to differentiate this population from the rest. Finally, DP thymocytes were an intermediate population between single-positive CD8+ and CD4+ T cells as biologically reported. To study the effect of DPs on the regulation of skin CD8+ T cells, we used a contact hypersensitivity model (CHS). Recipient animals were sensitized with Ovalbumin (OVA) in the flank a week prior to the injection of pure naïve DPs or OVA-immunized DPs. One day after T cell infusion, ears were challenged with an intradermal injection of OVA and thickness of the ears was measured as an indicator of CD8 T cell-induced inflammation. As expected, OVA-challenged ears from animals that received no cells showed a 40% increase in ear thickness compared with PBS-challenged control ears. Interestingly, the transfer of a low number of DPs significantly suppressed CD8+ T cell dependent inflammation. Moreover, the DP inhibitory phenotype depended on their activation status since only activated and not naïve DPs prevented skin inflammation. In summary, our results demonstrate that peripheral DPs do not originate from the direct seeding of immature DP thymocytes to the periphery, but from tissue-specific CD4+ T cells, which, after colonization of the organ, re-express CD8 potentially conferring functional changes. Additionally, we suggest a powerful immunoregulatory role for DPs in controlling CD8+ T cell function in the skin.