ADAM17 Deletion in Thymic Epithelial Cells Alters Aire Expression without Affecting T Cell Developmental Progression

Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs) are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach.We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted.In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified

Regulatory T cells limit induction of protective immunity and promote immune pathology following intestinal helminth infection

Foxp3+ regulatory T cells (Tregs) have a well-characterized role in limiting autoimmunity and dampening deleterious immune responses. However, a potential consequence of the immunosuppressive function of Tregs can be the limitation of protective immunity to infectious pathogens. Parasitic infections are a potent stimulus for the generation of Treg responses, which may be beneficial to both the parasite and the host by promoting persistence of infection and limiting immune-mediated pathology, respectively. In this study, we explore the functional role of Tregs post–low-dose infection with the intestinal helminth parasite Trichuris muris, which yields a chronic infection because of inefficient induction of Th2 responses. Early Treg depletion postinfection resulted in expedited worm clearance, and was associated with reduced Th1-mediated inflammation of the intestinal environment. Interestingly, this protective immunity was lost, and worm burden enhanced if Tregs were depleted later once the infection was established. Early and late Treg depletion resulted in enhanced Th2 and reduced Th1 cytokine and humoral responses. Blockade of the Th2 cytokine IL-4 resulted in a moderate increase in Th1 but had no effect on worm burden. Our findings suggest that Tregs preferentially limit Th2 cell expansion, which can impact infections where clear immune polarity has not been established. Thus, the impact of Treg depletion is context and time dependent, and can be beneficial to the host in situations where Th1 responses should be limited in favor of Th2 responses

Engineering of Saposin C Protein Chimeras for Enhanced Cytotoxicity and Optimized Liposome Binding Capability

Saposin C (sapC) is a lysosomal, peripheral-membrane protein displaying liposome fusogenic capabilities. Proteoliposomes of sapC and phosphatidylserine have been shown to be toxic for cancer cells and are currently on clinical trial to treat glioblastoma. As proof-of-concept, we show two strategies to enhance the applications of sapC proteoliposomes: (1) Engineering chimeras composed of sapC to modulate proteoliposome function; (2) Engineering sapC to modify its lipid binding capabilities. In the chimera design, sapC is linked to a cell death-inducing peptide: the BH3 domain of the Bcl-2 protein PUMA. We show by solution NMR and dynamic light scattering that the chimera is functional at the molecular level by fusing liposomes and by interacting with prosurvival Bcl-xL, which is PUMA’s known mechanism to induce cell death. Furthermore, sapC-PUMA proteoliposomes enhance cytotoxicity in glioblastoma cells compared to sapC. Finally, the sapC domain of the chimera has been engineered to optimize liposome binding at pH close to physiological values as protein–lipid interactions are favored at acidic pH in the native protein. Altogether, our results indicate that the properties of sapC proteoliposomes can be modified by engineering the protein surface and by the addition of small peptides as fusion constructs

CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8+ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8+ T cell (Tc1) response

CD8 Follicular T Cells Promote B Cell Antibody Class Switch in Autoimmune Disease

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality

Globule Leukocytes and Other Mast Cells in the Mouse Intestine

Only 2 major mast cell (MC) subtypes are commonly recognized in the mouse: the large connective tissue mast cells (CTMCs) and the mucosal mast cells (MMCs). Interepithelial mucosal inflammatory cells, most commonly identified as globule leukocytes (GLs), represent a third MC subtype in mice, which we term interepithelial mucosal mast cells (ieMMCs). This term clearly distinguishes ieMMCs from lamina proprial MMCs (lpMMCs) while clearly communicating their common MC lineage. Both lpMMCs and ieMMCs are rare in normal mouse intestinal mucosa, but increased numbers of ieMMCs are seen as part of type 2 immune responses to intestinal helminth infections and in food allergies. Interestingly, we found that increased ieMMCs were consistently associated with decreased mucosal inflammation and damage, suggesting that they might have a role in controlling helminth-induced immunopathology. We also found that ieMMC hyperplasia can develop in the absence of helminth infections, for example, in Treg-deficient mice, Arf null mice, some nude mice, and certain graft-vs-host responses. Since tuft cell hyperplasia plays a critical role in type 2 immune responses to intestinal helminths, we looked for (but did not find) any direct relationship between ieMMC and tuft cell numbers in the intestinal mucosa. Much remains to be learned about the differing functions of ieMMCs and lpMMCs in the intestinal mucosa, but an essential step in deciphering their roles in mucosal immune responses will be to apply immunohistochemistry methods to consistently and accurately identify them in tissue sections

IL-17A Increases Doxorubicin Efficacy in Triple Negative Breast Cancer

Due to lack of targetable receptors and intertumoral heterogeneity, triple negative breast cancer (TNBC) remains particularly difficult to treat. Doxorubicin (DOX) is typically used as nonselective neoadjuvant chemotherapy, but the diversity of treatment efficacy remains unclear. Comparable to variability in clinical response, an experimental model of TNBC using a 4T1 syngeneic mouse model was found to elicit a differential response to a seven-day treatment regimen of DOX. Single-cell RNA sequencing identified an increase in T cells in tumors that responded to DOX treatment compared to tumors that continued to grow uninhibited. Additionally, compared to resistant tumors, DOX sensitive tumors contained significantly more CD4 T helper cells (339%), γδ T cells (727%), Naïve T cells (278%), and activated CD8 T cells (130%). Furthermore, transcriptional profiles of tumor infiltrated T cells in DOX responsive tumors revealed decreased exhaustion, increased chemokine/cytokine expression, and increased activation and cytotoxic activity. γδ T cell derived IL-17A was identified to be highly abundant in the sensitive tumor microenvironment. IL-17A was also found to directly increase sensitivity of TNBC cells in combination with DOX treatment. In TNBC tumors sensitive to DOX, increased IL-17A levels lead to a direct effect on cancer cell responsiveness and chronic stimulation of tumor infiltrated T cells leading to improved chemotherapeutic efficacy. IL-17A's role as a chemosensitive cytokine in TNBC may offer new opportunities for treating chemoresistant breast tumors and other cancer types