THEMIS promotes T cell development and maintenance by rising the signaling threshold of the inhibitory receptor BTLA.

The current paradigm about the function of T cell immune checkpoints is that these receptors switch on inhibitory signals upon cognate ligand interaction. We here revisit this simple switch model and provide evidence that the T cell lineage protein THEMIS enhances the signaling threshold at which the immune checkpoint BTLA (B- and T-lymphocyte attenuator) represses T cell responses. THEMIS is recruited to the cytoplasmic domain of BTLA and blocks its signaling capacity by promoting/stabilizing the oxidation of the catalytic cysteine of the tyrosine phosphatase SHP-1. In contrast, THEMIS has no detectable effect on signaling pathways regulated by PD-1 (Programmed cell death protein 1), which depend mainly on the tyrosine phosphatase SHP-2. BTLA inhibitory signaling is tuned according to the THEMIS expression level, making CD8+ T cells more resistant to BTLA-mediated inhibition than CD4+ T cells. In the absence of THEMIS, the signaling capacity of BTLA is exacerbated, which results in the attenuation of signals driven by the T cell antigen receptor and by receptors for IL-2 and IL-15, consequently hampering thymocyte positive selection and peripheral CD8+ T cell maintenance. By characterizing the pivotal role of THEMIS in restricting the transmission of BTLA signals, our study suggests that immune checkpoint operability is conditioned by intracellular signal attenuators.

Lymphocyte migration and retention properties affected by ibrutinib in chronic lymphocytic leukemia.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients' samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.

A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage.

The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 in mouse models leads to proliferative defects associated with a blockade of T-cell development after ß-selection and of peripheral CD4+ T-cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1 deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.

THEMIS enhances the magnitude of normal and neuroinflammatory type 1 immune responses by promoting TCR-independent signals.

Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)-mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell-mediated responses.

CD5 signalosome coordinates antagonist TCR signals to control the generation of Treg cells induced by foreign antigens.

CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes.

PAX5-ELN oncoprotein promotes multistep B-cell acute lymphoblastic leukemia in mice.

PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (B-ALL) and is involved in various chromosomal translocations that fuse a part of PAX5 with other partners. However, the role of PAX5 fusion proteins in B-ALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human B-ALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in B-ALL development, we generated a knockin mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN-expressing mice efficiently developed B-ALL with an incidence of 80%. Leukemic transformation was associated with recurrent secondary mutations on Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrate that PAX5-ELN affected B-cell development in vitro and in vivo featuring an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Finally, our molecular and computational approaches identified PAX5-ELN-regulated gene candidates that establish the molecular bases of the preleukemic state to drive B-ALL initiation. Hence, our study provides a new in vivo model of human B-ALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development.

PAX5A and PAX5B isoforms are both efficient to drive B cell differentiation.

Pax5 is the guardian of the B cell identity since it primes or enhances the expression of B cell specific genes and concomitantly represses the expression of B cell inappropriate genes. The tight regulation of Pax5 is therefore required for an efficient B cell differentiation. A defect in its dosage can translate into immunodeficiency or malignant disorders such as leukemia or lymphoma. Pax5 is expressed from two different promoters encoding two isoforms that only differ in the sequence of their first alternative exon. Very little is known regarding the role of the two isoforms during B cell differentiation and the regulation of their expression. Our work aims to characterize the mechanisms of regulation of the expression balance of these two isoforms and their implication in the B cell differentiation process using murine ex vivo analyses. We show that these two isoforms are differentially regulated but have equivalent function during early B cell differentiation and may have functional differences after B cell activation. The tight control of their expression may thus reflect a way to finely tune Pax5 dosage during B cell differentiation process.

Thymic-Specific Serine Protease Limits Central Tolerance and Exacerbates Experimental Autoimmune Encephalomyelitis.

The genetic predisposition to multiple sclerosis (MS) is most strongly conveyed by MHC class II haplotypes, possibly by shaping the autoimmune CD4 T cell repertoire. Whether Ag-processing enzymes contribute to MS susceptibility by editing the peptide repertoire presented by these MHC haplotypes is unclear. Thymus-specific serine protease (TSSP) is expressed by thymic epithelial cells and thymic dendritic cells (DCs) and, in these two stromal compartments, TSSP edits the peptide repertoire presented by class II molecules. We show in this article that TSSP increases experimental autoimmune encephalomyelitis severity by limiting central tolerance to myelin oligodendrocyte glycoprotein. The effect on experimental autoimmune encephalomyelitis severity was MHC class II allele dependent, because the lack of TSSP expression conferred protection in NOD mice but not in C57BL/6 mice. Importantly, although human thymic DCs express TSSP, individuals segregate into two groups having a high or 10-fold lower level of expression. Therefore, the level of TSSP expression by thymic DCs may modify the risk factors for MS conferred by some MHC class II haplotypes.

Grb2-Mediated Recruitment of USP9X to LAT Enhances Themis Stability following Thymic Selection.

Themis is a new component of the TCR signaling machinery that plays a critical role during T cell development. The positive selection of immature CD4+CD8+ double-positive thymocytes and their commitment to the CD4+CD8- single-positive stage are impaired in Themis-/- mice, suggesting that Themis might be important to sustain TCR signals during these key developmental processes. However, the analysis of Themis mRNA levels revealed that Themis gene expression is rapidly extinguished during positive selection. We show in this article that Themis protein expression is increased in double-positive thymocytes undergoing positive selection and is sustained in immature single-positive thymocytes, despite the strong decrease in Themis mRNA levels in these subsets. We found that Themis stability is controlled by the ubiquitin-specific protease USP9X, which removes ubiquitin K48-linked chains on Themis following TCR engagement. Biochemical analyses indicate that USP9X binds directly to the N-terminal CABIT domain of Themis and indirectly to the adaptor protein Grb2, with the latter interaction enabling recruitment of Themis/USP9X complexes to LAT, thereby sustaining Themis expression following positive selection. Together, these data suggest that TCR-mediated signals enhance Themis stability upon T cell development and identify USP9X as a key regulator of Themis protein turnover.

X-Chromosome complement and estrogen receptor signaling independently contribute to the enhanced TLR7-mediated IFN-α production of plasmacytoid dendritic cells from women.

Human plasmacytoid dendritic cells (pDCs) play a major role in innate immunity through the production of type I IFNs after TLR engagement by pathogens. Sex-based differences in the innate function of human pDCs have been established, with pDCs from women exhibiting enhanced TLR7-mediated IFN-α production as compared with pDCs from males. In mice, we recently provided evidence for a role of estrogens as a positive regulator of pDC innate functions through cell-intrinsic estrogen receptor α signaling, but did not exclude a role for other X-linked factors, particularly in human pDCs. In this study, we investigated the respective contribution of X chromosome dosage and sex hormones using a humanized mouse model in which male or female NOD-SCID-ß2m(-/-) were transplanted with human progenitor cells purified from either male or female cord blood cells. We showed that, in response to TLR7 ligands, the frequency of IFN-α- and TNF-α-producing pDCs from either sex was greater in female than in male host mice, suggesting a positive role for estrogens. Indeed, blockade of estrogen receptor signaling during pDC development in vitro inhibited TLR7-mediated IFN-α production by human pDCs, which expressed both ESR1 and ESR2 genes. Interestingly, we also found that X chromosome dosage contributed to this sex bias as female pDCs have an enhanced TLR7-mediated IFN-α response as compared with male ones, irrespective of the sex of the recipient mice. Together, these results indicate that female sex hormones, estrogens, and X chromosome complement independently contribute to the enhanced TLR7-mediated IFN-α response of pDCs in women.

Estradiol promotes functional responses in inflammatory and steady-state dendritic cells through differential requirement for activation function-1 of estrogen receptor α.

17ß-Estradiol (E2) has been shown to regulate GM-CSF- or Flt3 ligand-driven dendritic cell (DC) development through estrogen receptor (ER) α signaling in myeloid progenitors. ERα regulates transcription of target genes through two distinct activation functions (AFs), AF-1 and AF-2, whose respective involvement varies in a cell type- or tissue-specific manner. In this study, we investigated the role of ERα AFs in the development and effector functions of inflammatory DCs, steady-state conventional DCs, and plasmacytoid DCs (pDC), using mouse lacking either AF-1 or AF-2. In agreement with previous works, we showed that E2 fostered the differentiation and effector functions of inflammatory DCs through ERα-dependent upregulation of IFN regulatory factor (IRF)-4 in GM-CSF-stimulated myeloid progenitors. Interestingly, whereas AF-1 was required for early IRF-4 upregulation in DC precursors, it was dispensable to enhance IRF-4 expression in differentiated DCs to a level compatible with the development of the more functional Ly6C(-) CD11b(+) DC subset. Presence of E2 had no effect on progenitors from either knock-in mice with 7-aa deletion in helix 12 of ERα, lacking AF-2, or ERα(-/-) mice. By contrast, in Flt3 ligand-driven DC differentiation, activation of AF-1 domain was required to promote the development of more functionally competent conventional DCs and pDCs. Moreover, lack of ERα AF-1 blunted the TLR7-mediated IFN-α response of female pDCs in vivo. Thus, our study demonstrates that ERα uses AF-1 differently in steady-state and inflammatory DC lineages to regulate their innate functions, suggesting that selective ER modulators could be used to target specific DC subsets.

The TLR-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor α signaling.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I interferons (IFN-α/ß) in response to viral or endogenous nucleic acids through activation of their endosomal Toll-like receptors (TLR-7 and TLR-9). Enhanced TLR-7-mediated IFN-α production by pDCs in women, compared with men, has been reported, but whether sex hormones, such as estrogens, are involved in this sex-based difference is unknown. Here we show, in humanized mice, that the TLR-7-mediated response of human pDCs is increased in female host mice relative to male. In a clinical trial, we establish that treatment of postmenopausal women with 17ß-estradiol markedly enhances TLR-7- and TLR-9-dependent production of IFN-α by pDCs stimulated by synthetic ligands or by nucleic acid-containing immune complexes. In mice, we found exogenous and endogenous estrogens to promote the TLR-mediated cytokine secretion by pDCs through hematopoietic expression of estrogen receptor (ER) α. Genetic ablation of ERα gene in the DC lineage abrogated the enhancing effect of 17ß-estradiol on their TLR-mediated production of IFN-α, showing that estrogens directly target pDCs in vivo. Our results uncover a previously unappreciated role for estrogens in regulating the innate functions of pDCs, which may account for sex-based differences in autoimmune and infectious diseases.