Regulatory T-cell dysfunctions are associated with increase in tumor necrosis factor α in autoimmune hemolytic anemia and participate in Th17 polarization.

Warm autoimmune hemolytic anemia (wAIHA) is a rare acquired autoimmune disease mediated by antibodies targeting red blood cells. The involvement of CD4 T-helper cells has been scarcely explored, with most findings extrapolated from animal models. Here, we performed quantification of both effector T lymphocytes (Teff) and regulatory T cells (Treg), associated with functional and transcriptomic analyses of Treg in human wAIHA. We observed a shift of Teff toward a Th17 polarization concordant with an increase in serum interleukin-17 concentration that correlates with red blood cell destruction parameters, namely lactate dehydrogenase and bilirubin levels. A decrease in circulating Treg, notably effector Treg, associated with a functional deficiency, as represented by their decrease capability to inhibit Teff proliferation, were also observed. Treg deficiency was associated with a reduced expression of Foxp3, the master transcription factor known to maintain the Treg phenotype stability and suppressive functions. Transcriptomic profiling of Treg revealed activation of the tumor necrosis facto (TNF)-α pathway, which was linked to increased serum TNF-α concentrations that were twice as high as in controls. Treg transcriptomic profiling also suggested that post-translational mechanisms possibly accounted for Foxp3 downregulation and Treg dysfunctions. Since TNF-α participates in the rupture of immune tolerance during wAIHA, its inhibition could be of interest. To this end, the effects of fostamatinib, a SYK inhibitor, were investigated in vitro, and we showed that besides the inhibition of erythrocyte phagocytosis by monocytes, fostamatinib is also able to dampen TNF-α production, thus appearing as a promising multitargeting therapy in wAIHA (clinicaltrials gov. Identifier: NCT02158195).

Human Monocyte-Derived Suppressor Cell Supernatant Induces Immunoregulatory Effects and Mitigates xenoGvHD.

Recently developed cell-based therapies have shown potential for graft-versus-host disease (GvHD) mitigation. Our team previously developed a protocol to generate human monocyte-derived suppressor Cells (HuMoSC), a subpopulation of CD33+ suppressor cells of monocytic origin. CD33+HuMoSC successfully reduced xenoGvHD severity in NOD/SCID/IL-2Rγc-/- (NSG) mice. While CD33+ HuMoSC culture supernatant inhibits T cell activation and proliferation, the recovery of CD33+ HuMoSC immunosuppressive cells and the subsequent production of their supernatant is limited. An attractive solution would be to use both the CD33+ and the large number of CD14+ cells derived from our protocol. Here, we assessed the immunoregulatory properties of the CD14+HuMoSC supernatant and demonstrated that it inhibited both CD4 and CD8 T cell proliferation and decreased CD8 cytotoxicity. In vivo, injection of CD14+HuMoSC supernatant reduced xenoGvHD in NSG mice. Furthermore, CD14+HuMoSC supernatant maintained its immunoregulatory properties in an inflammatory environment. Proteomic and multiplex analyses revealed the presence of immunosuppressive proteins such as GPNMB, galectin-3 and IL-1R(A) Finally, CD14+HuMoSC supernatant can be produced using good manufacturing practices and be used as complement to current immunosuppressive drugs. CD14+HuMoSC supernatant is thus a promising therapy for preventing GvHD. .

Improvement of Treg immune response after treatment with tocilizumab in giant cell arteritis.

OBJECTIVES: To study the percentage, suppressive function and plasticity of Treg in giant cell arteritis (GCA), and the effects of glucocorticoids and tocilizumab. METHODS: Blood samples were obtained from 40 controls and 43 GCA patients at baseline and after treatment with glucocorticoids + IV tocilizumab (n = 20) or glucocorticoids (n = 23). Treg percentage and phenotype were assessed by flow cytometry. Suppressive function of Treg was assessed by measuring their ability to inhibit effector T-cell (Teff) proliferation and polarisation into Th1 and Th17 cells. RESULTS: Treg (CD4+CD25highFoxP3+) frequency in total CD4+ T cells was decreased in active GCA patients when compared to controls (2.5% vs. 4.7%, P < 0.001) and increased after treatment with tocilizumab but worsened after treatment with glucocorticoids alone. Treg lacking exon 2 of FoxP3 were increased in GCA patients when compared to controls (23% vs. 10% of total Treg, P = 0.0096) and normalised after treatment with tocilizumab + glucocorticoids but not glucocorticoids alone. In GCA patients, Treg were unable to control Teff proliferation and induced ˜50% increase in the amount of IL-17+ Teff, which was improved after in vitro blockade of the IL-6 pathway by tocilizumab. CONCLUSION: This study reports quantitative and functional disruptions in the regulatory immune response of GCA patients and demonstrates that, unlike glucocorticoids, tocilizumab improves Treg immune response.

Mucosal-associated invariant T cells in Giant Cell Arteritis.

This study aimed to assess the implication of mucosal-associated invariant T (MAIT) cells in GCA. Blood samples were obtained from 34 GCA patients (before and after 3 months of treatment with glucocorticoids (GC) alone) and compared with 20 controls aged >50 years. MAIT cells, defined by a CD3+CD4-TCRγδ-TCRVα7.2+CD161+ phenotype, were analyzed by flow cytometry. After sorting, we assessed the ability of MAIT cells to proliferate and produce cytokines after stimulation with anti CD3/CD28 microbeads or IL-12 and IL-18. MAIT were stained in temporal artery biopsies (TAB) by confocal microscopy. MAIT cells were found in the arterial wall of positive TABs but was absent in negative TAB. MAIT frequency among total αß-T cells was similar in the blood of patients and controls (0.52 vs. 0.57%; P = 0.43) and not modified after GC treatment (P = 0.82). Expression of IFN-γ was increased in MAIT cells from GCA patients compared to controls (44.49 vs. 32.9%; P = 0.029), and not modified after 3 months of GC therapy (P = 0.82). When they were stimulated with IL-12 and IL-18, MAIT from GCA patients produced very high levels of IFN-γ and displayed a stronger proliferation compared with MAIT from controls (proliferation index 3.39 vs. 1.4; P = 0.032). In GCA, the functional characteristics of MAIT cells are modified toward a pro-inflammatory phenotype and a stronger proliferation capability in response to IL-12 and IL-18, suggesting that MAIT might play a role in GCA pathogenesis. Our results support the use of treatments targeting IL-12/IL-18 to inhibit the IFN-γ pathway in GCA.

Efficiency of human monocyte-derived suppressor cell-based treatment in graft-versus-host disease prevention while preserving graft-versus-leukemia effect.

Background: Immunosuppressive cell-based therapy is a recent strategy for controlling Graft-versus-Host Disease (GvHD). Such cells ought to maintain their suppressive function in inflammatory conditions and in the presence of immunosuppressive agents currently used in allogeneic hematopoietic cell transplantation (allo-HCT). Moreover, these therapies should not diminish the benefits of allo-HCT, the Graft-versus-Leukemia (GvL) effect. We have previously reported on a novel subset of human monocyte-derived suppressor cells (HuMoSC) as a prospective approach for controlling GvHD.Objective.The objective of this study was to explore the therapeutic relevance of the HuMoSC in clinical conditions. Methods: Immune regulatory functions of HuMoSC were assessed in inflammatory conditions and in the presence of immunosuppressants. The therapeutic efficiency of the association of HuMoSC with immunosuppressants was evaluated in an experimental model of GvHD induced by human PBMC in NOD/SCID/IL2-Rγc-/- (NSG) mice.Interestingly, the inhibitory functions of HuMoSC against T lymphocytes and their ability to polarize Treg are preserved, in vitro, in inflammatory environments and are not affected by immunosuppressive agents. In vivo, the association of HuMoSC-based treatment with an immunosuppressive drug showed a synergistic effect for controlling GvHD. Furthermore, HuMoSC control GvHD while preserving GvL effect in a xeno-GvHD conditioned mouse model with cell neoplasm (CAL-1). HuMoSC are generated according to good manufacturing practices (GMP) and we demonstrated that these cells tolerate long-term preservation with unaltered phenotype and function.Conclusion.HuMoSC-based therapy represents a promising approach for controlling GvHD and could be quickly implemented in clinical practice.