RORγt+ c-Maf+ Vγ4+ γδ T cells are generated in the adult thymus but do not reach the periphery.

T cell receptor (TCR) Vγ4-expressing γδ T cells comprise interferon γ (IFNγ)- and interleukin-17 (IL-17)-producing effector subsets, with a preference for IL-17 effector fate decisions during early ontogeny. The existence of adult-thymus-derived IL-17+ T cells (γδ17) remains controversial. Here, we use a mouse model in which T cells are generated exclusively in the adult thymus and employ single-cell chromatin state analysis to study their development. We identify adult-thymus-derived Vγ4 T cells that have all the molecular programs to become IL-17 producers. However, they have reduced IL-17 production capabilities and rarely reach the periphery. Moreover, this study provides high-resolution profiles of Vγ4 T cells in the adult thymus and lymph nodes and identifies Zeb1 as a potential γδ17 cell regulator. Together, this study provides valuable insights into the developmental traits of Vγ4 T cells during adulthood and supports the idea of age-specific signals required for thymic export and/or peripheral maturation of γδ17 cells.

ADAP deficiency combined with costimulation blockade synergistically protects intestinal allografts.

Adhesion and degranulation promoting adapter protein (ADAP) plays an important role in T cell activation. ADAP deficiency was recently found to prolong heart graft survival in mice. We investigated the role of ADAP in intestinal transplantation and the synergistic effect of ADAP deficiency and Costimulation blockade (CB). T cell proliferation and cytotoxic T lymphocyte (CTL) activity were determined. MHC mismatched intestinal allografts was transplanted heterotopically. Anti-CD40L antibody was applied to the recipient. Upon stimulation with allogenic dendritic cells (DC), ADAP-deficient (ADAP-/-) T cells displayed impaired proliferative responses compared with that of wild-type (WT) T cells. In contrast, the CTL activity in ADAP-/- mice was comparable with that of WT mice. Rejection of intestinal allografts was ameliorated, but not prevented in ADAP-/- mice. Although CB alone was not sufficient to mitigate the rejection, the combination of CB and ADAP deficiency profoundly inhibited rejection. This was accompanied by less infiltration and activation of host lymphocytes in the gut-associated lymphoid tissue of intestinal allografts. ADAP deficiency combined with CB protected the intestinal allografts synergistically. ADAP could be a novel target in the induction phase of the immune responses in organ transplantation.

Heterogeneous expression of the adhesion receptor CD226 on murine NK and T cells and its function in NK-mediated killing of immature dendritic cells.

The adhesion receptor CD226 (DNAM-1) is a member of the Ig superfamily possessing two extracellular V-like domains. In humans, CD226 was shown to be expressed by NK as well as T cells. During T cell priming, CD226-mediated costimulatory signals may skew the subsequent differentiation into the Th1 pathway. In addition, CD226 expressed on NK and cytotoxic T cells is engaged by its counter-receptor CD155, present on target cells, thereby triggering their elimination. We established mAb specifically recognizing mCD226, demonstrating that CD226 is expressed by precursor and mature but not developing T cells. In contrast, NK cells are distinguished by a rather heterogeneous CD226 expression profile. In addition, expression of CD226 appears coupled to that of other NK cell receptors, as high expression of CD226 was found to correlate with decreased proportions of Ly49D and H positive NK cells. Upon injection into mice, the anti-CD226 antibodies caused selective depletion of CD8(+) T cells. Moreover, these antibodies as well as a naturally occurring CD226 splice variant lacking the outermost V-like domain were instrumental in determining that CD226 adheres to CD155 via its first domain. In addition, antibodies were identified as capable of blocking the CD226/CD155 interaction and to prevent NK-driven killing of immature DC. CD226 is thus the first mNK receptor identified to be essential for the elimination of this particular cell type.

CD96 interaction with CD155 via its first Ig-like domain is modulated by alternative splicing or mutations in distal Ig-like domains.

The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes.

Transcoronary delivery of bone marrow cells to the infarcted murine myocardium: feasibility, cellular kinetics, and improvement in cardiac function.

Efficient strategies for labelling and delivery of bone marrow derived stem cells (BMCs) are required to elucidate the cellular kinetics and therapeutic effects after BMC transfer for myocardial infarction (MI). Lineage negative (lin-) BMCs, labelled ex vivo in a simple procedure with the cell tracker dye tetramethyl-rhodamine (TAMRA), were reliably detected by fluorescence microscopy with higher specificity than retroviral enhanced green fluorescence protein (EGFP) marking and detection. Only few cells entered the ischemic myocardium after intravenous (i.v.) application, but this number increased more than 18-fold after transcoronary delivery. Time course and kinetic analysis over 12 h revealed that myocardial colonization seems to be a biphasic process of first order decay with different elimination half-lives. Most cells are eliminated rapidly during the first 2 h (t1/2 40 min), but the remaining cells are retained significantly longer in the ischemic heart (t1/2 5.2 h). In contrast, BMC colonization of the spleen increased rather in a linear fashion. Although transcoronary BMC transfusion did not alter infarct size, it increased capillary density in the infarct border zone and improved LV function 4 weeks after MI. In conclusion, BMCs delivered by transcoronary injection increase capillary density and improve LV function after MI although homing to the ischemic heart is only transient.