Florence Vasseur

Immune Regulation by Self-Reactive T Cells is Antigen Specific

Immune regulation plays an important role in the establishment and maintenance of self-tolerance. Nevertheless, it has been difficult to conclude whether regulation is Ag specific because studies have focused on polyclonal populations of regulatory T cells. We have used in this study a murine transgenic model that generates self-reactive, regulatory T cells of known Ag specificity to determine their capacity to suppress naive T cells specific for other Ags. We show that these regulatory cells can regulate the responses of naive T cells with the same TCR specificity, but do not inhibit T cell proliferation or differentiation of naive T cells specific for other Ags. These results demonstrate that immune regulation may be more Ag specific than previously proposed.

Thymocytes may persist and differentiate without any input from bone marrow progenitors

New thymus transplant experiments reveal that in the absence of competing bone marrow progenitors, existing thymocytes can self-renew, guaranteeing thymus cellularity and the rapid reconstitution of the peripheral T cell pools.

Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation

Precursor CD4–CD8– (DN) thymocytes rearrange their TCR‐β genes, and only those which succeed in β‐selection subsequently expand and differentiate into immature CD4+CD8+ (DP) thymocytes. The cell subsets corresponding to the successive steps of this transition can be defined in terms of CD44 and CD25 expression. We partially synchronized the differentiation process by eliminating cycling cells with the anti‐mitotic agent demecolcine. Using in vivo pulse labeling with bromodeoxyuridine, we determined the order of entry into DNA synthesis of the different DN and transitory (CD4–/lo CD8+) cell subsets. Two independent proliferation phases were identified. The first cells to enter the cell cycle were CD44–CD25lo, and CD4/CD8/TCR‐/BrdU four‐color staining showed that they all expressed a low density of the TCR‐β chain, an element of the pre‐TCR (the TCR‐α locus is still in germ‐line configuration at this stage). Cycling of CD44+CD25+ cells was detected later, and no starting point was observed at the CD44–CD25hi stage. CD8 expression was immediately detectable in cycling cells, but they took 24 h to reach the DP stage. The study of TCR‐Cα‐deficient mice showed that β gene rearrangement occurred once proliferation had ceased at the DP stage, and that it had no influence on the DN‐DP transition. These data show that precursor thymocytes undergo two independent waves of expansion, and that the second wave is restricted to cells capable of pre‐TCR expression.

Regulation and kinetics of premigrant thymocyte expansion.

Normal mature thymocytes proliferate before emigrating to the periphery, and continuous bromodeoxyuridine labeling showed that more than 30 % of fully mature thymic emigrants have replicated DNA in the 24 h before exit. The percentage of DNA‐synthetizing singlepositive (SP) thymocytes is transiently augmented during the postnatal period, with peaks on days 2 and 4 for CD4 and CD8 cells, respectively. Similar kinetics were observed in mouse chimeras made by transfer of normal bone marrow cells into RAG‐2‐deficient mice. These data show that proliferation of mature thymocytes is developmentally regulated. The proliferation peaks (on days 16 and 18 post transfer) observed in simple bone marrow chimeras were abolished when lymph node T cells were mixed with the bone marrow cell inoculum, suggesting that the peripheral pool controls the late thymic expansion. The phenotype of cycling SP thymocytes is atypical: they do not regulate activation and adhesion surface molecules like peripheral activated T cells.

Tolerance induction to self antigens by peripheral dendritic cells

It has been suggested, but not formally demonstrated, that peripheral dendritic cells (DC) alone are capable of tolerance induction by clonal deletion and/or anergy. To resolve such an issue, it is important to develop in vivo systems where DC are the only cells capable of presenting antigen and where a T cell population with a known antigen specificity can be followed. Here we use a transgenic murine model, which expresses the influenza virus hemagglutinin (HA) on B cells and on CD8α+ and CD8α– DC but not on macrophages. If these mice are on a RAG–/– background, one has a model in which only DC present the HA antigen. In these mice, HA‐specific T cells are deleted very efficiently in the thymus and those remaining in the periphery cannot respond to further antigenic stimulation in vitro and cannot eliminate antigen in vivo. By performing adoptive transfers, we show for the first time that self‐antigen presentation exclusively by peripheral DC results in very efficient clonal deletion of the majority of antigen‐specific T cells with the remaining ones in an anergic state. This model will permit us to further address the mechanisms by which DC tolerize or prime T cells and to investigate whether anergy induction by DC is similar to anergy induction by B cells.

Quantitative and Qualitative Adjustment of Thymic T Cell Production by Clonal Expansion of Premigrant Thymocytes

In normal mice, single-positive thymocytes proliferate before being exported into the peripheral T cell pool. We measured the in vivo proliferation rates of mature thymocytes in several TCR transgenic mice. Different monoclonal TCR transgenic single-positive thymocytes proliferated at different rates in a given MHC context. Conversely, mature thymocytes expressing a given TCR, generated in mice of different MHC haplotypes, also showed different rates of proliferation. In p59fyn-deficient mice, the proliferation rate of mature thymocytes was diminished. Thus, premigrant thymocyte expansion is TCR mediated and depends on TCR affinity for self peptide/MHC ligands. In addition, we show that mature thymocyte expansion is clonotypic, increases the daily thymic T cell output, and modifies the TCR repertoire of newly produced T cells.

Cognate Antigen Stimulation Generates Potent CD8+ Inflammatory Effector T Cells

Inflammatory reactions are believed to be triggered by innate signals and have a major protective role by recruiting innate immunity cells, favoring lymphocyte activation and differentiation, and thus contributing to the sequestration and elimination of the injurious stimuli. Although certain lymphocyte types such as TH17 cells co-participate in inflammatory reactions, their generation from the naïve pool requires the pre-existence of an inflammatory milieu. In this context, inflammation is always regarded as beginning with an innate response that may be eventually perpetuated and amplified by certain lymphocyte types. In contrast, we here show that even in sterile immunizations or in MyD88-deficient mice, CD8 T cells produce a burst of pro-inflammatory cytokines and chemokines. These functions follow opposite rules to the classic CD8 effector functions since they are generated prior to cell expansion and decline before antigen elimination. As few as 56 CD8+ inflammatory effector cells in a lymph node can mobilize 107 cells in 24 h, including lymphocytes, natural killer cells, and several accessory cell types involved in inflammatory reactions. Thus, although inflammation modulates cognate responses, CD8 cognate responses also initiate local inflammatory reactions.

Fas receptor signaling is requisite for B cell differentiation.

The Fas/Fas ligand (FasL) pathway has been largely implicated in the homeostasis of mature cells. However, it is still unclear whether it plays a role at the progenitor level. To address this issue, we created chimeric mice by transferring C57BL/6 bone marrow (BM) cells of the lpr (Fas−FasL+) or gld (Fas+FasL−) genotype into Rag‐2−/− hosts of the same genetic background. In this model, the consequences of a deficient Fas/FasL pathway on lymphoid differentiation could be evaluated without endogenous competition. Analysis of the chimerism revealed a differential sensitivity of hematopoietic lineages to the lack of Fas receptor signaling. While donor‐derived myelo‐monocytic cells were similarly distributed in all chimeric mice, mature B cells were deleted in the BM and the spleen of lpr chimera, leading to the absence of the marginal zone (MZ) as detected by immunohistology. In contrast, B cell hematopoiesis was complete in gld chimera but MZ macrophages undetectable. These defects suggest a direct and determinant dual role of FasL regulation in negative selection of B cells and in maintenance of the MZ.

Selective involvement of the Fas (CD95)/Fas ligand pathway in bone marrow B cell progenitors

B lymphocyte generation in bone marrow (BM) compensates for cell loses. The Fas / Fas ligand (FasL) pathway has been implicated in apoptosis of various cell types. Abnormalities of the Fas receptor or of FasL expression are associated with excessive T cell proliferation and autoimmunity. To examine the role of the Fas / FasL system in B cell differentiation, we created double‐chimeric mice by transferring both C57BL / 6 (B6)‐Fas+ and lpr‐FasL+ BM cells into RAG‐2– / – hosts. Equal numbers of stem cells were co‐injected into sublethally irradiated recipients, and their progeny were studied by using antibodies directed against the B6‐Ly5.1+5.2+ and lpr‐Ly5.1–5.2+ populations. A longitudinal study lasting for up to 6 months revealed that cells of the lpr phenotype dominated the B6 phenotype in the BM, as a result of their active proliferation. Analysis of the B cell compartment showed more lpr than B6 cells among immature HSAhiB220lo populations. In contrast, the lpr and B6 phenotypes were equally represented among mature B cells. BM transfer to second hosts indicated that B6‐derived B cell progenitors were absent from the first host. These data suggest that activation of the Fas / FasL pathway disturbs the early steps of B cell development and might therefore contribute to the onset of autoimmune disorders.

Thy-1 modulation and cell proliferation at early steps of intrathymic bone marrow cell differentiation

Intrathymic (IT) transfer of bone marrow (BM) precursor cells in sublethally irradiated hosts has been widely used to study T cell differentiation and maturation. In this report we have used double congenic mice Ly 5.1 Thy 1.1 (host) and Ly 5.2 Thy 1.2 (donor) and detected cycling Ly 5.2+ BM cells by in vivo bromodeoxyuridine incorporation, before induction of the Thy 1.2 antigen. Until Day 9 post-transfer, some donor type cells express a high level of Thy 1.2 together with macrophage and granulocyte markers. A few days later, a Thy 1.2low population transiently B220+ was detected. Thereafter, donor type cells expressed an intermediate Thy 1.2 brightness; this population then persisted and surpassed the other subsets. Our findings permitted to establish a relationship between cell cycle and Thy 1 fluorescence intensity according to the sequence: Thy 1low resting, Thy 1low cycling, Thy 1high cycling, Thy 1high resting. Moreover, we have shown that cells from the myeloïd and B lineages can, in vivo, transiently express the Thy 1 antigen, develop and differentiate within the thymus microenvironment.