Sylvie Brochu

Identification of an immunodominant mouse minor histocompatibility antigen (MiHA). T cell response to a single dominant MiHA causes graft-versus-host disease.

T cell responses to non-MHC antigens are targeted to a restricted number of immunodominant minor histocompatibility antigens whose identity remains elusive. Here we report isolation and sequencing of a novel immunodominant minor histocompatibility antigen presented by H-2Db on the surface of C57BL/6 mouse cells. This nonapeptide (AAPDNRETF) shows strong biologic activity in cytotoxic T lymphocyte sensitization assays at concentrations as low as 10 pM. C3H.SW mice primed with AAPDNRETF in incomplete Freunds adjuvant generated a potent anti-C57BL/6 T cell-mediated cytotoxic activity, and T lymphocytes from AAPDNRETF-primed mice caused graft-versus-host disease when transplanted in irradiated C57BL/6 recipients. These results (a) provide molecular characterization of a mouse dominant minor histocompatibility antigen, (b) identify this peptide as a potential target of graft-versus-host disease and, (c) more importantly, demonstrate that a single dominant minor antigen can cause graft-versus-host disease. These findings open new avenues for the prevention of graft-versus-host disease and should further our understanding of the mechanisms of immunodominance in T cell responses to minor histocompatibility antigens.

Transcriptome sequencing of neonatal thymic epithelial cells

In order to gain novel insights into thymus biology, we analysed the whole transcriptome of cortical and medullary thymic epithelial cells (cTECs and mTECs) and of skin epithelial cells (ECs). Consistent with their ability to express ectopic genes, mTECs expressed more genes than other cell populations. Out of a total of 15,069 genes expressed in TECs, 25% were differentially expressed by at least 5-fold in cTECs vs. mTECs. Genes expressed at higher levels in cTECs than mTECs regulate numerous cell functions including cell differentiation, cell movement and microtubule dynamics. Many positive regulators of the cell cycle were overexpressed in skin ECs relative to TECs. Our RNA-seq data provide novel systems-level insights into the transcriptional landscape of TECs, highlight substantial divergences in the transcriptome of TEC subsets and suggest that cell cycle progression is differentially regulated in TECs and skin ECs.

Why T Cells of Thymic Versus Extrathymic Origin Are Functionally Different

Age-related thymic involution severely impairs immune responsiveness. Strategies to generate T cells extrathymically are therefore being explored with intense interest. We have demonstrated that T cells produced extrathymically were functionally deficient relative to thymus-derived T cells. The main limitation of extrathymic T cells is their undue susceptibility to apoptosis; they thus do not expand properly when confronted with pathogens. Using oncostatin M-transgenic mice, we found that in the absence of lymphopenia, T cells of extrathymic origin constitutively undergo excessive homeostatic proliferation that leads to overproduction of IL-2 and IFN-γ. IFN-γ up-regulates Fas and FasL on extrathymic CD8 T cells, thereby leading to their demise by Fas-mediated apoptosis. Moreover, IFN-γ and probably IL-2 curtail survival of extrathymic CD4 T cells by down-regulating IL-7Rα and Bcl-2, and they support a dramatic accumulation of FoxP3+ T regulatory cells. Additionally, we show that wild-type thymus-derived T cells undergoing homeostatic proliferation in a lymphopenic host shared key features of extrathymic T cells. Our work explains how excessive lymphopenia-independent homeostatic proliferation renders extrathymic T cells functionally defective. Based on previous work and data presented herein, we propose that extrathymic T cells undergo constitutive homeostatic proliferation because they are positively selected by lymph node hemopoietic cells rather than by thymic epithelial cells.

Chronic B-cell lymphocytosis

Persistent elevation of lymphocyte counts is usually associated with a malignant monoclonal lymphoproliferative disease. Over the last 8 years, amongst patients investigated in our center for undetermined persistent lymphocytosis, a diagnosis of malignant lymphoproliferation was excluded in 6 cases as studies of surface membrane immunoglobulin light chains showed that they presented a polyclonal expansion of their B‐lymphocyte pool. All patients were young‐to‐middle aged women presenting peculiar immunohematologic findings characterized by 1) persistent (2–7 yr) elevation of lymphocyte counts (4–14 × 109/l), 2) presence of characteristic binucleated B cells on peripheral blood smears, 3) a normal bone marrow histology, 4) a polyclonal increase of serum IgM with low‐to‐normal IgG and IgA levels. Histologic examination of the spleen in 2 patients and lymph nodes in 1 showed a benign follicular lymphoid hyperplasia. The evolution was benign in every case. We suggest that chronic polyclonal B‐cell lymphocytosis is a distinct clinicopathologic entity that should not be confused with malignant lymphoproliferative disorders.

Immunodomination results from functional differences between competing CTL

The presence of dominant epitopes suppresses generation of CTL activity toward other non‐dominant epitopes found on the same antigen‐presenting cell (APC). This phenomenon, termed immunodomination, drastically restricts the diversity of the repertoire of CTL responses. Under various experimental conditions we assessed the in vivo expansion by tetramer staining and function by expression of O‐glycans and intracellular perforin of CTL specific for a dominant (B6dom1) and a non‐dominant (HY) H2Db‐restricted epitope. Immunodomination abrogated expansion rather than differentiation of HY‐specific CTL. When immunodomination was precluded because HY was presented alone or because high numbers of antigen‐bearing APC were present, the numbers of HY‐specific T cells detected after antigen priming were similar to those of B6dom1‐specific T cells. The main difference between T cells that recognized B6dom1 versus HY was functional rather than quantitative. The key feature of T cells specific for B6dom1 is that they show striking up‐regulation of molecules involved in CTL effector activity rather than accumulating to particularlyhigh levels, as assessed by tetramer staining. These results support the emerging concept that following antigen priming, CTL populations of similar size can display important differences in effector function, and suggest that these functional differences are instrumental in shaping the repertoire of CTL responses.

Adult Thymic Epithelium Contains Nonsenescent Label-Retaining Cells

Progress in our understanding of thymic epithelial cell (TEC) renewal and homeostasis is hindered by the lack of markers for TEC progenitors. Stem and progenitor cell populations display remarkable diversity in their proliferative behavior. In some but not all tissues, stemness is associated with quiescence. The primary goal of our study was to discover whether quiescent cells were present in neonatal and adult TECs. To this end, we used a transgenic label-retaining cell (LRC) assay in which a histone H2B-GFP fusion protein is expressed under the control of the reverse tetracycline-controlled transactivator and the tetracycline operator minimal promoter. In adult mice, we found that both cortical and medullary TECs (cTECs and mTECs) proliferated more actively in females than males. Moreover, we observed three main differences between neonatal and adult TECs: 1) neonatal TECs proliferated more actively than adult TECs; 2) whereas cTECs and mTECs had similar turnover rates in young mice, the turnover of mTECs was more rapid than that of cTECs in adults; and 3) although no LRCs could be detected in young mice, LRCs were detectable after a 16-wk chase in adults. In female mice, LRCs were found almost exclusively among cTECs and expressed relatively low levels of p16INK4a, p19ARF, and Serpine1, and high levels of Bmi1, Foxn1, Trp63, and Wnt4. We conclude that LRCs in adult TECs are not senescent postmitotic cells and may represent the elusive progenitors responsible for TEC maintenance in the adult thymus.

Wnt4 regulates thymic cellularity through the expansion of thymic epithelial cells and early thymic progenitors

Thymus atrophy is the most common immunopathology in humans, and its occurrence is hastened by several factors that coalesce in patients receiving chemotherapy and most of all in recipients of hematopoietic cell transplantation. We have shown previously that posthematopoietic cell transplantation thymic function was improved by retroviral overexpression of Wnt4 in donor hematopoietic cells. Here, by using both conventional and conditional null mutant mice, we show that Wnt4 regulates steady-state thymic cellularity by a thymic epithelial cell (TEC)-dependent mechanism. The absence of Wnt4 suppressed fetal and postnatal thymic expansion and resulted in decreased TEC numbers, an alteration of the medullary-to-cortical TEC ratio, and a disproportionate loss of the most immature cKit(hi) thymocyte precursors. Wnt4 also is implicated in the maintenance of adult thymopoiesis, although the impact of its deletion once thymic involution has been initiated is more subtle. Together, our results show that Wnt4 controls thymic size by modulating TEC expansion and the earliest, TEC-dependent steps of thymocyte development both in the fetal and postnatal thymus. Wnt4 and its downstream signaling pathways could thus represent interesting candidates to improve thymic output in subjects with thymic atrophy.

SMAD3 prevents graft-versus-host disease by restraining Th1 differentiation and granulocyte-mediated tissue damage

Gene expression profiling of human donor T cells before allogeneic hematopoietic cell transplantation revealed that expression of selected genes correlated with the occurrence of graft-versus-host disease (GVHD) in recipients. The gene with the best GVHD predictive accuracy was SMAD3, a core component of the transforming growth factor-β signaling pathway, whose expression levels vary more than a 6-fold range in humans. The putative role of SMAD3 in the establishment of graft-host tolerance remained elusive. We report that SMAD3-KO mice present ostensibly normal lymphoid and myeloid cell subsets. However, the lack of SMAD3 dramatically increased the frequency and severity of GVHD after allogeneic hematopoietic cell transplantation into major histocompatibility complex-identical recipients. Lethal GVHD induced by SMAD3-KO donors affected mainly the intestine and resulted from massive tissue infiltration by T-bet(+) CD4 T cells and granulocytes that caused tissue damage by in situ release of Th1 cytokines and oxidative-nitrosative mediators, respectively. Our report reveals the nonredundant roles of SMAD3 in the development of tolerance to the host. Furthermore, our data support the concept that SMAD3 levels in donor cells dictate the risk of GVHD and that SMAD3 agonists would be attractive for prevention of GVHD.

Differential effects of γc cytokines on postselection differentiation of CD8 thymocytes

The primary consequence of positive selection is to render thymocytes responsive to cytokines and chemokines expressed in the thymic medulla. In the present study, our main objective was to discover which cytokines could support the differentiation of positively selected thymocytes. To this end, we have developed an in vitro model suitable for high-throughput analyses of positive selection and CD8 T-cell differentiation. The model involves coculture of TCR(hi)CD5(int)CD69(-) double-positive (DP) thymocytes with peptide-pulsed OP9 cells and γc-cytokines. We report that IL-4, IL-7, and IL-21 have nonredundant effects on positively selected DP thymocytes. IL-7 signaling phosphorylates STAT5 and ERK; induces Foxo1, Klf2, and S1pr1; and supports the differentiation of classic CD8 T cells. IL-4 activates STAT6 and ERK and supports the differentiation of CD8(int)PD-L1(hi)CD44(hi)EOMES(+) innate CD8 T cells. IL-21 is produced by thymic epithelial cells and the IL-21 receptor-α is strongly induced on DP thymocytes undergoing positive selection. IL-21 signaling phosphorylates STAT3 and STAT5, but not ERK, and does not support CD8 T-cell differentiation. However, IL-21 has a unique ability to up-regulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Our data suggest that injection of recombinant IL-21 might enhance thymic output in subjects with age- or disease-related thymic atrophy.

Differential Features of AIRE-Induced and AIRE-Independent Promiscuous Gene Expression in Thymic Epithelial Cells

Establishment of self-tolerance in the thymus depends on promiscuous expression of tissue-restricted Ags (TRA) by thymic epithelial cells (TEC). This promiscuous gene expression (pGE) is regulated in part by the autoimmune regulator (AIRE). To evaluate the commonalities and discrepancies between AIRE-dependent and -independent pGE, we analyzed the transcriptome of the three main TEC subsets in wild-type and Aire knockout mice. We found that the impact of AIRE-dependent pGE is not limited to generation of TRA. AIRE decreases, via non–cell autonomous mechanisms, the expression of genes coding for positive regulators of cell proliferation, and it thereby reduces the number of cortical TEC. In mature medullary TEC, AIRE-driven pGE upregulates non-TRA coding genes that enhance cell–cell interactions (e.g., claudins, integrins, and selectins) and are probably of prime relevance to tolerance induction. We also found that AIRE-dependent and -independent TRA present several distinctive features. In particular, relative to AIRE-induced TRA, AIRE-independent TRA are more numerous and show greater splicing complexity. Furthermore, we report that AIRE-dependent versus -independent TRA project nonredundant representations of peripheral tissues in the thymus.