CMIP is a negative regulator of T cell signaling.

Upon their interaction with cognate antigen, T cells integrate different extracellular and intracellular signals involving basal and induced protein-protein interactions, as well as the binding of proteins to lipids, which can lead to either cell activation or inhibition. Here, we show that the selective T cell expression of CMIP, a new adapter protein, by targeted transgenesis drives T cells toward a naïve phenotype. We found that CMIP inhibits activation of the Src kinases Fyn and Lck after CD3/CD28 costimulation and the subsequent localization of Fyn and Lck to LRs. Video microscopy analysis showed that CMIP blocks the recruitment of LAT and the lipid raft marker cholera toxin B at the site of TCR engagement. Proteomic analysis identified several protein clusters differentially modulated by CMIP and, notably, Cofilin-1, which is inactivated in CMIP-expressing T cells. Moreover, transgenic T cells exhibited the downregulation of GM3 synthase, a key enzyme involved in the biosynthesis of gangliosides. These results suggest that CMIP negatively impacts proximal signaling and cytoskeletal rearrangement and defines a new mechanism for the negative regulation of T cells that could be a therapeutic target.

A novel combination of chemotherapy and immunotherapy controls tumor growth in mice with a human immune system.

Mice reconstituted with a human immune system and bearing human tumors represent a promising model for developing novel cancer immunotherapies. Here, we used mass cytometry and multi-parametric flow cytometry to characterize human leukocytes infiltrating a human breast cancer tumor model in immunocompromised NOD.SCID.γc-null mice reconstituted with a human immune system and compared it to samples of breast cancer patients. We observed highly activated human CD4+ and CD8+ T cells in the tumor, as well as minor subsets of innate immune cells in both settings. We also report that ICOS+ CD4+ regulatory T cells (Treg) were enriched in the tumor relative to the periphery in humanized mice and patients, providing a target to affect Treg and tumor growth. Indeed, administration of a neutralizing mAb to human ICOS reduced Treg proportions and numbers and improved CD4 + T cell proliferation in humanized mice. Moreover, a combination of the anti-ICOS mAb with cyclophosphamide reduced tumor growth, and that was associated with an improved CD8 to Treg ratio. Depletion of human CD8+ T cells or of murine myeloid cells marginally affected the effect of the combination therapy. Altogether, our results indicate that a combination of anti-ICOS mAb and chemotherapy controls tumor growth in humanized mice, opening new perspectives for the treatment of breast cancer. One sentence summary: Targeting ICOS in combination with chemotherapy is a promising strategy to improve tumor immunity in humans.

Expression of CMIP in podocytes is restricted to specific classes of lupus nephritis.

Lupus glomerulopathies are classified into various histological patterns, which probably result from different pathophysiological origins. Podocyte injury can be demonstrated in lupus nephritis but its clinical relevance is far little appreciated and is often masked by proliferative lesions and inflammatory cell infiltrations. Two patterns of podocyte lesions may be considered, either occurring in the context of renal inflammation or reflecting podocyte dysfunction in non-proliferative and non-inflammatory glomerulopathies. This distinction remains elusive since no reliable biomarker discriminates between both entities. CMIP was recently found induced in some glomerular disease but its expression in different lupus nephritis classes has not been investigated. Twenty-four adult patients with lupus nephritis, including non-proliferative (n = 11) and proliferative (n = 13) glomerulopathies were analyzed. Clinical, biological and immunological data were compared with immunomorphological findings. We analyzed by quantitative and qualitative methods the expression of CMIP in different histological classes. We found CMIP abundance selectively increased in podocytes in class II and class V glomerulopathies, while in proliferative forms (class III and class IV), CMIP was rarely detected. CMIP was not expressed in cellular crescents, endothelial cells or mesangial cells. CMIP colocalized with some subsets of B and T cells within glomerular or interstitial mononuclear cell infiltrates but never with macrophages. Hematuria is rarely present in lupus glomerulopathies expressing CMIP. There was no correlation between classical immunological markers and CMIP expression. Thus, CMIP induction in lupus nephritis seems restricted to non-proliferative glomerulopathies and may define a specific pattern of podocyte injury.

NEPHRUTIX: A randomized, double-blind, placebo vs Rituximab-controlled trial assessing T-cell subset changes in Minimal Change Nephrotic Syndrome.

Minimal-change nephrotic syndrome (MCNS) is an immune-mediated glomerular disease. We have analyzed the modifications on T-cell subsets in twenty-three patients who were highly steroid/calcineurin inhibitor and/or mycophenolate mofetil-dependent for frequently relapsing nephrotic syndrome (FRNS) and who were enrolled in a multicenter, double-blind, randomized, placebo vs Rituximab-controlled trial. Patients with FRNS entered the trial at remission and were randomly assigned to receive either Rituximab or placebo. In both groups, patient blood samples were analyzed at inclusion and then monthly until six months post-perfusion. Disclosure of patient's allocation code occurred in relapse or at the end of the trial. All patients under placebo displaying relapse were subsequently treated with Rituximab. Despite the significant decrease of immunosuppressive drugs, remission was maintained in all patients included in the Rituximab group, except one (n = 9/10). On the other hand, relapses occurred within a few weeks (means ≈ 7.3 weeks) in all patients receiving placebo (n = 13). At inclusion, before rituximab therapy, the frequency of different T-cell subsets were highly similar in both groups, except for CD8+ and invariant TCRVα24 T-cell subsets, which were significantly increased in patients of the Placebo group ((p = 0,0414 and p = 0.0428, respectively). Despite the significant decrease of immunosuppressive drugs, remission was maintained in all patients included in the Rituximab group (n = 10), except one. Relapses were associated with a significant decrease in CD4+CD25highFoxP3high Tregulatory cells (p = 0.0005) and IL2 expression (p = 0.0032), while CMIP abundance was significantly increased (p = 0.03). Remissions after Rituximab therapy were associated in both groups with significant decrease in the frequency of CD4+CD45RO+CXCR5+, invariant natural killer T-cells (INKT) and CD4-CD8- (double-negative, DN) T-cells expressing the invariant Vα24 chain (DN-TCR Vα24) T-cells, suggesting that MCNS involves a disorder of innate and adaptive immune response, which can be stabilized by Rituximab treatment.

Inhibition of the JAK/STAT Signaling Pathway in Regulatory T Cells Reveals a Very Dynamic Regulation of Foxp3 Expression.

The IL-2/JAK3/STAT-5 signaling pathway is involved on the initiation and maintenance of the transcription factor Foxp3 in regulatory T cells (Treg) and has been associated with demethylation of the intronic Conserved Non Coding Sequence-2 (CNS2). However, the role of the JAK/STAT pathway in controlling Foxp3 in the short term has been poorly investigated. Using two different JAK/STAT pharmacological inhibitors, we observed a detectable loss of Foxp3 after 10 min. of treatment that affected 70% of the cells after one hour. Using cycloheximide, a general inhibitor of mRNA translation, we determined that Foxp3, but not CD25, has a high turnover in IL-2 stimulated Treg. This reduction was correlated with a rapid reduction of Foxp3 mRNA. This loss of Foxp3 was associated with a loss in STAT-5 binding to the CNS2, which however remains demethylated. Consequently, Foxp3 expression returns to normal level upon restoration of basal JAK/STAT signaling in vivo. Reduced expression of several genes defining Treg identity was also observed upon treatment. Thus, our results demonstrate that Foxp3 has a rapid turn over in Treg partly controlled at the transcriptional level by the JAK/STAT pathway.

Immunopathogenesis of idiopathic nephrotic syndrome with relapse.

Idiopathic change nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults, is characterized by heavy proteinuria and a relapsing remitting course. Although the mechanisms underlying the pathophysiology of proteinuria remain unclear, clinical and experimental observations suggest that lymphocyte and podocyte disturbances are two sides of the disease. The current hypothesis suggests that immune cells release a putative factor, which alters podocyte function resulting in nephrotic proteinuria. Besides T-cell abnormalities, recent evidence of B-cell depletion efficacy in sustained remissions added a new challenge in understanding the immunological mechanisms of INS. In this review, we discuss recent insights related to podocyte disorders occurring in INS and their relevance in human diseases.

Expression patterns of RelA and c-mip are associated with different glomerular diseases following anti-VEGF therapy.

Renal toxicity constitutes a dose-limiting side effect of anticancer therapies targeting vascular endothelial growth factor (VEGF). In order to study this further, we followed up 29 patients receiving this treatment, who experienced proteinuria, hypertension, and/or renal insufficiency. Eight developed minimal change nephropathy/focal segmental glomerulopathy (MCN/FSG)-like lesions and 13 developed thrombotic microangiopathy (TMA). Patients receiving receptor tyrosine kinase inhibitors (RTKIs) mainly developed MCN/FSG-like lesions, whereas TMA complicated anti-VEGF therapy. There were no mutations in factor H, factor I, or membrane cofactor protein of the complement alternative pathway, while plasma ADAMTS13 activity persisted and anti-ADAMTS13 antibodies were undetectable in patients with TMA. Glomerular VEGF expression was undetectable in TMA and decreased in MCN/FSG. Glomeruli from patients with TMA displayed a high abundance of RelA in endothelial cells and in the podocyte nuclei, but c-mip was not detected. Conversely, MCN/FSG-like lesions exhibited a high abundance of c-mip, whereas RelA was scarcely detected. RelA binds in vivo to the c-mip promoter and prevents its transcriptional activation, whereas RelA knockdown releases c-mip activation. The RTKI sorafenib inhibited RelA activity, which then promoted c-mip expression. Thus, our results suggest that c-mip and RelA define two distinct types of renal damage associated with VEGF-targeted therapies.

Upregulation of c-mip is closely related to podocyte dysfunction in membranous nephropathy.

Membranous nephropathy is a glomerular disease typified by a nephrotic syndrome without infiltration of inflammatory cells or proliferation of resident cells. Although the cause of the disease is unknown, the primary pathology involves the generation of autoantibodies against antigen targets on the surface of podocytes. The mechanisms of nephrotic proteinuria, which reflect a profound podocyte dysfunction, remain unclear. We previously found a new gene, c-mip (c-maf-inducing protein), that was associated with the pathophysiology of idiopathic nephrotic syndrome. Here we found that c-mip was not detected in the glomeruli of rats with passive-type Heymann nephritis given a single dose of anti-megalin polyclonal antibody, yet immune complexes were readily present, but without triggering of proteinuria. Rats reinjected with anti-megalin develop heavy proteinuria a few days later, concomitant with c-mip overproduction in podocytes. This overexpression was associated with the downregulation of synaptopodin in patients with membranous nephropathy, rats with passive Heymann nephritis, and c-mip transgenic mice, while the abundance of death-associated protein kinase and integrin-linked kinase was increased. Cyclosporine treatment significantly reduced proteinuria in rats with passive Heymann nephritis, concomitant with downregulation of c-mip in podocytes. Thus, c-mip has an active role in the podocyte disorders of membranous nephropathy.