Description

Peripheral CD4⁺CD8⁺ T-cells arise from tissue specific CD4⁺ T-cells

Jazmina L. Gonzalez-Cruz, Margaret Veitch, Zewen K. Tuong, Nana H. Overgaard, Jennifer A. Bridge, Jessica He, Katherine Watson, Ian H. Frazer, Nicole L. La Gruta, Raymond J. Steptoe and James W. Wells

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 a novel 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 VB7 and VB6-expression by DP cells in the lymph-nodes was sufficient to differentiate this population from the rest. Finally, DP thymocytes were more related to single-positive CD8⁺ T cells than DPs in the spleen or lymph-nodes. 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.