Actin dynamics regulation by TTC7A/PI4KIIIα limits DNA damage and cell death under confinement.

BACKGROUND: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. OBJECTIVES: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. METHODS: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. RESULTS: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. CONCLUSIONS: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.

DOCK11 deficiency in patients with X-linked actinopathy and autoimmunity.

Dedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho-guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients' autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients' lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.

Ttc7a regulates hematopoietic stem cell functions while controlling the stress-induced response.

The molecular machinery that regulates the balance between self-renewal and differentiation properties of hematopoietic stem cells (HSC) has yet to be characterized in detail. Here we found that the tetratricopeptide repeat domain 7 A (Ttc7a) protein, a putative scaffold protein expressed by HSC, acts as an intrinsic regulator of the proliferative response and the self-renewal potential of murine HSC in vivo Loss of Ttc7a consistently enhanced the competitive repopulating ability of HSC and their intrinsic capacity to replenish the hematopoietic system after serial cell transplantations, relative to wildtype cells. Ttc7a-deficient HSC exhibit a different transcriptomic profile for a set of genes controlling the cellular response to stress, which was associated with increased proliferation in response to chemically induced stress in vitro and myeloablative stress in vivo Our results therefore revealed a previously unrecognized role of Ttc7a as a critical regulator of HSC stemness. This role is related, at least in part, to regulation of the endoplasmic reticulum stress response.

Mesenchymal stem cells and their immunosuppressive role in transplantation tolerance.

Since they were first described, mesenchymal stem cells (MSCs) have been shown to have important effector mechanisms and the potential for use in cell therapy. A great deal of research has been focused on unveiling how MSCs contribute to anti-inflammatory responses, including describing several cell populations involved and identifying soluble and other effector molecules. In this review, we discuss some of the contemporary evidence for use of MSCs in the field of immune tolerance, with a special emphasis on transplantation. Although considerable effort has been devoted to understanding the biological function of MSCs, additional resources are required to clarify the mechanisms of their induction of immune tolerance, which will undoubtedly lead to improved clinical outcomes for MSC-based therapies.

IL-33 enhances retinoic acid signaling on CD4+ T cells.

Several molecules have been described as CD4+ T cells differentiation modulators and among them retinoic acid (RA) and more recently, IL-33, have been studied. Due to the similarities in T helper cell skewing properties between RA and IL-33, we asked whether IL-33 intersects, directly or indirectly, the RA signaling pathway. Total CD4+ T cells from DR5-luciferase mice were activated in the presence of RA with or without IL-33, and RA signaling was visualized using ex vivo imaging. Our results demonstrate that IL-33 itself is able to trigger RA signaling on CD4+ T cells, which is highly increased when IL-33 is added in conjunction with RA. This study presents IL-33 as a potential player that may synergize with RA in controlling T cell differentiation, and suggests that IL-33 may be an attractive target in controlling T cell differentiation in vivo.

Exogenous interleukin-33 targets myeloid-derived suppressor cells and generates periphery-induced Foxp3⁺ regulatory T cells in skin-transplanted mice.

Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4(+) T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3(+) regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4(+) T-cell expansion and conversion of CD4(+)  Foxp3(-) T cells into CD4(+)  Foxp3(+) Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3(+) Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.

Rapamycin-conditioned dendritic cells activated with monophosphoryl lipid-A promote allograft acceptance in vivo.

AIM: To date, there is no human dendritic cell (DC) based therapy to prevent allograft rejection in transplanted patients. Here, we evaluate a potential protocol using a murine in vivo transplant model. MATERIALS & METHODS: We generated murine bone marrow-derived DCs (BM-DCs), modulated with rapamycin (Rapa) and activated with monophosphoryl lipid A (Rapamycin-treated and monophosphoryl lipid A-matured DCs [Rapa-mDCs]). DCs phenotype was evaluated by flow cytometry, cytokine production by ELISA and their T-cell stimulatory ability was tested in co-cultures with CD4(+) T cells. Using an in vivo skin graft model, we evaluated DCs tolerogenicity. RESULTS: In vitro, Rapa-mDCs exhibit a semi-mature phenotype given by intermediate levels of co-stimulatory molecules and cytokines, and inhibit CD4(+) T-cell proliferation. In vivo, skin-grafted mice treated with Rapa-mDCs show high allograft survival, accumulation of Foxp3(+) Tregs and cytokine pattern modification. CONCLUSION: Rapa-mDCs re-educate the inflammatory microenvironment, promoting skin-allograft survival.

Retinaldehyde dehydrogenase activity is triggered during allograft rejection and it drives Th1/Th17 cytokine production.

Retinoic acid (RA), a vitamin A metabolite, has been attributed to relevant functions in adaptive immunity. On T cells, the disruption on RA signaling alters both CD4+ and CD8+ T cells effector function. In this study, we evaluated the contribution of RA synthesis during the immune response using an in vivo skin transplantation model. Our data indicates that the frequency and number of cells containing an active retinaldehyde dehydrogenase (RALDH), a key enzyme for RA synthesis, is increased during skin transplant rejection. In addition, we found that the expression of the mRNA coding for the isoform RALDH2 is up-regulated on graft rejecting draining lymph nodes (dLNs) cells. Lastly, we observed that IFN-γ and IL-17 production by ex vivo re-stimulated dLNs cells is greatly increased during rejection, which it turns depends on RA synthesis, as shown in experiments using a specific RALDH inhibitor. Altogether, our data demonstrate that RA synthesis is incremented during the immune response against an allograft, and also indicates that the synthesis of RA is required for cytokine production by dLNs resident T cells.

Neuropilin-1+ regulatory T cells promote skin allograft survival and modulate effector CD4+ T cells phenotypic signature.

During allograft rejection, several immune cell types, including dendritic cells, CD4(+) and CD8(+) T cells among others, recirculate between the graft and the nearest draining lymph node, resulting in immunity against the 'foreign' tissue. Regulatory CD4(+) T cells are critical for controlling the magnitude of the immune response and may act to promote or maintain tolerance. They are characterized by the expression of CD25 and Foxp3, and more recently, Neuropilin-1 (Nrp1). The role of these suppressor cells during allograft rejection is not well understood. Our work shows that during graft rejection, there is an increase in the frequency of total CD4(+) T cells expressing Nrp1, but the expression of this molecule is downregulated in the regulatory CD4(+) T-cell compartment. Interestingly, the expression of the transcription factor Eos, which renders cell function stability, is also reduced. In adoptive transfer experiments, we observed that during allograft rejection: (i) natural regulatory CD4(+) T cells maintain high levels of Nrp1 expression, (ii) effector CD4(+) T cells (Nrp1(-)) become Nrp1(+)Eos(+) and (iii) the transfer of regulatory CD4(+) T cells (Nrp1(+)) can promote allograft survival, and also enhance the gain of Nrp1 and Eos on T-effector cells. Together, these data suggest that rejection occurs, at least in part, through the loss of Nrp1 expression on regulatory CD4(+) T cells, their stability or both. Additionally, the transfer of regulatory CD4(+) T cells (based on Nrp1 expression) permits the acceptance of the allograft, placing Nrp1 as a new target for immune therapy.

Neuropilin-1 in transplantation tolerance.

In the immune system, Neuropilin-1 (Nrp1) is a molecule that plays an important role in establishing the immunological synapse between dendritic cells (DCs) and T cells. Recently, Nrp1 has been identified as a marker that seems to distinguish natural T regulatory (nTreg) cells, generated in the thymus, from inducible T regulatory (iTreg) cells raised in the periphery. Given the crucial role of both nTreg and iTreg cells in the generation and maintenance of immune tolerance, the ability to phenotypically identify each of these cell populations in vivo is needed to elucidate their biological properties. In turn, these properties have the potential to be developed for therapeutic use to promote immune tolerance. Here we describe the nature and functions of Nrp1, including its potential use as a therapeutic target in transplantation tolerance.