Microbiota stimulation generates LCMV-specific memory CD8+ T cells in SPF mice and determines their TCR repertoire during LCMV infection.

Both mouse and human harbour memory phenotype CD8+ T cells specific for antigens in hosts that have not been previously exposed to these antigens. The origin and the nature of the stimuli responsible for generation of CD44hi CD8+ T cells in specific pathogen-free (SPF) mice remain controversial. It is known that microbiota plays a crucial role in the prevention and resolution of systemic infections by influencing myelopoiesis, regulating dendritic cells, inflammasome activation and promoting the production of type I and II interferons. By contrast, here we suggest that microbiota has a direct effect on generation of memory phenotype CD44hiGP33+CD8+ T cells. In SPF mice, it generates a novel GP33+CD44hiCD8+ T cell sub-population associating the properties of innate and genuine memory cells. These cells are highly enriched in the bone marrow, proliferate rapidly and express immediate effector functions. They dominate the response to LCMV and express particular TCRß chains. The sequence of these selected TCRß chains overlaps with that of GP33+CD8+ T cells directly selected by microbiota in the gut epithelium of SPF mice, demonstrating a common selection mechanism in gut and peripheral CD8+ T cell pool. Therefore microbiota has a direct role in priming T cell immunity in SPF mice and in the selection of TCRß repertoires during systemic infection. We identify a mechanism that primes T cell immunity in SPF mice and may have a major role in colonization resistance and protection from infection.

Notch signaling in group 3 innate lymphoid cells modulates their plasticity.

The Notch signaling pathway is conserved throughout evolution, and it controls various processes, including cell fate determination, differentiation, and proliferation. Innate lymphoid cells (ILCs) are lymphoid cells lacking antigen receptors that fulfill effector and regulatory functions in innate immunity and tissue remodeling. Type 3 ILCs (ILC3s) reinforce the epithelial barrier and maintain homeostasis with intestinal microbiota. We demonstrated that the population of natural cytotoxicity receptor-positive (NCR(+)) ILC3s in mice is composed of two subsets that have distinct developmental requirements. A major subset depended on the activation of Notch2 in NCR(-) ILC3 precursors in the lamina propria of the small intestine to stimulate expression of the genes encoding the transcription factors T-bet, RORγt, and aryl hydrocarbon receptor (AhR). Notch signaling contributed to the transition of NCR(-) cells into NCR(+) cells, the more proinflammatory subset, in a cell-autonomous manner. In the absence of Notch signaling, this subset of NCR(-) ILC3s did not acquire the gene expression profile of NCR(+) ILC3s. A second subset of NCR(+) ILC3s did not depend on Notch for their development or for increased transcription factor abundance; however, their production of cytokines and cell surface abundance of NCRs were decreased in the absence of Notch signaling. Together, our data suggest that Notch is a regulator of the plasticity of ILC3s by controlling NCR(+) cell fate.

Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus.

The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that thymopoiesis was initiated by a first wave of T cell lineage-restricted progenitor cells with limited capacity for population expansion but accelerated differentiation into mature T cells. They gave rise to αß and γδ T cells that constituted Vγ3(+) dendritic epithelial T cells. Thymopoiesis was subsequently maintained by less-differentiated progenitor cells that retained the potential to develop into B cells and myeloid cells. In that second wave, which started before birth, progenitor cells had high proliferative capacity but delayed differentiation capacity and no longer gave rise to embryonic γδ T cells. Our work reconciles conflicting hypotheses on the nature of thymus-settling progenitor cells.

Origin, trafficking, and intraepithelial fate of gut-tropic T cells.

The small intestine epithelium (SI-Ep) harbors millions of unconventional (γδ and CD4(-) CD8(-) NK1.1(-) TCRαß) and conventional (CD8αß and CD4) T cells, designated intraepithelial lymphocytes (IELs). Here, we identified the circulating pool of SI-Ep-tropic T cells and studied their capacity to colonize the SI-Ep under steady-state conditions in SPF mice. Developmentally regulated levels of α4ß7 endowed recent thymic emigrants (RTEs) of unconventional types with higher SI-Ep tropism than their conventional homologues. SI-Ep-tropic RTEs, which in all lineages emerged naive, homed to the SI-Ep, but this environment was inadequate to stimulate them to cycle. In contrast, conventional and, unexpectedly, unconventional T cells, particularly Vγ7(+) (hallmark of γδ IELs), previously stimulated to cycle in the gut-associated lymphoid tissue (GALT), proliferated in the SI-Ep. Cycling unconventional SI-Ep immigrants divided far more efficiently than their conventional homologues, thereby becoming predominant. This difference impacted on acquisition of high Granzyme B content, which required extensive proliferation. In conclusion, SI-Ep-tropic T cells follow a thymus-SI-Ep or a GALT-SI-Ep pathway, the latter generating highly competitive immigrants that are the sole precursors of cytotoxic IELs. These events occur continuously as part of the normal IEL dynamics.

Gamma c cytokines condition the progressive differentiation of CD4+ T cells.

After their initial antigen encounter in the secondary lymphoid organs, activated T cells must receive additional signals in the peripheral tissues to fully differentiate. Here, we provide evidence that gamma(c) cytokines are critical during this process. Using the Marilyn (Ml) T cell antigen receptor (TCR) transgenic model, we show that male skin grafts are tolerated in the absence of gamma(c), but that Ml CD4(+) T cells proliferate normally in response to antigen, traffic to the graft site and recruit an inflammatory response [including natural killer (NK) cells, neutrophils, and macrophages] that is independent of T cell gamma(c) expression. Whereas wild-type T cells demonstrate a progressive differentiation phenotype from the spleen to the tissues, skin-infiltrating effector T cells (CD44(hi)CD62L(lo)) from gamma(c)(-) mice were phenotypically abnormal with reduced ICOS, NKG2D, granzyme B, and IFN-gamma expression. These defects could be mapped to deficiencies in IL-2 and, surprisingly, IL-15. These results define a late checkpoint in T cell differentiation in the tissues where gamma(c) cytokines, including IL-15, authenticate CD4(+) T cell effector functions.

Impaired accumulation of antigen-specific CD8 lymphocytes in chemokine CCL25-deficient intestinal epithelium and lamina propria.

CCL25 and CCR9 constitute a chemokine/receptor pair involved in T cell development and in gut-associated immune responses. In this study, we generated CCL25(-/-) mice to answer questions that could not be addressed with existing CCR9(-/-) mice. Similar phenotypes were observed for both CCL25(-/-) and CCR9(-/-) mice, consistent with the notion that CCL25 and CCR9 interact with each other exclusively. We assessed the requirement for CCL25 in generating CCR9(high) CD8 intestinal memory-phenotype T cells and the subsequent accumulation of these cells within effector sites. TCR-transgenic naive CD8 T cells were transferred into wild-type or CCL25-deficient hosts. Oral sensitization with Ag allowed these naive donor cells to efficiently differentiate into CCR9(high) memory-phenotype cells within the mesenteric lymph nodes of wild-type hosts. This differentiation event occurred with equal efficiency in the MLN of CCL25-deficient hosts, demonstrating that CCL25 is not required to induce the CCR9(high) memory phenotype in vivo. However, we found that CCL25 deficiency severely impaired the Ag-dependent accumulation of donor-derived CD8 T cells within both lamina propria and epithelium of the small intestine. Thus, although CCL25 is not necessary for generating memory-phenotype CD8 T cells with "gut-homing" properties, this chemokine is indispensable for their trafficking to the small intestine.

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127.

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice.

In the absence of thymopoiesis, T lymphocytes are nevertheless present, mainly in the gut epithelium. Ontogeny of the extrathymic pathway and the extent of its involvement in euthymic mice are controversial. These questions have been addressed by assessing the expression of recombinase activating gene (RAG) through the use of green fluorescent protein RAG2 transgenic mouse models. In athymic mice, T lymphopoiesis occurs mainly in the mesenteric lymph node and less in the Peyer's patches. Ontogenic steps of this lymphopoiesis resemble those of thymopoiesis, but with an apparent bias toward gamma delta T cell production and with a paucity of oligoclonal alpha beta T cells possibly resulting from a deficit in positive selection. Whether in athymic or euthymic mice, neither T intraepithelial lymphocytes (IEL) nor cryptopatch cells (reported to contain precursors of IEL) displayed fluorescence indicating recent RAG protein synthesis. Newly made T cells migrate from the mesenteric node into the thoracic duct lymph to reach the gut mucosa. In euthymic mice, this extrathymic pathway is totally repressed, except in conditions of severe lymphocytic depletion. Thus, in normal animals, all gut T IEL, including CD8 alpha alpha(+) cells, are of thymic origin, CD8 alpha alpha(+) TCR alpha beta(+) IEL being the likely progeny of double negative NK1-1(-) thymocytes, which show polyclonal V alpha and V beta repertoires.

Gut intraepithelial lymphocyte development.

CD8alphabeta(+) and CD4(+) intraepithelial lymphocytes, the progeny of double-positive thymocytes, are oligoclonal T-cell populations that have accumulated in the gut wall as the result of repeated antigenic stimulations, which lead to rounds of traffic through the lymph/blood circuit ending in an alpha4beta7-integrin-driven homing all along the gut mucosa. In contrast, CD8alphaalpha(+) intraepithelial lymphocytes, which may be TCRgammadelta(+) or alphabeta(+), result in part from local differentiation in the gut, but studies comparing euthymic and athymic mice suggest a thymic double-negative origin for many of them.