Lymph Node CD4 Research Articles

Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Abstract 6734: Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC

Abstract Background: Stimulator of interferon genes (STING) agonists elicit potent innate immune enhancement and mobilize robust adaptive immune responses. Given the potential risk of immunotoxicity from associated cytokine release due to STING agonist treatment, there is a need to identify mitigation strategies that minimize immune-related toxicity while offering a therapeutic window for STING-mediated anti-tumor effects. This strategy is particularly relevant for myeloid-targeted immunotherapies, as cytokine release syndrome (CRS) can be myeloid cell-mediated. We utilized preclinical models to evaluate the pharmacological impact of pretreatment with agents routinely used for management of CRS in advance of delivering TAK-500, a novel, systemic, immune-cell directed STING agonist antibody drug conjugate (iADC) that selectively targets CCR2+ myeloid cells. Methods: Dexamethasone and anti-IL-6R antibodies were tested at clinically relevant doses 1 hour prior to treatment with TAK-500 or mTAK-500, a murine surrogate, to study immunomodulatory and anti-tumor effects in preclinical models. Induction of innate and adaptive immune responses and cytokine release was evaluated in human, ex vivo, and in tumor-bearing mouse models to characterize potential impact of premedication strategies on drug pharmacology and anti-tumor efficacy. Results: TAK-500 triggered dose-dependent monocyte activation and cytokine secretion ex vivo. Monocyte activation was minimally impacted by dexamethasone or anti-IL-6R pretreatment. Suppression of IL-6 and IFN-ƴ and increase in IL-10 were observed following dexamethasone pretreatment, whereas anti-IL-6R maintained a more proinflammatory milieu. In syngeneic mouse models, pretreatment with dexamethasone and anti-mouse IL-6R had minimal impact on mTAK-500 anti-tumor efficacy. Key pharmacodynamic effects, including innate immune cell activation in periphery and tumor-draining lymph node and intratumoral CD8+ T cell accumulation, were minimally impacted by either pretreatment agent. Serum cytokine analyses demonstrated suppression of IL-6, IFN-ƴ and increases in IL-10 with dexamethasone, but not with anti-IL-6R, similar to ex vivo findings. Conclusion: Premedication of TAK-500 and mTAK-500 with dexamethasone or anti-IL-6R antibodies does not substantially impact STING-mediated innate immune activation, mobilization of adaptive immune responses or anti-tumor efficacy, despite altered cytokine profiles compared to (m)TAK-500 without premedication. Clinically, these premedication strategies have been effective in mitigating immunotherapy-associated CRS with T cell therapeutics, and here resulted in maintained anti-tumor activity when used in combination with TAK-500, suggesting a viable potential strategy for mitigation of STING-induced immunotoxicities in patients. Citation Format: Emily Rosentrater, Michelle L. Ganno, Tiquella Hatten, Henry Mack, Arjun Reddy Bollampalli, Dong Mei Zhang, Alex Parent, Jeffrey Raizer, Richard C. Gregory, Neil Lineberry, Vicky Appleman. Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6734.

LOXL1 promotes tumor cell malignancy and restricts CD8 + T cell infiltration in colorectal cancer

BackgroundColorectal cancer (CRC) is a leading cause of cancer mortality globally. Lymph node metastasis and immunosuppression are main factors of poor prognosis in CRC patients. Lysyl oxidase like 1 (LOXL1), part of the lysyl oxidase (LOX) family, plays a yet unclear role in CRC. This study aimed to identify effective biomarkers predictive of prognosis and efficacy of immunotherapy in CRC patients, and to elucidate the prognostic value, clinical relevance, functional and molecular features, and immunotherapy predictive role of LOXL1 in CRC and pan-cancer.MethodsWeighted gene co-expression network analysis (WGCNA) was employed to explore gene modules related to tumor metastasis and CD8 + T cell infiltration. LOXL1 emerged as a hub gene through differential gene expression and survival analysis. The molecular signatures, functional roles, and immunological characteristics affected by LOXL1 were analyzed in multiple CRC cohorts, cell lines and clinical specimens. Additionally, LOXL1's potential as an immunotherapy response indicator was assessed, along with its role in pan-cancer.ResultsTurquoise module in WGCNA analysis was identified as the hub module associated with lymph node metastasis and CD8 + T cell infiltration. Aberrant elevated LOXL1 expression was observed in CRC and correlated with poorer differentiation status and prognosis. Molecular and immunological characterization found that LOXL1 might mediate epithelial-mesenchymal transition (EMT) process and immunosuppressive phenotypes of CRC. Functional study found that LOXL1 enhanced tumor cell proliferation, migration and invasion. Moreover, high LOXL1 levels corresponded to reduced CD8 + T cell infiltration and predicted poor clinical outcomes of immunotherapy. Similar trends were also observed at the pan-cancer level.ConclusionsOur findings underscore the critical role of LOXL1 in modulating both malignancy and immunosuppression in CRC. This positions LOXL1 as a promising biomarker for predicting prognosis and the response to immunotherapy in CRC patients.Graphical

Dissecting the Protective Effect of CD8+ T Cells in Response to SARS-CoV-2 mRNA Vaccination and the Potential Link with Lymph Node CD8+ T Cells.

SARS-CoV-2 vaccines have played a crucial role in effectively reducing COVID-19 disease severity, with a new generation of vaccines that use messenger RNA (mRNA) technology being administered globally. Neutralizing antibodies have featured as the heroes of vaccine-induced immunity. However, vaccine-elicited CD8+ T cells may have a significant impact on the early protective effects of the mRNA vaccine, which are evident 12 days after initial vaccination. Vaccine-induced CD8+ T cells have been shown to respond to multiple epitopes of SARS-CoV-2 and exhibit polyfunctionality in the periphery at the early stage, even when neutralizing antibodies are scarce. Furthermore, SARS-CoV-2 mRNA vaccines induce diverse subsets of memory CD8+ T cells that persist for more than six months following vaccination. However, the protective role of CD8+ T cells in response to the SARS-CoV-2 mRNA vaccines remains a topic of debate. In addition, our understanding of CD8+ T cells in response to vaccination in the lymph nodes, where they first encounter antigen, is still limited. This review delves into the current knowledge regarding the protective role of polyfunctional CD8+ T cells in controlling the virus, the response to SARS-CoV-2 mRNA vaccines, and the contribution to supporting B cell activity and promoting immune protection in the lymph nodes.

Abstract 3472: A tumor draining lymph node CD8 T cell memory response is pivotal for a decrease in recurrence after neoadjuvant anti PD-1 therapy for NSCLC

Abstract Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Even at early stages, death rates remain disproportionately high due to metastatic recurrence. Recently, blockade of the PD-1/PD-L1 axis has shown impressive increases in recurrence-free survival in a subset of patients. While efforts to decrease relapse have focused on decreasing residual micrometastatic disease at the time of definitive treatment, PD-1 directed therapy may allow for an adaptive immune response and the establishment of anti-tumor immunosurveillance. Building on results from a clinically relevant murine model of metastatic relapse after tumor resection and robust assays on systematically characterized tumor, lymph node and blood samples of early-stage lung cancer patients, we posit that immunologic CD8 T cell memory may be a primary mechanism of metastatic protection. Methods: We utilized a syngeneic murine model of metastatic lung adenocarcinoma (344SQ) after resection of tumor, tumor draining lymph node (tdLN) and non-draining lymph nodes (ndLN). Using bioluminescent imaging (BLI) and flow cytometric analysis we monitored T cell kinetics and anti-tumor activity after administration of anti-PD-1 antibody and/or KD033, a fusion antibody combining a high affinity anti-PD-L1 IgG1 antibody with an IL-15Rα sushi binding domain. Tumor/tdLN and ndLN T cells were characterized for PD-1/TCF-1/CD62L/CD44/CXCR5 memory phenotypes. In parallel, we analyzed resected tumor, tdLN and ndLN from early-stage NSCLC patients. Results: tdLN from murine models and early-stage patients maintain robust PD-1+ CXCR5+ CD8 T cell memory phenotypes not significantly found in the primary tumor, ndLN and peripheral blood. Neoadjuvant treatment with PD-1 blockade alone had a heterogenous response, with robust proliferation of T cell central and stem cell memory populations (CM & SCM) at the tdLN in responders compared to non-responders (p<0.05). Combination therapy with IL-15Rα agonist (KD033) potentiated diverse PD-1+ CXCR5+ CD8 stem cell like memory subsets in the tdLN and subsequent response at the primary tumor (100%) as well as displayed superior protection against metastatic recurrence up to 200 days compared to PD-1 inhibition or KD033 alone (median survival undetermined vs. 120d, 161d respectively p<0.05). Removal of tdLN or blockade of lymph node migration with FTY720 prior to therapy decreased a population of SCM and CM CD8 T cells found in the primary tumor, decreased primary tumor response and altered systemic memory subpopulations in the ndLN (p<0.05). Conclusions: Our data strongly points to a T cell memory response within the tumor draining lymph node as a possible driver of protection from systemic cancer recurrence, laying the groundwork for a new therapeutic strategy aimed at establishing CD8 immunosurveillance for protection from cancer recurrence. Citation Format: Tatiana Delgado Cruz, Geoffrey J. Markowitz, Mitchell Martin, Arshdeep Singh, Shelley Yang Bai, Nasser Altorki, Timothy McGraw, Vivek Mittal, Jonathan Villena-Vargas. A tumor draining lymph node CD8 T cell memory response is pivotal for a decrease in recurrence after neoadjuvant anti PD-1 therapy for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3472.

HIV-1 DNA and Immune Activation Levels Differ for Long-Lived T-Cells in Lymph Nodes, Compared with Peripheral Blood, during Antiretroviral Therapy.

Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4+ and CD8+ T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4+ T-cell subsets. Immune activation was significantly higher in all CD8+ T-cell subsets, and memory CD8+ T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8+ T-cells. The differences seen in CM and TM CD4+ T-cell subsets were more pronounced among participants with CD4+ T-cell counts of <500 cells/μL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4+ T-cell recovery during antiretroviral therapy. IMPORTANCE This study provides new insights into the contributions of different CD4+ and CD8+ T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4+ T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4+ and CD8+ T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.

CD169+ sinus macrophages in regional lymph nodes do not predict mismatch-repair status of patients with colorectal cancer.

Mismatch-repair deficiency and microsatellite instability-high (dMMR/MSI-H) colorectal cancer (CRC) is treated with programmed death (PD)-1 antibody regardless of PD-ligand (L)1 expression in tumor cells. We previously found that abundant CD169+ macrophages in regional lymph node (RLN) sinuses and CD8+ tumor-infiltrating lymphocytes (TILs) positively correlated in CRC and were associated with a favorable prognosis. However, associations between dMMR/MSI-H CRC and CD8+ TILs or prognoses vary among studies. In this study, we attempted to compare the association between MMR status, CD169+ macrophages in RLNs, CD8+ TILs, PD-L1 scores, and prognoses in CRC. We immunostained 83 surgically resected CRC tumors that we previously analyzed for MMR proteins, and identified 9 that were dMMR. The number of CD169+ macrophages in RLNs and CD8+ TILs significantly correlated with overall survival, whereas MMR status did not. The number of cells positive for the TIL markers CD3, CD4, CD8, and TIA-1, and macrophage markers CD68 and CD169 in RLNs did not significantly differ between groups according to MMR status. Furthermore, combined positive scores (CPS) for PD-L1 expression in five of nine dMMR CRCs were all <1. We found that dMMR in CRC did not correlate with numbers of CD169+ macrophages in RLNs or CD8+ TILs. CRC with CD169+ macrophages in RLNs and abundant CD8+ TILs indicates a better prognosis and it should be immunologically classified as a different antitumor group from dMMR CRC.

Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation.

Histotripsy is a novel focused ultrasound tumor ablation modality with potent immunostimulatory effects. To measure the spatiotemporal kinetics of local andabscopal responses to histotripsy, C57BL/6 mice bearing bilateral flank B16 melanoma or Hepa1-6 hepatocellular carcinoma tumors were treated with unilateral sham or partial histotripsy. Treated and contralateral untreated (abscopal) tumors were analyzed using multicolor immunofluorescence, digital spatial profiling, RNA sequencing (RNASeq), and flow cytometry. Unilateral histotripsy triggered abscopal tumor growth inhibition. Within the ablation zone, early high mobility group box protein 1 (HMGB1) release and necroptosis were accompanied by immunogenic cell death transcriptional responses in tumor cells and innate immune activation transcriptional responses in infiltrating myeloid and natural killer (NK) cells. Delayed CD8+ T cell intratumoral infiltration was spatiotemporally aligned with cancer cell features of ferroptosis; this effect was enhanced by CTLA-4 blockade and recapitulated in vitro when tumor-draining lymph node CD8+ T cells were co-cultured with tumor cells. Inoculation with cell-free tumor fractions generated by histotripsy but not radiation or freeze/thaw conferred partial protection from tumor challenge. We propose that histotripsy may evoke local necroptotic immunogenic cell death, priming systemic adaptive immune responses and abscopal ferroptotic cancer cell death.

Opposing Roles of DCs and iNKT Cells in the Induction of Foxp3 Expression by MLN CD25+CD4+ T Cells during IFNγ-Driven Colitis

We have previously shown that a deficiency of CD1d-restricted invariant natural killer T (iNKT) cells exacerbates dextran sulfate sodium (DSS)-induced colitis in Yeti mice that exhibit IFNγ-mediated hyper-inflammation. Although iNKT cell-deficiency resulted in reduced Foxp3 expression by mesenteric lymph node (MLN) CD4+ T cells in DSS-treated Yeti mice, the cellular mechanisms that regulate Foxp3 expression by CD25+CD4+ T cells during intestinal inflammation remain unclear. We found that Foxp3-CD25+CD4+ T cells expressing Th1 and Th17 phenotypic hallmarks preferentially expanded in the MLNs of DSS-treated Yeti/CD1d knockout (KO) mice. Moreover, adoptive transfer of Yeti iNKT cells into iNKT cell-deficient Jα18 KO mice effectively suppressed the expansion of MLN Foxp3-CD25+CD4+ T cells during DSS-induced colitis. Interestingly, MLN dendritic cells (DCs) purified from DSS-treated Yeti/CD1d KO mice promoted the differentiation of naive CD4+ T cells into Foxp3-CD25+CD4+ T cells rather than regulatory T (Treg) cells, indicating that MLN DCs might mediate Foxp3+CD25+CD4+ T cell expansion in iNKT cell-sufficient Yeti mice. Furthermore, we showed that Foxp3-CD25+CD4+ T cells were pathogenic in DSS-treated Yeti/CD1d KO mice. Our result suggests that pro-inflammatory DCs and CD1d-restricted iNKT cells play opposing roles in Foxp3 expression by MLN CD25+CD4+ T cells during IFNγ-mediated intestinal inflammation, with potential therapeutic implications.

The Effects of Tangning Ziyabitusi on Gut Microbiota and T lymphocyte Subsets in Impaired Glucose Regulation Rats.

Impaired glucose regulation (IGR) represents the prediabetic state and is associated with gut microbiota (GM) dysbiosis and chronic inflammation. Tangning Ziyabitusi Tablet (TZT) is a Chinese Uyghur herbal medicine with preventative and therapeutic effects on diabetes, but its hypoglycemic mechanisms are unclear. Thirty-six male Wistar rats were divided into the normal diet (ND) and IGR groups. The IGR group was given a high-fat diet (HFD). After the IGR model establishment, the ND group was divided into ND and ND+TZT groups, and the IGR group into IGR and IGR+TZT groups. After 8 weeks of TZT administration, 16S rRNA sequencing and untargeted metabolomics were performed on fecal samples. Mesenteric lymph nodes were also collected, and T lymphocytes separated after rats were sacrificed. Flow cytometry was used to characterize different CD4+ T cell subsets in mesenteric lymph nodes. Finally, we analyzed the correlation between GM and characteristic fecal metabolites. Impaired glucose tolerance and insulin resistance were improved in the IGR+TZT group when compared with the IGR group. Bacterial 16S rRNA sequencing results showed that Sobs and Chao1 indices in the IGR group were significantly decreased, but were increased in the IGR+TZT group. The relative abundance of Bacteroidetes was decreased while the relative abundance of Firmicutes was increased in the IGR group. Adlercreutzia abundance was decreased after TZT administration, while the abundance of Christensenellaceae_R-7_group, norank_f_norank_o_Clostridia_UCG-014, UCG-005, and Eubacterium_nodatum_group was increased in the IGR+TZT group. Lymph node CD4+ T cell proportions in the IGR group were significantly increased, while they were significantly decreased in the IGR+TZT group. Correlation analysis showed that tumor necrosis factor-α, interleukin-6, T helper cells (Th1, Th2, Treg), and insulin had a greater impact on GM community structure. TZT improved glucose tolerance and ameliorated GM dysbiosis in IGR rats. Additionally, TZT significantly modulated CD4+ T cell subset proportions in rat mesenteric lymph nodes and fecal metabolism. Moreover, correlation analysis showed that key microbiota was closely related to IGR indices. Thus, TZT modulated GM composition and immune functions of the intestinal mucosa. We provide useful information for the investigation of active mechanisms and the clinical application of TZT.

Effects of Different Routes and Forms of Vitamin D Administration on Mesenteric Lymph Node CD4+ T Cell Polarization and Intestinal Injury in Obese Mice Complicated with Polymicrobial Sepsis.

This study compared the efficacies of enteral cholecalciferol and/or intravenous (IV) calcitriol administration on mesenteric lymph node (MLN) cluster-of-differentiation-4-positive (CD4+) T cell distribution and intestinal barrier damage in obese mice complicated with sepsis. Mice were fed a high-fat diet for 16 weeks and then sepsis was induced by cecal ligation and puncture (CLP). Mice were divided into the following sepsis groups: without vitamin D (VD) (S); with oral cholecalciferol 1 day before CLP (G); with IV calcitriol 1 h after CLP (V); and with both cholecalciferol before and IV calcitriol after CLP (GV). All mice were sacrificed at 12 or 24 h after CLP. The findings show that the S group had a higher T helper (Th)17 percentage than the VD-treated groups at 12 h after CLP. The V group exhibited a higher Th1 percentage and Th1/Th2 ratio than the other groups at 24 h, whereas the V and GV groups had a lower Th17/regulatory T (Treg) ratio 12 h post-CLP in MLNs. In ileum tissues, the VD-treated groups had higher tight junction protein and cathelicidin levels, and higher mucin gene expression than the S group at 24 h post-CLP. Also, aryl hydrocarbon receptor (AhR) and its associated cytochrome P450 1A1 and interleukin 22 gene expressions were upregulated. In contrast, levels of lipid peroxides and inflammatory mediators in ileum tissues were lower in the groups with VD treatment after CLP. These results suggest that IV calcitriol seemed to have a more-pronounced effect on modulating the homeostasis of Th/Treg subsets in MLNs. Both oral cholecalciferol before and IV calcitriol after CLP promoted cathelicidin secretion, alleviated intestinal inflammation, and ameliorated the epithelial integrity in obese mice complicated with sepsis possibly via VD receptor and AhR signaling pathways.

Exenatide enhanced the antitumor efficacy on PD-1 blockade by the attenuation of neutrophil extracellular traps

Neutrophil extracellular traps (NETs) are extracellular webs of DNA, histones, and granular contents, such as myeloperoxidase (MPO) and elastase, which are released by neutrophils. Reactive oxygen species (ROS) are involved in NETs formation that promote tumor progress. Exenatide could downregulate ROS production in some cell types. However, it is unknown whether Exenatide could influence tumor progress through NETs. Here, we constructed the LLC-based lung cancer and MC38-based colon cancer models and found that Exenatide treatment decreased tumor infiltrated NETs and peripheral MPO-DNA complex and elastase. In addition, the in vitro study showed that Exenatide decreased NETs formation and release. Furthermore, flow cytometry analysis showed that Exenatide treatment reduced ROS production in tumor infiltrated and in vitro neutrophils. However, the ROS inhibitor DPI counteracted the decease of tumor infiltrated and in vitro NETs formation and release by Exenatide. Functionally, the Exenatide/αPD-1 combination therapy was superior to single therapy in restricting tumor growth. Removement of NETs by DNase I weaken the enhancement of αPD-1 treatment by Exenatide. The enriched tumor infiltrated, spleen and lymph node CD8+ T cells from combination therapy group secreted higher concentration of IFN-γ than single treatment. In addition, Exenatide exhibited no direct influence on IFN-γ secretion while purified NETs decreased IFN-γ secretion by CD8+ T cells. The rechallenge study showed that the combination therapy activated long-term tumor rejection. In summary, our findings suggested that Exenatide might be a promising therapeutic candidate for enhancing PD-1 blockade in tumor treatment.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the β-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

CD103 Expression on Regulatory and Follicular T Cells in Lymph Nodes, Bronchoalveolar Lavage Fluid and Peripheral Blood of Sarcoidosis Patients.

(1) Background: Sarcoidosis is a chronic multisystem disorder of unknown aetiology, driven by a T-cell mechanism allowing T-cell attachment and transmigration through the endothelium, and endorsed by the expression of an integrin alpha-E beta-7 (CD103). This study aimed to analyse the different distribution and compartmentalisation of CD103 expression on T cell subsets in BAL, peripheral blood mononuclear cells (PBMC) and lymph nodes (LLN) from sarcoidosis patients. (2) Patients: We consecutively and prospectively enrolled 14 sarcoidosis patients. We collected PBMC, LLN and BAL at the same time from all patients. Through flow cytometric analysis, we analysed the expression of CD103 on regulatory and follicular T cell subsets. (3) Results: All patients were in radiological Scadding stage II. The multivariate analysis found that the variables which most influenced the peripheral blood compartment were high CD8+ and low ThReg, CD8+CD103+ and Tfh cell percentages. A principal component analysis plot performed to distinguish LLN, BAL and PBMC showed that they separated on the basis of CD4+, CD4+CD103+, CD8+, CD8+CD103+, TcEffector, TcNaive, ThNaive, ThEffector, Threg, ThregCD103+, Tfh, TcfCXC5+ and CD4+CD103+/CD4+ with 65.96% of the total variance. (4) Conclusions: Our study is the first to report a link between the imbalance in circulating, alveolar and lymph node CD8+ and CD8+CD103+ T cells, ThReg, Tfh and ThNaive and the CD103+CD4+/CD4+ T cell ratio in the development of sarcoidosis. These findings shine a spotlight on the pathogenesis of sarcoidosis and may offer new predictors for diagnosis. Our study provides additional understanding for a personalised, and hopefully more effective treatment of sarcoidosis.

Development of uveitis in a mouse model of spontaneous autoimmunity correlates with frequency of autoantigen-specific regulatory T cells

Abstract B6 AireGW/+Lyn−/− mice are a model of spontaneously autoimmunity in which 50% of mice develop uveitis by 9 weeks of age, and the remaining mice do not develop autoimmunity. Uveitis results from CD4+ T cells responding to the retina protein interphotoreceptor retinoid-binding protein (IRBP). To define cellular mechanisms that determine whether mice developed autoimmunity or not, we did single-cell RNA sequencing (scRNA-seq) of eye-draining lymph node (LN) CD4+ T cells that recognize the P2 epitope of IRBP (amino acid 271–290). Uniform manifold approximation and projection (UMAP) analysis showed that these CD4+ T cells included distinct subsets that were Ly6c1hi, Pdcd1hi and Treg, with the Treg population being overrepresented in the mice without disease. Pseudotime analysis indicated that the Ly6c1hi T cell subset represented an earlier stage in activation, whereas the Pdcd1hi subset represented a later stage. The two types of effector CD4+ T cells of mice with and without disease largely clustered together relative to their gene expression. Similar analysis of P2-specific CD4+ T cells from retinas with inflammation indicated a progression from proliferative (Mki67hi) cells to a Lag3hi subset to a Lag3hi/Pdcd1hi subset. In mice without uveitis, depletion of Treg by treatment of AireGW/+Lyn−/− Foxp3DTR+/Y mice with diphtheria toxin resulted in rapid expansion of P2–specific CD4+T cells in the draining LN, development of inflammation in the retina, and immune activation in other locations leading to lethality. Ongoing experiments are further testing the hypothesis that the fraction of Treg within the IRBP-specific CD4+ T cells determines whether or not these genetically susceptible mice develop eye-specific autoimmunity. Supported by AI138479

Interleukin-10 contributes to reservoir establishment and persistence in SIV-infected macaques treated with antiretroviral therapy.

Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Intravenous calcitriol administration modulates mesenteric lymph node CD4+ T-cell polarization and attenuates intestinal inflammation in obese mice complicated with polymicrobial sepsis.

Sepsis is a lethal syndrome with T-cell dysregulation, imbalanced inflammatory reactions, and gastrointestinal dysfunction. Obesity coexistent with sepsis can cause more-deleterious disease outcomes. Vitamin D is a nutrient with immunomodulatory ability and helps maintain intestinal homeostasis. This study investigated treatment with calcitriol on mesenteric lymph node (MLN) CD4+ T-cell polarization and intestinal injury in obese mice with sepsis. Mice received a high-fat diet for 10 weeks; then, mice were separated into an obese control group without sepsis and sepsis groups that underwent cecal ligation and puncture (CLP). Septic mice were subdivided into a group that was injected with saline (SS group) or a group that was injected with calcitriol (SD group) via a tail vein 1 h after CLP. Obese mice with sepsis were euthanized at 12 or 24 h post CLP. Sepsis resulted in increased percentages of type 2 T helper (Th2), Th17, and regulatory T (Treg) cells in MLNs. Also, inflammation-associated genes were upregulated and tight junction genes downregulated in the intestines after CLP. Compared with the SS group, the SD group exhibited reduced Th2, Th17, and Treg percentages in MLNs. Also, intestinal inflammatory chemokine expressions were reduced, whereas MUC2, ZO-1, and occludin had increased after CLP. Lower inflammatory cytokine levels in peritoneal lavage fluid in the ileum were also noted in the SD group. Intravenous calcitriol treatment after sepsis can elicit more-balanced CD4 T-cell subsets in lymph nodes near the intestines and alleviate intestinal inflammation and injury in obese mice complicated with sepsis.

T Lymphocyte Maturation Profile in the EBUS-TBNA Lymph Node Depending on the DLCO Parameter in Patients with Pulmonary Sarcoidosis.

Sarcoidosis (SA) is a systemic granulomatous disorder of unknown etiology with lung and mediastinal lymph nodes (LNs) as the main location. T lymphocytes play important role in the formation of granulomas in SA, but still little is known about the role of maturation profile in the development of inflammatory changes. The aim of this study was to determine the CD4+ and CD8+ T cells maturation profile in LNs and in peripheral blood (PB) and its relation to disease severity expressed by diffusing capacity of the lung for carbon monoxide (DLCO). 29 patients with newly pulmonary SA were studied. Flow cytometry was used for cells evaluation in EBUS-TBNA samples. We observed lower median proportion of T lymphocytes, CD4+ T and CD8+ T cells in patients with DLCO< 80% than in patients with normal diffusion (DLCO > 80%). Patients with DLCO < 80% had lower median proportion of effector and higher median proportion of central memory CD4+ and CD8+ T cells than patients with DLCO > 80%. We reported for the first time that LNs CD4+ and CD8+ T cells maturation differs depending on the DLCO value in sarcoidosis. Lymphocytes profiles in LNs may reflect the immune status of patients with SA and can be analysed by flow cytometry of EBUS-TBNA samples.

Erb‐(IL10)2 induces abscopal antitumor effects of radiotherapy through the activation and recruitment of lymph node CD8+ T cells

AbstractObjectiveAlthough radiotherapy (RT) has been widely used in cancer treatment, it provides limited benefits in patients with metastatic cancers due to rare abscopal antitumor effects. Recent progress in cancer immunotherapy provides a potential new strategy to boost the abscopal antitumor effects of RT.MethodsWe fused interleukin 10 (IL10) dimer onto the anti‐epidermal growth factor receptor antibody cetuximab (Erbitux) to form a new bispecific protein Erb‐(IL10)2. The antitumor effect and biological activity of Erb‐(IL10)2 were measured in the B16‐EGFR‐OVA tumor model. In vivo cell depletion and flow cytometry analyses were used to assess the mechanism of antitumor effects.ResultsErb‐(IL10)2 treatment alone showed modest tumor growth inhibition, while local single dose RT (10 Gy) retarded irradiated tumor growth without affecting the growth of nonirradiated tumors. Notably, the combination therapy of RT and Erb‐(IL10)2 not only additively inhibited irradiated tumor growth but also induced abscopal antitumor responses. In vivo depletion of CD8+ T cells abrogated the combinational antitumor effects, while blocking lymphocyte trafficking through FTY720 treatment abolished the abscopal antitumor responses without affecting the antitumor effects on the irradiated tumor sites.ConclusionThis study provides evidence for the radiosensitive role of Erb‐(IL10)2 in the B16‐EGFR‐OVA tumor model. Our findings suggest a novel strategy to elicit the abscopal antitumor effects of RT by combining tumor‐targeted therapy with IL10.