Specific ablation of CD4+ T-cells promotes heart regeneration in juvenile mice.
ABSTRACT: Unlike adult cardiomyocytes, neonatal cardiomyocytes can readily proliferate that contributes to a transient regenerative potential after myocardial injury in mice. We have recently reported that CD4+ regulatory T-cells promote this process; however, the role of other CD4+ T-cell subsets as well as CD8+ T-cells in postnatal heart regeneration has been less studied. Methods: by comparing the regenerating postnatal day (P) 3 and the non-regenerating P8 heart after injury, we revealed the heterogeneity of CD4+ and CD8+ T-cells in the myocardium through single cell analysis. We also specifically ablated CD4+ and CD8+ T-cells using the lytic anti-CD4 and -CD8 monoclonal antibodies, respectively, in juvenile mice at P8 after myocardial injury. Results: we observe significantly more CD4+FOXP3- conventional T-cells in the P8 heart when compared to that of the P3 heart within a week after injury. Surprisingly, such a difference is not seen in CD8+ T-cells that appear to have no function as their depletion does not reactivate heart regeneration. On the other hand, specific ablation of CD4+ T-cells contributes to mitigated cardiac fibrosis and increased cardiomyocyte proliferation after injury in juvenile mice. Single-cell transcriptomic profiling reveals a pro-fibrotic CD4+ T-cell subset in the P8 but not P3 heart. Moreover, there are likely more Th1 and Th17 cells in the P8 than P3 heart. We further demonstrate that cytokines of Th1 and Th17 cells can directly reduce the proliferation and increase the apoptosis of neonatal cardiomyocytes. Moreover, ablation of CD4+ T-cells can directly or indirectly facilitate the polarization of macrophages away from the pro-fibrotic M2-like signature in the juvenile heart. Nevertheless, ablation of CD4+ T-cells alone does not offer the same protection in the adult heart after myocardial infarction, suggesting a developmental change of immune cells including CD4+ T-cells in the regulation of age-related mammalian heart repair. Conclusions: our results demonstrate that ablation of CD4+ but not CD8+ T-cells promotes heart regeneration in juvenile mice; and CD4+ T-cells play a distinct role in the regulation of heart regeneration and repair during development.
Project description:Observations in patients with ANCA-associated vasculitis suggest that CD8<sup>+</sup> T cells participate in disease, but there is no experimental functional evidence of pathologic involvement for these cells. Myeloperoxidase (MPO) is a well defined autoantigen in ANCA-associated vasculitis. Studies in experimental models of anti-MPO GN suggest that, after ANCA-induced neutrophil localization, deposited MPO within glomeruli is recognized by autoreactive T cells that contribute to injury. We tested the hypothesis that CD8<sup>+</sup> T cells mediate disease in experimental ANCA-associated vasculitis. CD8<sup>+</sup> T cell depletion in the effector phase of disease attenuated injury in murine anti-MPO GN. This protection associated with decreased levels of intrarenal IFN-?, TNF, and inflammatory chemokines and fewer glomerular macrophages. Moreover, we identified a pathogenic CD8<sup>+</sup> T cell MPO epitope (MPO<sub>431-439</sub>) and found that cotransfer of MPO<sub>431-439</sub>-specific CD8<sup>+</sup> T cell clones exacerbated disease mediated by MPO-specific CD4<sup>+</sup> cells in Rag1<sup>-/-</sup> mice. Transfer of MPO<sub>431-439</sub>-specific CD8<sup>+</sup> cells without CD4<sup>+</sup> cells mediated glomerular injury when MPO was planted in glomeruli. These results show a pathogenic role for MPO-specific CD8<sup>+</sup> T cells, provide evidence that CD8<sup>+</sup> cells are a therapeutic target in ANCA-associated vasculitis, and suggest that a molecular hotspot within the MPO molecule contains important CD8<sup>+</sup>, CD4<sup>+</sup>, and B cell epitopes.
Project description:In this study, we observe that the ischemic tissues of type-2 diabetic (T2D) patients and mice have significantly more CD8<sup>+</sup> T-cells than that of their normoglycemic counterparts, respectively. However, the role of CD8<sup>+</sup> T-cells in the pathogenesis of diabetic vascular complication has been less studied. <b>Methods</b>: We employed loss-of-function studies in mouse models using the non-lytic anti-CD8 antibody that blocks tissue infiltration of CD8<sup>+</sup> T-cells into the injured tissue. We also performed genome-wide, single-cell RNA-sequencing of CD8<sup>+</sup> T-cells to uncover their role in the pathogenesis of diabetic vascular diseases. <b>Results</b>: The vascular density is negatively correlated with the number of CD8<sup>+</sup> T-cells in the ischemic tissues of patients and mice after injury. CD8<sup>+</sup> T-cells or their supernatant can directly impair human and murine angiogenesis. Compared to normoglycemic mice that can regenerate their blood vessels after injury, T2D mice fail in this regeneration. Treatment with the CD8 checkpoint blocking antibody increases the proliferation and function of endothelial cells in both Lepr<sup>db/db</sup> mice and diet-induced diabetic <i>Cdh5-Cre;Rosa-YFP</i> lineage-tracing mice after ischemic injury<i>.</i> Furthermore, single-cell transcriptomic profiling reveals that CD8<sup>+</sup> T-cells of T2D mice showed a <i>de novo</i> cell fate change from the angiogenic, tissue-resident memory cells towards the effector and effector memory cells after injury. Functional revascularization by CD8 checkpoint blockade is mediated through unleashing such a poised lineage commitment of CD8<sup>+</sup> T-cells from T2D mice. <b>Conclusion</b>: Our results reveal that CD8<sup>+</sup> T-cell plasticity regulates vascular regeneration; and give clinically relevant insights into the potential development of immunotherapy targeting vascular diseases associated with obesity and diabetes.
Project description:Ischemia reperfusion injuries (IRI) are unavoidable in solid organ transplantation. IRI augments alloimmunity but the mechanisms involved are poorly understood. Herein, we examined the effect of IRI on antigen specific alloimmunity. We demonstrate that ischemia promotes alloimmune activation, leading to more severe histological features of rejection, and increased CD4<sup>+</sup> and CD8<sup>+</sup> T cell graft infiltration, with a predominantly CD8<sup>+</sup> IFN?<sup>+</sup> infiltrate. This process is dependent on the presence of alloreactive CD4<sup>+</sup> T cells, where depletion prevented infiltration of ischemic grafts by CD8<sup>+</sup> IFN?<sup>+</sup> T cells. IL-6 is a known driver of ischemia-induced rejection. Herein, depletion of donor antigen-presenting cells reduced ischemia-induced CD8<sup>+</sup> IFN?<sup>+</sup> allograft infiltration, and improved allograft outcomes. Following prolonged ischemia, accelerated rejection was observed despite treatment with CTLA4Ig, indicating that T cell costimulatory blockade failed to overcome the immune activating effect of IRI. However, despite severe ischemic injury, treatment with anti-IL-6 and CTLA4Ig blocked IRI-induced alloimmune injury and markedly improved allograft survival. We describe a novel pathway where IRI activates innate immunity, leading to upregulation of antigen specific alloimmunity, resulting in chronic allograft injury. Based on these findings, we describe a clinically relevant treatment strategy to overcome the deleterious effect of IRI, and provide superior long-term allograft outcomes.
Project description:N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) RNA modification, a dynamic and reversible process, is essential for tissue development and pathogenesis. However, the potential involvement of m<sup>6</sup>A in the regulation of cardiomyocyte (CM) proliferation and cardiac regeneration remains unclear. In this study, we aimed to investigate the essential role of m<sup>6</sup>A modification in heart regeneration during postnatal and adult injury. <b>Methods and results:</b> In this study, we identified the downregulation of m<sup>6</sup>A demethylase ALKBH5, an m6A "eraser" that is responsible for increased m<sup>6</sup>A methylation, in the heart after birth. Notably, <i>ALKBH5</i> knockout mice exhibited decreased cardiac regenerative ability and heart function after neonatal apex resection. Conversely, forced expression of ALKBH5 via adeno-associated virus-9 (AAV9) delivery markedly reduced the infarct size, restored cardiac function and promoted CM proliferation after myocardial infarction in juvenile (7 days old) and adult (8-weeks old) mice. Mechanistically, ALKBH5-mediated m<sup>6</sup>A demethylation improved the mRNA stability of YTH N<sup>6</sup>-methyladenosine RNA-binding protein 1 (YTHDF1), thereby increasing its expression, which consequently promoted the translation of Yes-associated protein (YAP). The modulation of ALKBH5 and YTHDF1 expression in human induced pluripotent stem cell-derived cardiomyocytes consistently yielded similar results. <b>Conclusion:</b> Taken together, our findings highlight the vital role of the ALKBH5-m<sup>6</sup>A-YTHDF1-YAP axis in the regulation of CMs to re-enter the cell cycle. This finding suggests a novel potential therapeutic strategy for cardiac regeneration.
Project description:Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4<sup>+</sup> cells into cytotoxic effectors required the presence of CD8<sup>+</sup> cells. Serial exposures to DART led to the exponential expansion of CD4 <b><sup>+</sup></b> and CD8 <b><sup>+</sup></b> cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.
Project description:The cardiovascular and immune systems undergo profound and intertwined alterations with aging. Recent studies have reported that an accumulation of memory and terminally differentiated T cells in elderly subjects can fuel myocardial aging and boost the progression of heart diseases. Nevertheless, it remains unclear whether the immunological senescence profile is sufficient to cause age-related cardiac deterioration or merely acts as an amplifier of previous tissue-intrinsic damage. Herein, we sought to decompose the causality in this cardio-immune crosstalk by studying young mice harboring a senescent-like expanded CD4<sup>+</sup> T cell compartment. Thus, immunodeficient NSG-DR1 mice expressing HLA-DRB1*01:01 were transplanted with human CD4<sup>+</sup> T cells purified from matching donors that rapidly engrafted and expanded in the recipients without causing xenograft reactions. In the donor subjects, the CD4<sup>+</sup> T cell compartment was primarily composed of naïve cells defined as CCR7<sup>+</sup>CD45RO<sup>-</sup>. However, when transplanted into young lymphocyte-deficient mice, CD4<sup>+</sup> T cells underwent homeostatic expansion, upregulated expression of PD-1 receptor and strongly shifted towards effector/memory (CCR7<sup>-</sup> CD45RO<sup>+</sup>) and terminally-differentiated phenotypes (CCR7<sup>-</sup>CD45RO<sup>-</sup>), as typically seen in elderly. Differentiated CD4<sup>+</sup> T cells also infiltrated the myocardium of recipient mice at comparable levels to what is observed during physiological aging. In addition, young mice harboring an expanded CD4<sup>+</sup> T cell compartment showed increased numbers of infiltrating monocytes, macrophages and dendritic cells in the heart. Bulk mRNA sequencing analyses further confirmed that expanding T-cells promote myocardial inflammaging, marked by a distinct age-related transcriptomic signature. Altogether, these data indicate that exaggerated CD4<sup>+</sup> T-cell expansion and differentiation, a hallmark of the aging immune system, is sufficient to promote myocardial alterations compatible with inflammaging in juvenile healthy mice.
Project description:The immune system plays a central role in cancer development, showing both anti-tumor and pro-tumor activities depending on the immune cell subsets and the disease context. While CD8 T cells are associated with a favorable outcome in most cancers, only T helper type 1 (Th1) CD4 T cells play a protective role, in contrast to Th2 CD4 T cells. Double positive (DP) CD4<sup>+</sup>CD8<sup>+</sup> T cells remain understudied, although they were already described in human cancers, with conflicting data regarding their role. Here, we quantified and phenotypically/functionally characterized DP T cells in blood from urological cancer patients. We analyzed blood leukocytes of 24 healthy donors (HD) and 114 patients with urological cancers, including bladder (<i>n</i> = 54), prostate (<i>n</i> = 31), and kidney (<i>n</i> = 29) cancer patients using 10-color flow cytometry. As compared to HD, levels of circulating DP T cells were elevated in all urological cancer patients, which could be attributed to increased frequencies of both CD4<sup>high</sup>CD8<sup>low</sup> and CD4<sup>+</sup>CD8<sup>high</sup> DP T-cell subsets. Of note, most CD4<sup>high</sup>CD8<sup>low</sup> DP T cells show a CD8?? phenotype, whereas CD4<sup>+</sup>CD8<sup>high</sup> cells express both CD8? and CD8? subunits. Functional properties were investigated using <i>ex-vivo</i> generated DP T-cell clones. DP T cells from patients were skewed toward an effector memory phenotype, along with enhanced Th2 cytokine production. Interestingly, both CD8?? and CD8?? DP T cells were able to trigger Th2 polarization of naïve CD4 T cells, while restraining Th1 induction. Thus, these data highlight a previously unrecognized immunoregulatory mechanism involving DP CD4<sup>+</sup>CD8<sup>+</sup> T cells in urological cancers.
Project description:Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4<sup>+</sup>CD8<sup>+</sup> thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4<sup>+</sup>CD8<sup>+</sup> cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4<sup>+</sup>CD8<sup>+</sup> cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4<sup>+</sup>CD8<sup>+</sup> cells and such an expression is regulated by Notch3 through ?-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4<sup>+</sup>CD8<sup>+</sup> cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4<sup>+</sup>CD8<sup>+</sup> cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4<sup>+</sup>CD8<sup>+</sup> cells, by a "thymus-autonomous" mechanism.
Project description:Tendon injuries are a common clinical condition with limited treatment options. The cellular components of the innate immune system, such as neutrophils and macrophages, have been studied in tendon injuries. However, the adaptive immune system, comprising specialized lymphocytes, plays an important role in orchestrating the healing of numerous tissues, but less is known about these cells in tendon healing. To gain a greater understanding of the biological processes that regulate tendon healing, we determined how the cellular components of the adaptive and innate immune system respond to a tendon injury using two-month-old male mice. We observed that lymphatic vasculature is present in the epitenon and superficial regions of Achilles tendons, and that the lymphatics drain into the popliteal lymph node. We then created an acute Achilles tenotomy followed by repair, and collected tendons and popliteal lymph nodes 1, 2, and 4 wk after injury. Tendon injury resulted in a robust adaptive immune cell response that followed an initial innate immune cell response in tendons and lymph nodes. Monocytes, neutrophils, and macrophages initially accumulated at 1 wk after injury in tendons, while dendritic cells and CD4<sup>+</sup> T cells peaked at 2 wk after injury. B cells and CD8<sup>+</sup> T cells progressively increased over time. In parallel, immune cells of the popliteal lymph node demonstrated a similarly coordinated response to the injury. These results suggest that there is an adaptive immune response to tendon injury, and adaptive immune cells may play a role in regulating tendon healing.<b>NEW & NOTEWORTHY</b> While the innate immune system, consisting of macrophages and related hematopoietic cells, has been studied in tendon injury, less is known about the adaptive immune system. Using a mouse model of Achilles tendon tenotomy and repair, we observed an adaptive immune cell response, consisting of CD4<sup>+</sup> and CD8<sup>+</sup> T cells, and B cells, which occur through 4 wk after tendon injury. This response appeared to be coordinated by the draining popliteal lymph node.
Project description:<h4>Background</h4>Lung CD4<sup>+</sup> T-cell depletion and dysfunction, CD8<sup>+</sup> T-cell alveolitis, smoking, and poor control of human immunodeficiency virus (HIV) are features of HIV-associated chronic obstructive pulmonary disease (COPD), but these changes have not been evaluated in smokers at risk for COPD. We evaluated the impact of viral suppression following initiation of antiretroviral therapy (ART) on HIV-specific immunity and the balance of the CD4<sup>+</sup> T-cell to CD8<sup>+</sup> T-cell ratio in the lung.<h4>Methods</h4>Using flow cytometry, we assessed the T-cell immune response in lung and blood specimens obtained from 12 actively smoking HIV-positive patients before ART initiation and after ART-associated viral suppression.<h4>Results</h4>HIV suppression resulted in enhanced lung and systemic HIV-specific CD4<sup>+</sup> T-cell immune responses without significant changes in CD8<sup>+</sup> T-cell responses. We observed an increase in lung ratios of CD4<sup>+</sup> T cells to CD8<sup>+</sup> T cells and CD4<sup>+</sup> T-cell frequencies, decreased CD8<sup>+</sup> T-cell numbers, and resolution of CD8<sup>+</sup> T-cell alveolitis after ART in 9 of 12 individuals. Viral suppression reduced Fas receptor and programmed death 1 expression in lung CD4<sup>+</sup> T cells, correlating with enhanced effector function and reduced susceptibility to apoptosis. HIV suppression rescued peripheral but not lung HIV-specific CD4<sup>+</sup> T-cell proliferation, resulting in augmented effector multifunction.<h4>Discussion</h4>Together, our results demonstrate that HIV suppression restores lung mucosal HIV-specific CD4<sup>+</sup> T-cell multifunctional immunity and balance in the ratio of CD4<sup>+</sup> T cells to CD8<sup>+</sup> T cells, often resolving CD8<sup>+</sup> T-cell alveolitis in active smokers. Peripheral expansion and redistribution of CD4<sup>+</sup> T cells and increased resistance to apoptosis are 2 mechanisms contributing to immunologic improvement following viral suppression in patients at risk for HIV-associated COPD.