Peripheral Thy1⁺ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Abstract

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of ab and cd T lymphocytes. Surprisingly, using a new model of LAT-deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat Inv/Inv ) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1 1 cells. Thy1 dull cells expressed markers of NK lymphocytes and contained low frequency of TCR-c gene rearrangements without detectable TCR-d rearrangements. Thy1 high cells resembled immature CD44 1 CD25 1 thymocytes and contained high frequency of non-productive TCR-c and TCR-d rearrangements, indicating that cells displaying molecular signatures of commitment toward cd T-cell lineage can develop and populate lymphoid tissues of LAT-deficient mice. Phenotypically similar Thy1 high cells were also found in lymph nodes of lymphocyte-deficient (Rag2 À/À ) mice but not in T lymphocyte proficient, heterozygous Lat 1/Inv mice suggesting that Thy1 high cells of LAT-deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.Key words: Extrathymic lymphopoiesis . LAT . Thymopoiesis . Thy1 . g/d T cells Supporting Information available online IntroductionLinker for activation of T cells (LAT) belongs to a family of transmembrane adaptor proteins involved in transduction of immunoreceptor-mediated signals [1,2].It is expressed in a number of hematopoietic cell lineages including T cells [3], pre-B cells [4], NK lymphocytes [5], megakaryocytes [6] and mast cells [7]. However, physiological roles of LAT adaptor in these cell lineages seem variable. In LAT knockout mice, NK lymphocytes, megakaryocytes and mast cells show seemingly normal development [8] with only discrete deficiencies of particular signaling pathways [5][6][7]. During B-cell maturation LAT deficiency causes a partial developmental block at the pre-B-cell stage [9,10]. In contrast, development of ab and gd T cells is entirely dependent on LAT adaptor, which is essential for signaling at the pre-TCR and ''TCR-gd quality control'' checkpoints [8,11]. Neither ab nor gd T cells have previously been found to develop and/or colonize the periphery of LAT knockout mice.In the thymus the incoming earliest CD4 À CD8 À double negative (DN) T-cell precursors progress through discrete 2596developmental stages, which may be characterized by cell surface expression of c-kit (CD117), CD25 and CD44 molecules and rearrangement status of g,. Transitions from the most immature and heterogeneous DN1 stage (c-kit high CD25 À CD44 1 ) through DN2 stage (c-kit int CD25 1 CD44 1 ) toward DN3 stage (c-kit low CD25 1 CD44 À ) are TCR independent, since only at DN3 stage most cells committed to T-cell lineages express TCR/CD3 complexes and undergo gd or b selection resulting in transition to DN4 (c-kit...

Complexity of transcriptional regulation within the Rag locus: identification of a second Nwc promoter region within the Rag2 intron

“…Cell suspensions were prepared by standard procedures and, after staining with antibodies, were analyzed as described (Miazek et al 2009). Bone marrow cell suspension was depleted of mature B cells with dynabeads coupled with anti-mouse Ig (Dynal).…”

Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription

“… 1 , 2 During ontogeny, it is first detectable within CD4−CD8−CD25+CD44+ (DN2) thymocytes and is upregulated during CD4−CD8− (DN) to CD4+CD8+ (DP) transition. 3 , 4 Targeted deletion of LAT arrests development of Tαβ and Tγδ thymocytes at the CD4−CD8−CD25+CD44− (DN3) stage, which coincides with the insufficient pre-T-cell receptor (TCR) signaling. 5 , 6 Forced expression of p56Lck kinase from its proximal promoter allows for DN-to-DP transition in LAT-deficient mice and further maturation of conventional LAT-deficient Tαβ cells.…”