Characterization of T Cell Differentiation in the Murine Gut

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-ε, recombination activating gene (Rag)-1, and pre-Tα mRNAs expression at single cell level; (c) we characterized TCR-β, -γ, and -α locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Tα chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 ε/δ and pre-Tα deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut

Tumor-Infiltrating CD3- NK Cells Are More Effective than CD3+ T Cells in Killing Autologous Melanoma Cells

We have studied the phenotype and functional activity of tumor-infiltrating lymphocytes (TIL) derived from eight human melanomas cultured for up to 60 d in the presence of recombinant IL-2. In the early period of the cultures, TIL were predominantly T cells of CD8+ phenotype and contained 10–30% of CD3- cells. Four of the five early TIL cultures tested in a cytotoxicity assay displayed a degree of MHC-unrestricted lysis on a series of autologous and allogenic melanoma cell lines as well as the K562 natural killer-sensitive target. With longer periods of time in culture, all TIL lines showed a decrease in lytic activity that was associated with the loss of CD3- cells. Thus, most of the killing of short-term TIL cultures appeared to be mediated by CD3- natural killer cells, whereas CD3+ T cells were found to be weak anti-tumor effectors. Even though the CD3+ T cells were not cytotoxic on K562 targets, their lytic activity (even weak) against melanoma cells appeared to be non-MHC restricted and was blocked by anti-CD3 antibodies. In addition, cytotoxicity of the CD3+ TIL cultures was compared to that of a CD3-/NKH1+ cell line purified from peripheral blood. It was found that natural killer cells were much more potent than CD3+ TIL on the melanoma cell lines tested

The cyclin D1 carboxyl regulatory domain controls the division and differentiation of hematopoietic cells

International audienceAbstractBackgroundThe family of D cyclins has a fundamental role in cell cycle progression, but its members (D1, D2, D3) are believed to have redundant functions. However, there is some evidence that contradicts the notion of mutual redundancy and therefore this concept is still a matter of debate.ResultsOur data show that the cyclin D1 is indispensable for normal hematopoiesis. Indeed, in the absence of D1, either in genetic deficient mice, or after acute ablation by RNA interference, cyclins D2 and D3 are also not expressed preventing hematopoietic cell division and differentiation at its earliest stage. This role does not depend on the cyclin box, but on the carboxyl regulatory domain of D1 coded by exons 4–5, since hematopoietic differentiation is also blocked by the conditional ablation of this region.ConclusionThese results demonstrate that not all functions of individual D cyclins are redundant and highlight a master role of cyclin D1 in hematopoiesis

Correction: Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

[This corrects the article DOI: 10.1371/journal.pone.0157822.]

On the Role of the Pre–T Cell Receptor in αβ versus γδ T Lineage Commitment

AbstractThe role of the pre–T cell receptor (TCR) in lineage commitment to the γδ versus αβ lineage of T cells was addressed by analyzing TCRβ chain rearrangements in γδ T cells from wild-type and pre-TCR-deficient mice by single cell polymerase chain reaction. Results show that the pre-TCR selects against γδ T cells containing rearranged Vβ genes and that γδ T cell precursors but not γδ T cells express the pre-TCRα protein. Furthermore, pre-TCR-induced proliferation could not be detected in γδ T cells. We propose that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism that has largely replaced an evolutionary more ancient stochastic mechanism of lineage commitment

Extrathymic T Cell Lymphopoiesis

International audienceIn 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 ␥␦ T cell production and with a paucity of oligoclonal ␣␤ 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 ␣␣ ϩ cells, are of thymic origin, CD8 ␣␣ ϩ TCR ␣␤ ϩ IEL being the likely progeny of double negative NK1-1 Ϫ thymocytes, which show polyclonal V ␣ and V ␤ repertoires

Regulatory T cells prevent CD8 T cell maturation by inhibiting CD4 th cells at tumor sites

Natural regulatory T cells (Tregs) are present in high frequencies among tumor-infiltrating lymphocytes and in draining lymph nodes, supposedly facilitating tumor development. To investigate their role in controlling local immune responses, we analyzed intratumoral T cell accumulation and function in the presence or absence of Tregs. Tumors that grew in normal BALB/c mice injected with the 4T1 tumor cell line were highly infiltrated by Tregs, CD4 and CD8 cells, all having unique characteristics. Most infiltrating Tregs expressed low levels of CD25Rs and Foxp3. They did not proliferate even in the presence of IL-2 but maintained a strong suppressor activity. CD4 T cells were profoundly anergic and CD8 T cell proliferation and cytotoxicity were severely impaired. Depletion of Tregs modified the characteristics of tumor infiltrates. Tumors were initially invaded by activated CD4CD25 T cells, which produced IL-2 and IFN-γ. This was followed by the recruitment of highly cytotoxic CD8 T cells at tumor sites leading to tumor rejection. The beneficial effect of Treg depletion in tumor regression was abrogated when CD4 helper cells were also depleted. These findings indicate that the massive infiltration of tumors by Tregs prevents the development of a successful helper response. The Tregs in our model prevent Th cell activation and subsequent development of efficient CD8 T cell activity required for the control of tumor growth