Macroautophagy in lymphatic endothelial cells inhibits T cell–mediated autoimmunity

Lymphatic endothelial cells (LECs) present peripheral tissue antigens to induce T cell tolerance. In addition, LECs are the main source of sphingosine-1-phosphate (S1P), promoting naive T cell survival and effector T cell exit from lymph nodes (LNs). Autophagy is a physiological process essential for cellular homeostasis. We investigated whether autophagy in LECs modulates T cell activation in experimental arthritis. Whereas genetic abrogation of autophagy in LECs does not alter immune homeostasis, it induces alterations of the regulatory T cell (T reg cell) population in LNs from arthritic mice, which might be linked to MHCII-mediated antigen presentation by LECs. Furthermore, inflammation-induced autophagy in LECs promotes the degradation of Sphingosine kinase 1 (SphK1), resulting in decreased S1P production. Consequently, in arthritic mice lacking autophagy in LECs, pathogenic Th17 cell migration toward LEC-derived S1P gradients and egress from LNs are enhanced, as well as infiltration of inflamed joints, resulting in exacerbated arthritis. Our results highlight the autophagy pathway as an important regulator of LEC immunomodulatory functions in inflammatory conditions.     

Adenosine A3 agonists reverse neuropathic pain via T cell–mediated production of IL-10

The A₃ adenosine receptor (A₃AR) has emerged as a therapeutic target with A₃AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A₃ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A₃AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A₃AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4⁺ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A₃AR agonist-mediated anti-allodynia in Rag-KO mice. CD4⁺ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4⁺ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A₃AR activation. Notably, A₃AR agonism reduced DRG neuron excitability when cocultured with CD4⁺ T cells in an IL-10–dependent manner. A₃AR action on CD4⁺ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A₃AR on CD4⁺ T cells to release IL-10 is required and sufficient evidence for the use of A₃AR agonists as therapeutics.     

Anti-TNF immunotherapy reduces CD8+ T cell–mediated antimicrobial activity against Mycobacterium tuberculosis in humans

The incidence of tuberculosis is increased during treatment of autoimmune diseases with anti-TNF antibodies. This is a significant clinical complication, but also provides a unique model to study immune mechanisms in human tuberculosis. Given the key role for cell-mediated immunity in host defense against Mycobacterium tuberculosis, we hypothesized that anti-TNF treatment impairs T cell–directed antimicrobial activity. Anti-TNF therapy reduced the expression in lymphocytes of perforin and granulysin, 2 components of the T cell–mediated antimicrobial response to intracellular pathogens. Specifically, M. tuberculosis–reactive CD8⁺CCR7^–CD45RA⁺ effector memory T cells (T_EMRA cells) expressed the highest levels of granulysin, lysed M. tuberculosis, and infected macrophages and mediated an antimicrobial activity against intracellular M. tuberculosis. Furthermore, T_EMRA cells expressed cell surface TNF and bound the anti-TNF therapeutic infliximab in vitro, making them susceptible to complement-mediated lysis. Immune therapy with anti-TNF was associated with reduced numbers of CD8⁺ T_EMRA cells and decreased antimicrobial activity against M. tuberculosis, which could be rescued by the addition of CD8⁺ T_EMRA cells. These results suggest that anti-TNF therapy triggers a reduction of CD8⁺ T_EMRA cells with antimicrobial activity against M. tuberculosis, providing insight into the mechanism whereby key effector T cell subsets contribute to host defense against tuberculosis.     

Myeloid cell–targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell–mediated immunity

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8⁺ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage–related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell–targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8⁺ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV⁺ mEERL and HPV^– MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8⁺ and CD4⁺ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8⁺ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.     

Critical roles of Bim in T cell activation and T cell–mediated autoimmune inflammation in mice

Bim, the B cell lymphoma 2–interacting (Bcl2-interacting) mediator, maintains immunological tolerance by deleting autoreactive lymphocytes through apoptosis. We report here that Bim is also, paradoxically, required for the activation of autoreactive T cells. Deletion of Bim in hematopoietic cells rendered mice resistant to autoimmune encephalomyelitis and diabetes, and Bim-deficient T cells had diminished cytokine production. Upon T cell receptor activation, Bim-deficient T cells exhibited severe defects in both calcium release and dephosphorylation of nuclear factor of activated T cells (NFAT) but maintained normal levels of activation of NF-κB and MAPKs. The defective calcium signaling in Bim-deficient T cells was associated with a significant increase in the formation of an inhibitory complex containing Bcl2 and the inositol triphosphate receptor (IP₃R). Thus, in addition to mediating the death of autoreactive T cells, Bim also controlled T cell activation through the IP₃R/calcium/NFAT pathway. These results indicate that a single protein is used to control both the activation and apoptosis of autoreactive T cells and may explain why Bim-deficient mice do not reject their own organs despite lacking thymic negative selection.     

CD4+ T cell–independent vaccination against Pneumocystis carinii in mice

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. Moreover, deficient CD4+ T lymphocytes impair vaccination approaches to prevent opportunistic infection. Therefore, we investigated a CD4+ T cell-independent vaccine approach to a prototypic AIDS-defining infection, Pneumocystis carinii (PC) pneumonia. Here, we demonstrate that bone marrow-derived dendritic cells (DCs) expressing the murine CD40 ligand, when pulsed ex vivo by PC antigen, elicited significant titers of anti-PC IgG in CD4-deficient mice. Vaccinated animals demonstrated significant protection from PC infection, and this protection was the result of an effective humoral response, since adoptive transfer of CD4-depleted splenocytes or serum conferred this protection to CD4-deficient mice. Western blot analysis of PC antigen revealed that DC-vaccinated, CD4-deficient mice predominantly reacted to a 55-kDa PC antigen. These studies show promise for advances in CD4-independent vaccination against HIV-related pathogens.     

The regulation of T cell homeostasis and autoimmunity by T cell–derived LIGHT

Costimulatory molecules on antigen-presenting cells (APCs) play an important role in T cell activation and expansion. However, little is known about the surface molecules involved in direct T-T cell interaction required for their activation and expansion. LIGHT, a newly discovered TNF superfamily member (TNFSF14), is expressed on activated T cells and immature dendritic cells. Here we demonstrate that blockade of LIGHT activity can reduce anti-CD3-mediated proliferation of purified T cells, suggesting that T cell-T cell interaction is essential for this proliferation. To test the in vivo activity of T cell-derived LIGHT in immune homeostasis and function, transgenic (Tg) mice expressing LIGHT in the T cell lineage were generated. LIGHT Tg mice have a significantly enlarged T cell compartment and a hyperactivated peripheral T cell population. LIGHT Tg mice spontaneously develop severe autoimmune disease manifested by splenomegaly, lymphadenopathy, glomerulonephritis, elevated autoantibodies, and severe infiltration of various peripheral tissues. Furthermore, the blockade of LIGHT activity ameliorates the severity of T cell-mediated diseases. Collectively, these findings establish a crucial role for this T cell-derived costimulatory ligand in T cell activation and expansion; moreover, the dysregulation of T cell-derived LIGHT leads to altered T cell homeostasis and autoimmune disease.     

HLA-E–restricted HIV-1–specific CD8+ T cell responses in natural infection

CD8⁺ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E–restricted CD8⁺ T cell responses in HIV infection, however, remains unknown. In this study, CD8⁺ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E–restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8⁺ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8⁺ T cells. Importantly, bulk CD8⁺ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E–restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E–restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8⁺ T cell responses in HIV infection.     

Myeloid cell–derived PROS1 inhibits tumor metastasis by regulating inflammatory and immune responses via IL-10

Stimulation of TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases promotes tumor progression through numerous cellular mechanisms. TAM cognate ligands GAS6 and PROS1 (for TYRO3 and MERTK) are secreted by host immune cells, an interaction which may support tumor progression. Here, we revealed an unexpected antimetastatic role for myeloid-derived PROS1: suppressing metastatic potential in lung and breast tumor models. Pros1 deletion in myeloid cells led to increased lung metastasis, independent of primary tumor infiltration. PROS1-cKO bone marrow–derived macrophages (BMDMs) led to elevated TNF-α, IL-6, Nos2, and IL-10 via modulation of the Socs3/NF-κB pathway. Conditioned medium from cKO BMDMs enhanced EMT, ERK, AKT, and STAT3 activation within tumor cells and promoted IL-10–dependent invasion and survival. Macrophages isolated from metastatic lungs modulated T cell proliferation and function, as well as expression of costimulatory molecules on DCs in a PROS1-dependent manner. Inhibition of MERTK kinase activity blocked PROS1-mediated suppression of TNF-α and IL-6 but not IL-10. Overall, using lung and breast cancer models, we identified the PROS1/MERTK axis within BMDMs as a potent regulator of adaptive immune responses with a potential to suppress metastatic seeding and revealed IL-10 regulation by PROS1 to deviate from that of TNF-α and IL-6.     

Cul4A-DDB1–mediated monoubiquitination of phosphoglycerate dehydrogenase promotes colorectal cancer metastasis via increased S-adenosylmethionine

Although serine metabolism plays a crucial role in the proliferation and survival of tumor cells, how it supports tumor cell migration remains poorly understood. Phosphoglycerate dehydrogenase (PHGDH) catalyzes the oxidation of 3-phosphoglycerate to 3-phosphonooxypyruvate, the first committed step in de novo serine biosynthesis. Here we show that PHGDH was monoubiquitinated by cullin 4A–based E3 ligase complex at lysine 146 in colorectal cancer (CRC) cells, which enhanced PHGDH activity by recruiting a chaperone protein, DnaJ homolog subfamily A member 1, to promote its tetrameric formation, thereby increasing the levels of serine, glycine, and S-adenosylmethionine (SAM). Increased levels of SAM upregulated the expression of cell adhesion genes (laminin subunit gamma 2 and cysteine rich angiogenic inducer 61) by initiating SET domain containing 1A–mediated trimethylation of histone H3K4, thereby promoting tumor cell migration and CRC metastasis. Intriguingly, SAM levels in tumors or blood samples correlated with the metastatic recurrence of patients with CRC. Our finding not only reveals a potentially new role and mechanism of SAM-promoted tumor metastasis but also demonstrates a regulatory mechanism of PHGDH activity by monoubiquitination.     

SARS-CoV-2–specific CD8+ T cell responses in convalescent COVID-19 individuals

Characterization of the T cell response in individuals who recover from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8⁺ T cell recognition in a cross-sectional sample of 30 coronavirus disease 2019 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis and from humoral and inflammatory responses. There were 132 SARS-CoV-2–specific CD8⁺ T cell responses detected across 6 different HLAs, corresponding to 52 unique epitope reactivities. CD8⁺ T cell responses were detected in almost all convalescent individuals and were directed against several structural and nonstructural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2–specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8⁺ T cell response. Overall, T cells exhibited distinct differentiation into stem cell and transitional memory states (subsets), which may be key to developing durable protection.     

NVX-CoV2373 vaccination induces functional SARS-CoV-2–specific CD4+ and CD8+ T cell responses

NVX-CoV2373 is an adjuvanted recombinant full-length SARS-CoV-2 spike trimer protein vaccine demonstrated to be protective against COVID-19 in efficacy trials. Here we demonstrate that vaccinated individuals made CD4⁺ T cell responses after 1 and 2 doses of NVX-CoV2373, and a subset of individuals made CD8⁺ T cell responses. Characterization of the vaccine-elicited CD8⁺ T cells demonstrated IFN-γ production. Characterization of the vaccine-elicited CD4⁺ T cells revealed both circulating T follicular helper (cTfh) cells and Th1 cells (IFN-γ⁺, TNF-α⁺, and IL-2⁺) were detectable within 7 days of the primary immunization. Spike-specific CD4⁺ T cells were correlated with the magnitude of the later SARS-CoV-2–neutralizing antibody titers, indicating that robust generation of CD4⁺ T cells, capable of supporting humoral immune responses, may be a key characteristic of NVX-CoV2373 that utilizes Matrix-M adjuvant.     

CD4+ T Cell–mediated Granulomatous Pathology in Schistosomiasis Is Downregulated by a B Cell–dependent Mechanism Requiring Fc Receptor Signaling

The effector functions of CD4⁺ T lymphocytes are generally thought to be controlled by distinct populations of regulatory T cells and their soluble products. The role of B cells in the regulation of CD4-dependent host responses is less well understood. Hepatic egg granuloma formation and fibrosis in murine schistosomiasis are dependent on CD4⁺ lymphocytes, and previous studies have implicated CD8⁺ T cells or cross-regulatory cytokines produced by T helper (Th) lymphocytes as controlling elements of this pathologic process. In this report, we demonstrate that B cell–deficient (μMT) mice exposed to Schistosoma mansoni develop augmented tissue pathology and, more importantly, fail to undergo the spontaneous downmodulation in disease normally observed during late stages of infection. Unexpectedly, B cell deficiency did not significantly alter T cell proliferative response or cause a shift in the Th1/Th2 balance. Since schistosome-infected Fc receptor–deficient (FcR γ chain knockout) mice display the same exacerbated egg pathology as that observed in infected μMT mice, the B cell– dependent regulatory mechanism revealed by these experiments appears to require receptor-mediated cell triggering. Together, the data demonstrate that humoral immune response/FcR interactions can play a major role in negatively controlling inflammatory disease induced by CD4⁺ T cells.     

T淋巴细胞内mTOR/S6途径在难治/复发再生障碍性贫血发病机制中的作用

Objective To explore the activation status of signal pathway of mTOR/S6 in bone marrow(BM) T lymphocytes of refractory/relapsed aplastic anemia patients(AA),and the effects of rapamycin (RAPA)and CTLA-4 immunoglobulin(CTLA-4 Ig)on this pathway.Methods BM was collected from 13refractory/relapsed AA patients,8 newly diagnosed severe AA(SAA)patients and 10 iron deficiency anemia (IDA)(as controls)patients,and cocultured with RAPA and CTLA-4 Ig.The expression of p-mTOR,p-S6 and Interferon γ(IFN-γ)in CD3+ T cells was measured by flow cytometry(FCM).Results ①The expression of p-mTOR,P-S6 and IFN-γ in CD3+ T cells in refractory/relapsed AA group were significantly higher than those in controls(P＜0.01).②The expression of P-mTOR and p-S6 in T cels in newly diagnosed SAA group,Was similar to those in controls(P＞0.05),but significantly lower than those in refractory/relapsed AA group(P＜0.01).The expression level of IFN-γ in T cells were significantly higher than that in controls (P＜0.01).③On exposure to RAPA,The levels of p-mTOR,p-S6 and IFN-γ in T cells in refractory/relapsed AA patients were significantly lower than those before the exposure(all P＜0.05).And so were when exposed to CTLA-4 Ig(all P＜0.01).Conclusion ①The mTOR/S6 signal pathway is activated in refractory/relapsed AA.②The expression of p-mTOR,p-S6 and IFN-γ in refractory/relapsed AA Can be suppressed by RAPA or CTLA-4 Ig.③The signal pathway of CD28/mTOR/S6/IFN-γ might take part in immune pathogenesis of refractory/relapsed AA.     

Uridine diphosphate–glucose/P2Y14R axis is a nonchemokine pathway that selectively promotes eosinophil accumulation

Allergic asthma is a chronic inflammatory airway disease characterized by dysregulated type 2 immune responses, including degranulating airway eosinophils that induce tissue damage and airway hyperresponsiveness (AHR). The type 2 cytokines interleukin 5 (IL-5) and IL-13 and the eosinophil-specific chemokine CCL11/CCL24/CCL26 axis recruit, activate, and regulate eosinophils in the airways. In this issue of the JCI, Karcz et al. identified a mechanism involving the nucleotide sugar UDP-glucose (UDP-G) and the purinergic receptor P2Y₁₄R in amplifying eosinophil accumulation in the lung. During type 2 inflammation, UDP-G activates P2Y₁₄R on eosinophils, inducing the cells to move and migrate into the lung. Pharmacologically or genetically inhibiting P2Y₁₄R on eosinophils attenuated eosinophil infiltration and AHR. Future experiments, including identifying additional type 2 factors regulating P2Y₁₄R expression on lung eosinophils, are necessary to ascertain the impact of targeting P2Y₁₄R as an alternative or adjunctive therapy to current type 2 biologics for the treatment of asthma.     

T淋巴细胞内mTOR/S6途径在难治/复发再生障碍性贫血发病机制中的作用

目的 研究难治/复发再生障碍性贫血(AA)患者骨髓T淋巴细胞内雷帕霉素靶蛋白(mTOR)/核糖体蛋白S6途径活化情况,以及mTOR抑制剂雷帕霉素(RAPA)和CD28抗原阻断剂CTLA-4免疫球蛋白(CTLA-41g)对此途径的影响.方法 采集13例难治/复发AA、8例初治重型AA(SAA)以及1O例缺铁性贫血(IDA)患者(对照组)的骨髓标本,加入RAPA和CTLA-41g进行孵育,用流式细胞术检测加药前后每组患者CD3+T淋巴细胞胞质内磷酸化mTOR(p-mTOR)、磷酸化S6(P-S6)和干扰素-γ(IFN-γ)的表达水平,并分析其与疾病的关系.结果 ①难治/复发AA组患者骨髓CD3+T细胞胞内p-mTOR、P-S6及IFN-γ的表达水平较对照组显著升高(P值均＜0.01).②初治SAA患者p-mTOR和p-S6的表达水平低于难治/复发AA组(P值均＜0.01),而与对照组比较差异无统计学意义(P值均＞0.05);初治组IFN-γ的表达水平明显高于对照组(P值均＜0.01).③RAPA和CTLA-41g作用后,难治/复发AA患者p-mTOR、p-S6及IFN-γ的表达水平较用药前明显下降(P值均＜0.01).结论 CD3+T淋巴细胞内mTOR/S6途径在难治/复发AA患者中活化.RAPA和CTLA-41g均可抑制难洽/复发AA患者体内mTOR/S6途径,并下调IFN-γ.CD28/mTOR/S6和IFN-γ途径参与难治/复发AA的免疫发病机制,并且对RAPA和CTLA-41g敏感,以CD28、mTOR为治疗靶点,临床应用RAPA和CTLA-4Ig治疗此类AA患者值得探索.