Unconventional T Cells Influence Clinical Outcome After Allogeneic Hematopoietic Cell Transplantation.

We evaluated the impact of early recovery of mucosal-associated invariant T cells (MAIT) and gamma-delta (γδ) T cells, especially Vδ2+ T cells, on the clinical outcomes of 76 patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). MAIT cells were identified at day 20-30 post-transplant using flow cytometry and defined as CD3+ TCRVα7.2+CD161+. Two subsets of Vδ2+ T cells were analyzed according to the expression of CD26. The cytotoxicity profile of MAIT and Vδ2+ T cells was analyzed according to the intracellular expression of perforin and granzyme B, and intracellular IFN-γ was evaluated after in vitro activation. CD26+Vδ2+ T cells displayed higher intracellular levels of IFN-γ, whereas CD26- Vδ2+ T were found to be more cytotoxic. Moreover, MAIT cell frequency was correlated with the frequency of Vδ2+ T cells with a better correlation observed with Vδ2+CD26+ than with the Vδ2+CD26- T cell subset. By using the composite endpoint graft-versus-host disease (GvHD)-free, relapse-free survival (GRFS) as the primary endpoint, we found that patients with a higher MAIT cell frequency at day 20-30 after allo-HCT had a significantly increased GRFS and a better overall survival (OS) and disease-free survival (DFS). Moreover, patients with a low CD69 expression by MAIT cells had an increased cumulative incidence of grade 2-4 acute GvHD (aGvHD). These results suggest that MAIT cell reconstitution may provide mitigating effects early after allo-HCT depending on their activation markers and functional status. Patients with a high frequency of Vδ2+CD26+ T cells had a significantly higher GRFS, OS and DFS, but there was no impact on cumulative incidence of grade 2-4 aGVHD, non-relapse mortality and relapse. These results revealed that the impact of Vδ2+ T cells on the success of allo-HCT may vary according to the frequency of the CD26+ subset.

Interplay between plasmacytoid dendritic cells and tumor-specific T cells in peripheral blood influences long-term survival in non-small cell lung carcinoma.

Plasmacytoid dendritic cells (pDCs) represent a subset of antigen-presenting cells that play an ambivalent role in cancer immunity. Here, we investigated the clinical significance of circulating pDCs and their interaction with tumor-specific T cell responses in patients with non-small cell lung cancer (NSCLC, n = 126) . The relation between intratumoral pDC signature and immune checkpoint inhibitors efficacy was also evaluated. Patients with NSCLC had low level but activated phenotype pDC compared to healthy donors. In overall population, patients with high level of pDC (pDChigh) had improved overall survival (OS) compared to patients with pDClow, median OS 30.4 versus 20.7 months (P = 0.013). This clinical benefit was only observed in stage I to III patients, but not in metastatic disease. We showed that patients harboring pDChigh profile had high amount of Th1-diffentiation cytokine interleukin-12 (IL-12) in blood and had functional T cells directed against a broad range of tumor antigens. Furthermore, a high pDC signature in the tumor microenvironment was associated with improved clinical outcome in patients treated with anti-PD-(L)1 therapy. Overall, this study showed that circulating pDChigh is associated with long-term OS in NSCLC and highlighted the predictive value of intratumor pDC signature in the efficacy of immune checkpoint inhibitors.

6-Sulfo LacNAc monocytes are quantitatively and functionally disturbed in systemic sclerosis patients.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis, microangiopathy, and autoantibodies. We previously reported that circulating follicular helper T (cTfh) cells are increased in SSc and induce plasmablast differentiation. However, mechanisms leading to cTfh cell expansion and activation in SSc remain to be established. Tfh cells require IL-12 for their expansion and differentiation. 6-Sulfo LacNAc monocytes (slanMo), a subset of monocytes, have a higher capacity to produce IL-12 and to induce CD4+ T cell proliferation in comparison with dendritic cells (DC) or classical monocytes. The aim of this study was to perform a quantitative and functional analysis of monocytes and DC and to correlate them with cTfh cell expansion and clinical manifestations in SSc. Using flow cytometry, we analyzed different monocyte subsets including slanMo and DC from 36 SSc patients and 26 healthy controls (HC). In vitro culture experiments of sorted slanMo were performed for functional analysis and cytokine production. We observed that slanMo, intermediate and non-classical monocytes were increased in SSc in comparison with HC. Furthermore, the increase in slanMo cells was more potent in patients with diffuse SSc. We observed a significant positive correlation between slanMo and cTfh cell levels in SSc patients but not in HC. Other monocyte subsets did not correlate with cTfh cell expansion. In addition, we observed that in vitro, slanMo cells from SSc patients produced less IL-12 than slanMo from HC. SlanMo are increased in SSc and may participate in the activation of cTfh cells in SSc.

TFH cells in systemic sclerosis.

Systemic sclerosis is an autoimmune disease characterized by excessive dermal fibrosis with progression to internal organs, vascular impairment and immune dysregulation evidenced by the infiltration of inflammatory cells in affected tissues and the production of auto antibodies. While the pathogenesis remains unclear, several data highlight that T and B cells deregulation is implicated in the disease pathogenesis. Over the last decade, aberrant responses of circulating T follicular helper cells, a subset of CD4 T cells which are able to localise predominantly in the B cell follicles through a high level of chemokine receptor CXCR5 expression are described in pathogenesis of several autoimmune diseases and chronic graft-versus-host-disease. In the present review, we summarized the observed alteration of number and frequency of circulating T follicular helper cells in systemic sclerosis. We described their role in aberrant B cell activation and differentiation though interleukine-21 secretion. We also clarified T follicular helper-like cells involvement in fibrogenesis in both human and mouse model. Finally, because T follicular helper cells are involved in both fibrosis and autoimmune abnormalities in systemic sclerosis patients, we presented the different strategies could be used to target T follicular helper cells in systemic sclerosis, the therapeutic trials currently being carried out and the future perspectives from other auto-immune diseases and graft-versus-host-disease models.

Regulatory B cell imbalance correlates with Tfh expansion in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease with fibrosis, microangiopathy and immune dysfunction. B cell abnormalities characterised by autoantibody production and polyclonal B cell activation play an important role in the pathogenesis of SSc. We previously identified an expansion of functional and activated circulating T follicular helper (cTfh) cells in SSc patients. The aim of this study was to analyse the frequency of regulatory B (Breg) cell subsets and the correlation with Tfh in SSc patients. METHODS: Circulating Breg cells CD24hiCD38hi and CD27+CD24hi levels and cTfh cells CD4+CXCR5+PD1+ were determined by cytometry in 50 SSc patients and 32 healthy subjects. RESULTS: The frequency of Breg cells CD24hiCD38hi and CD24hiCD27+ was significantly reduced in patients with SSc as compared to controls (p=0.02 and p<0.001, respectively). In contrast, when examining the CD21low B cell subset, the frequency was significantly increased in SSc patients compared to healthy controls, (p<0.001). There was no difference in Breg cell levels in patients with diffuse SSc and limited SSc. However, CD24hiCD27+ Breg cell frequency was significantly decreased in SSc patients with pulmonary arterial hypertension (p=0.014), but not in patients with interstitial lung disease (p=0.058). Furthermore, we observed a negative correlation between cTfh and CD24hiCD27+ Breg cell levels in SSc patients but not in healthy controls (p=0.02). CONCLUSIONS: These results suggest that Breg cell subsets may participate in the regulation of cTfh and disease severity. Decreased CD24hiCD27+ Breg cell frequency may contribute to the development of SSc.

Clonal haematopoiesis is increased in early onset in systemic sclerosis.

OBJECTIVES: SSc is an autoimmune disease characterized by fibrosis, microangiopathy and immune dysfunctions including dysregulation of proinflammatory cytokines. Clonal haematopoiesis of indeterminate potential (CHIP) is defined by the acquisition of somatic mutations in haematopoietic stem cells leading to detectable clones in the blood. Recent data have shown a higher risk of cardiovascular disease in patients with CHIP resulting from increased production of proinflammatory cytokines and accelerated atherosclerosis. Eventual links between CHIP and autoimmune diseases are undetermined. The aim of our study was to evaluate the prevalence of CHIP in SSc patients and its association with clinical phenotype. METHODS: Forty-one genes frequently mutated in myeloid malignancies were sequenced in peripheral blood mononuclear cells from 90 SSc patients and 44 healthy donors. RESULTS: A total of 15 somatic variants were detected in 13/90 SSc patients (14%) and four somatic variants in 4/44 (9%) healthy donors (HD) (P = 0.58). The prevalence of CHIP was significantly higher in younger SSc patients than in HD: 25% (6/24) vs 4% (1/26) (P = 0.045) under 50 years and 17% (7/42) vs 3% (1/38) (P = 0.065) under 60 years. The prevalence of CHIP in patients over 70 years was similar in SSc patients and healthy donors. The most common mutations occurred in DNMT3A (seven variants). No major clinical differences were observed between SSc patients with or without CHIP. CONCLUSION: Whether CHIP increases the risk to develop SSc or is a consequence of a SSc-derived modified bone marrow micro-environment remains to be explored.

Distinct prognostic value of circulating anti-telomerase CD4+ Th1 immunity and exhausted PD-1+/TIM-3+ T cells in lung cancer.

BACKGROUND: Despite the critical roles of Th1-polarised CD4+ T cells in cancer immunosurveillance, the translation of their potential to clinical use remains challenging. Here, we investigate the clinical relevance of circulating antitumor Th1 immunity in non-small cell lung cancer (NSCLC). METHODS: The circulating antitumor Th1 response was assessed by the ELISpot assay in 170 NSCLC patients using a mixture of HLA class II-restricted peptides from telomerase (TERT). Phenotyping of blood immune cells was performed by flow cytometry. RESULTS: TERT-reactive CD4 T-cell response was detected in 35% of NSCLC patients before any treatment. Functional analysis showed that these cells were effector memory and Th1 polarised capable to produce effector cytokines, such as IFN-γ, TNF-α and IL-2. The presence of anti-TERT Th1 response was inversely correlated with the level of exhausted PD-1+/TIM-3+CD4 T cells. The level of these two immune parameters differentially affected the survival, so that increased level of anti-TERT Th1 response and low rate of exhausted PD-1+TIM-3+CD4+ T cells were associated with a better prognosis. CONCLUSIONS: Systemic anti-TERT Th1 response plays a strong antitumor protective role in NSCLC. This study underlines the potential interest of monitoring circulating antitumor Th1 response for patients' stratification and therapy decision.

Circulating follicular helper T cells are increased in systemic sclerosis and promote plasmablast differentiation through the IL-21 pathway which can be inhibited by ruxolitinib.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterised by widespread fibrosis, microangiopathy and autoantibodies. Follicular helper T (Tfh) cells CD4+CXCR5+PD-1+ cooperate with B lymphocytes to induce the differentiation of plasmocytes secreting immunoglobulins (Ig). Circulating Tfh (cTfh) cells are increased in several autoimmune diseases. However, there are no data about cTfh cells and their interaction with B cells in SSc. The aim of this study was to perform a quantitative and functional analysis of cTfh cells in SSc. METHODS: Using flow cytometry, we analysed cTfh cells from 50 patients with SSc and 32 healthy controls (HC). In vitro coculture experiments of sorted cTfh and B cells were performed for functional analysis. IgG and IgM production were measured by ELISA. RESULTS: We observed that cTfh cell numbers are increased in patients with SSc compared with HC. Furthermore, the increase in cTfh cells was more potent in patients with severe forms of SSc such as diffuse SSc and in the presence of arterial pulmonary hypertension. cTfh cells from patients with SSc present an activated Tfh phenotype, with high expression of BCL-6, increased capacity to produce IL-21 in comparison with healthy controls. In vitro, cTfh cells from patients with SSc had higher capacity to stimulate the differentiation of CD19+CD27+CD38hi B cells and their secretion of IgG and IgM through the IL-21 pathway than Tfh cells from healthy controls. Blocking IL-21R or using the JAK1/2 inhibitor ruxolitinib reduced the Tfh cells' capacity to stimulate the plasmablasts and decreased the Ig production. CONCLUSIONS: Circulating Tfh cells are increased in SSc and correlate with SSc severity. The IL-21 pathway or JAK1/2 blockade by ruxolitinib could be a promising strategy in the treatment of SSc.

Rituximab-based first-line treatment of cGVHD after allogeneic SCT: results of a phase 2 study.

Chronic graft-versus-host disease (cGVHD) is the main cause of late nonrelapse mortality and morbidity after allogeneic stem cell transplantation (allo-SCT). To improve such patients' outcomes, we conducted a phase 2, prospective, multicenter trial to test the efficacy of the addition of rituximab to corticosteroids (CSs) and cyclosporine A (CsA) as first-line therapy for newly diagnosed cGVHD after allo-SCT. Twenty-four patients (median age, 47 years) with mild (n = 2), moderate (n = 7), or severe (n = 15) cGVHD were included. All patients received rituximab 375 mg/m2 weekly for 4 weeks, followed by a second course 1 month later for patients with partial response. Twenty of 24 patients (83%) were in response at 1 year. Furthermore, among 19 evaluable patients, 14 (74%) were off CSs. The estimated 1-year overall survival was 83%, and the 1-year cumulative incidence of nonrelapse mortality was 14%. One patient died of progressive multifocal leukoencephalopathy. Although PD-L1hi naive B cells were significantly decreased at diagnosis of cGVHD, they increased after anti-CD20 B-cell depletion. In contrast, activated ICOShi PD-1hi circulating T follicular helper (Tfh) cells decreased after rituximab treatment. Overall, the addition of rituximab to corticosteroid and CsA appeared to be safe and effective for first-line treatment of cGVHD. Furthermore, our data suggest that this efficacy may be in part related to an effect on PD-L1hi B cells and Tfh cells. This study was registered at www.clinicaltrials.gov as identifier NCT01135641.

Larger number of invariant natural killer T cells in PBSC allografts correlates with improved GVHD-free and progression-free survival.

We studied the impact of a set of immune cells contained within granulocyte colony-stimulating factor-mobilized peripheral blood stem cell grafts (naïve and memory T-cell subsets, B cells, regulatory T cells, invariant natural killer T cells [iNKTs], NK cells, and dendritic cell subsets) in patients (n = 80) undergoing allogeneic stem cell transplantation (SCT), using the composite end point of graft-versus-host disease (GVHD)-free and progression-free survival (GPFS) as the primary end point. We observed that GPFS incidences in patients receiving iNKT doses above and below the median were 49% vs 22%, respectively (P= .007). In multivariate analysis, the iNKT dose was the only parameter with a significant impact on GPFS (hazard ratio = 0.48; 95% confidence interval, 0.27-0.85;P= .01). The incidences of severe grade III to IV acute GVHD and National Institutes of Health grade 2 to 3 chronic GVHD (12% and 16%, respectively) were low and associated with the use of antithymocyte globulin in 91% of patients. No difference in GVHD incidence was reported according to the iNKT dose. In conclusion, a higher dose of iNKTs within the graft is associated with an improved GPFS. These data may pave the way for prospective and active interventions aiming to manipulate the graft content to improve allo-SCT outcome.

A bit of sweetness for GVHD prevention.

In this issue of Blood, Parmar et al report on preclinical data suggesting that use of ex vivo fucosylated third-party human regulatory T cells (Tregs) could be an effective strategy for prevention of graft-versus-host disease (GVHD).

Human monocyte-derived suppressor cells control graft-versus-host disease by inducing regulatory forkhead box protein 3-positive CD8+ T lymphocytes.

BACKGROUND: Adoptive transfer of immunosuppressive cells has emerged as a promising strategy for the treatment of immune-mediated disorders. However, only a limited number of such cells can be isolated from in vivo specimens. Therefore efficient ex vivo differentiation and expansion procedures are critically needed to produce a clinically relevant amount of these suppressive cells. OBJECTIVE: We sought to develop a novel, clinically relevant, and feasible approach to generate ex vivo a subpopulation of human suppressor cells of monocytic origin, referred to as human monocyte-derived suppressive cells (HuMoSCs), which can be used as an efficient therapeutic tool to treat inflammatory disorders. METHODS: HuMoSCs were generated from human monocytes cultured for 7 days with GM-CSF and IL-6. The immune-regulatory properties of HuMoSCs were investigated in vitro and in vivo. The therapeutic efficacy of HuMoSCs was evaluated by using a graft-versus-host disease (GvHD) model of humanized mice (NOD/SCID/IL-2Rγc(-/-) [NSG] mice). RESULTS: CD33+ HuMoSCs are highly potent at inhibiting the proliferation and activation of autologous and allogeneic effector T lymphocytes in vitro and in vivo. The suppressive activity of these cells depends on signal transducer and activator of transcription 3 activation. Of therapeutic relevance, HuMoSCs induce long-lasting memory forkhead box protein 3-positive CD8+ regulatory T lymphocytes and significantly reduce GvHD induced with human PBMCs in NSG mice. CONCLUSION: Ex vivo-generated HuMoSCs inhibit effector T lymphocytes, promote the expansion of immunosuppressive forkhead box protein 3-positive CD8+ regulatory T cells, and can be used as an efficient therapeutic tool to prevent GvHD.

Peripheral Blood Plasmacytoid Dendritic Cells at Day 100 Can Predict Outcome after Allogeneic Stem Cell Transplantation.

The rapidly increasing use of allogeneic stem cell transplantation (allo-SCT) emphasizes the need for identifying variables predictive of its outcome. Plasmacytoid dendritic cells (pDCs) play a major role in establishing immune competence and in several autoimmune diseases. Thus, we investigated whether pDCs might influence the outcome of patients after allo-SCT in 79 consecutive patients who underwent this procedure. pDCs were identified in the blood of patients at day 100 after allo-SCT by staining peripheral blood mononuclear cells for surface markers and intracellular cytokines and analyzing them on a flow cytometer. We found the pDC level at day 100 was not influenced by patient or graft characteristics, and only the absence of previous grades II to IV acute graft-versus-host disease was significantly associated with higher levels of blood pDCs after allo-SCT (OR, .67; 95% CI, .54 to .83; P = .0004). Using the median value of pDCs at day 100 to divide the patients into 2 distinct groups, we observed that a low pDC level was correlated with a worse overall survival (55% versus 86%, P = .007). In a multivariate analysis, only low pDC level (OR, 3.41; 95% CI, 1.19 to 9.79; P = .02) and older patient age (OR, 5.16; 95% CI, 1.15 to 23.14; P = .03) were significantly predictive of increased risk of death. We conclude that monitoring of pDC may be useful for patient management and may have a significant impact on the probability of a favorable outcome of allo-SCT.

Impact of donor hematopoietic cells mobilized with G-CSF and plerixafor on murine acute graft-versus-host-disease.

BACKGROUND AIMS: This study aimed to characterize the immune effectors contained in the grafts from donor mice mobilized by granulocyte colony-stimulating factor (G-CSF) and plerixafor and to evaluate their impact on the development of acute graft-versus-host-disease (aGVHD). METHODS: Mobilization was done with G-CSF alone or G-CSF plus plerixafor (G+P). RESULTS: In grafts collected after G+P mobilization, we observed a significantly higher proportion of c-kit(+)Sca-1(+) hematopoietic stem cells compared with G-CSF. A significant increase in the percentage of plasmacytoid dendritic cells was detected in the G+P graft compared with G-CSF graft. We also studied the ability of stem cell grafts mobilized with G+P to induce GVHD in a mouse model. We observed higher mortality (P < 0.001) associated with increased aGVHD clinical score (P < 0.0001) as well as higher pathology score in the intestine of mice receiving G+P as compared with G-CSF grafts (P < 0.001). Moreover, the exacerbated aGVHD severity was associated with upregulation of CCR6 expression on both CD4(+) and CD8(+) T cells from the G+P grafts, as well as on T cells from mice transplanted with G+P grafts. CONCLUSIONS: In conclusion, we showed that grafts mobilized with G+P exhibited functional features different from those mobilized with G-CSF alone, which increase the severity of aGVHD in the recipients.

Increased regulatory T-cell numbers are associated with farm milk exposure and lower atopic sensitization and asthma in childhood.

BACKGROUND: European cross-sectional studies have suggested that prenatal and postnatal farm exposure decreases the risk of allergic diseases in childhood. Underlying immunologic mechanisms are still not understood but might be modulated by immune-regulatory cells early in life, such as regulatory T (Treg) cells. OBJECTIVE: We sought to assess whether Treg cells from 4.5-year-old children from the Protection against Allergy: Study in Rural Environments birth cohort study are critical in the atopy and asthma-protective effect of farm exposure and which specific exposures might be relevant. METHODS: From 1133 children, 298 children were included in this study (149 farm and 149 reference children). Detailed questionnaires until 4 years of age assessed farming exposures over time. Treg cells were characterized as upper 20% CD4(+)CD25(+) forkhead box protein 3 (FOXP3)(+) (intracellular) in PBMCs before and after stimulation (with phorbol 12-myristate 13-acetate/ionomycin or LPS), and FOXP3 demethylation was assessed. Atopic sensitization was defined by specific IgE measurements; asthma was defined by a doctor's diagnosis. RESULTS: Treg cells were significantly increased in farm-exposed children after phorbol 12-myristate 13-acetate/ionomycin and LPS stimulation. Exposure to farm milk was defined as a relevant independent farm-related exposure supported by higher FOXP3 demethylation. Treg cell (upper 20% CD4(+)CD25(+), FOXP3(+) T cells) numbers were significantly negatively associated with doctor-diagnosed asthma (LPS stimulated: adjusted odds ratio, 0.26; 95% CI, 0.08-0.88) and perennial IgE (unstimulated: adjusted odds ratio, 0.21; 95% CI, 0.08-0.59). Protection against asthma by farm milk exposure was partially mediated by Treg cells. CONCLUSIONS: Farm milk exposure was associated with increased Treg cell numbers on stimulation in 4.5-year-old children and might induce a regulatory phenotype early in life, potentially contributing to a protective effect for the development of childhood allergic diseases.

Effects of bortezomib on the immune system: a focus on immune regulation.

Bortezomib, the first-in-class proteasome inhibitor, has become one of the standard treatments in multiple myeloma. The agent is thought to exert its antimyeloma effects through the inhibition of NF-κB. However, evidence suggests that bortezomib also affects additional cell survival pathways, such as the p44/42 mitogen-activated protein kinase pathway, and inhibitory effects on IL-6, TNF-α, and vascular endothelial growth factor have also been demonstrated. These diverse effects have prompted the investigation of bortezomib's activity in various immune and inflammatory processes. This review summarizes the data reported with bortezomib in the prevention of graft-versus-host disease, antibody-mediated graft rejection, and anti-angiogenesis and in the treatment of rheumatoid arthritis, multiple sclerosis, and other inflammatory diseases. The positive results obtained suggest a role for bortezomib in these different indications, and therefore further investigations are warranted.

Characterization of peripheral blood stem cell grafts mobilized by granulocyte colony-stimulating factor and plerixafor compared with granulocyte colony-stimulating factor alone.

BACKGROUND AIMS: This study aimed to characterize the immune effectors contained in apheresis samples obtained from patients with grafts mobilized with plerixafor and granulocyte colony-stimulating factor (G-CSF) (P+G) compared with grafts mobilized with G-CSF alone (G). METHODS: Aliquots of apheresis samples were obtained from 36 patients with malignant diseases after mobilization with G (n = 18) or P+G (n = 18). The phenotype and cytokine secretion profile of T cell and dendritic cell subsets were characterized by multicolor cytometry including intracellular cytokine staining. RESULTS: In grafts collected after mobilization with P+G, there was a significantly higher percentage of CD3(+) T cells compared with samples collected after mobilization with G alone. On a functional level, a significant increase of interferon-γ and tumor necrosis factor-α secreting CD8(+) T cells was observed in the P+G group compared with the G group. CD4(+)Foxp3(+) regulatory T cells were similar in both groups but exhibited a lower expression of inducible costimulatory molecule and a significantly higher expression of CD127 in the P+G group. Myeloid dendritic cells (MDCs) and BDCA3(+) dendritic cells were similar in both groups. In contrast, plasmacytoid dendritic cells (PDCs) (CD123(+)BDCA2(+)HLA-DR(+)) were significantly increased in the P+G grafts, leading to a higher PDC-to-MDC ratio. PDCs mobilized by P+G displayed different functional markers--a higher percentage of ILT7(+) PDCs and decreased expression of CD86--suggesting a potential regulatory capacity of PDCs mobilized by P+G. CONCLUSIONS: Grafts mobilized with P+G exhibited major different functional features compared with grafts mobilized with G alone, suggesting that such grafts may have an impact on patient outcome after autologous stem cell transplantation.

Plasmacytoid dendritic cells play a major role in apoptotic leukocyte-induced immune modulation.

Several APCs participate in apoptotic cell-induced immune modulation. Whether plasmacytoid dendritic cells (PDCs) are involved in this process has not yet been characterized. Using a mouse model of allogeneic bone marrow engraftment, we demonstrated that donor bone marrow PDCs are required for both donor apoptotic cell-induced engraftment and regulatory T cell (Treg) increase. We confirmed in naive mice receiving i.v. syngeneic apoptotic cell infusion that PDCs from the spleen induce ex vivo Treg commitment. We showed that PDCs did not interact directly with apoptotic cells. In contrast, in vivo macrophage depletion experiments using clodronate-loaded liposome infusion and coculture experiments with supernatant from macrophages incubated with apoptotic cells showed that PDCs required macrophage-derived soluble factors--including TGF-ß--to exert their immunomodulatory functions. Overall, PDCs may be considered as the major APC involved in Treg stimulation/generation in the setting of an immunosuppressive environment obtained by apoptotic cell infusion. These findings show that like other APCs, PDC functions are influenced, at least indirectly, by exposure to blood-borne apoptotic cells. This might correspond with an additional mechanism preventing unwanted immune responses against self-antigens clustered at the cell surface of apoptotic cells occurring during normal cell turnover.

TGF-beta-exposed plasmacytoid dendritic cells participate in Th17 commitment.

TGF-ß is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-ß on pDC, and to our knowledge, no study has assessed the capacity of TGF-ß-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-ß-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-ß/Smad signal, because TGF-ß treatment induced Smad2 phosphorylation in pDC and blockade of TGF-ß signaling with the SD208 TGF-ßRI kinase inhibitor abrogated Th17 commitment induced by TGF-ß-treated pDC. Moreover, TGF-ß mRNA synthesis and active TGF-ß release were induced in TGF-ß-treated pDC and anti-TGF-ß Ab blocked Th17 commitment. Unexpectedly, TGF-ß treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-ß-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-ß-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-ß-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-ß-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.

Pooled allogeneic faecal microbiota MaaT013 for steroid-resistant gastrointestinal acute graft-versus-host disease: a single-arm, multicentre phase 2 trial.

Background: Failure of gastrointestinal acute graft-versus-host disease (GI-aGvHD) to respond to steroid therapy is associated with limited further therapeutic options. We aimed to assess the safety and efficacy of the first-in-human use of the pooled allogeneic faecal microbiota, MaaT013, for the treatment of steroid-refractory GI-aGvHD. Methods: This prospective, international, single-arm, phase 2a study reports clinical outcomes from a 24-patient cohort with grade III-IV, steroid refractory GI-aGvHD treated with the pooled allogeneic faecal microbiota MaaT013. MaaT013 involved pooling faecal matter from 3 to 8 screened donors then transplanting the pooled batches into patients to treat GI-aGVHD. The 24 patients were treated in the HERACLES study (Aug 2018 to Nov 2020) at 26 sites in Europe and an additional 52 patients were treated in a compassionate use/expanded access program (EAP) in France (July 2018 to April 2021). The primary endpoint was GI response at day 28, defined as the proportion of patients with GI-aGvHD who had a complete response (CR) or very good partial response (VGPR). GvHD grading and staging were assessed according to the revised Glucksberg criteria. Adverse events and severe adverse events were monitored for 6 months and 12 months, respectively. The HERACLES study was registered with ClinicalTrials.gov (NCT03359980). Findings: Compared with single donors, MaaT013 is characterised by higher microbial richness and reduced variability across batches. At day 28 (D28), the GI-overall response rate (ORR) was 38% in the prospective population, including 5 complete responses (CR), 2 very good partial responses (VGPR) and 2 partial responses (PR). In the EAP, the GI-ORR was 58% (17 CR, 9 VGPR and 4 PR). The 12-month overall survival (OS) was 25% in the prospective study and 38% in the EAP. Regarding safety, five infectious complications, including 3 sepsis, could not be excluded from being related to the study procedure in HERACLES. Shotgun sequencing analyses of the identified strains suggest that none were found in MaaT013. In the EAP, 18 pharmacovigilance cases were reported among 52 treated patients, including 11 bacteraemia/sepsis. In HERACLES, we observed in stools from responding patients at D28 a higher microbiota richness and increased levels of beneficial bacteria, in particular butyrate producers, along with increased levels of short-chain fatty acid and bile acids. In contrast, stools from non-responding (NR) patients displayed increased levels of pathogenic pro-inflammatory bacteria along with increased systemic inflammatory parameters. Interpretation: Overall, MaaT013 was safe in this population of highly immunocompromised patients and was associated with responses in some patients with GI-aGvHD and deserves further investigation. Funding: MaaT Pharma.