Co-stimulators CD40-CD40L, a potential immune-therapy target for atherosclerosis: A review.

The interaction between CD40 and CD40 ligand (CD40L) a crucial co-stimulatory signal for activating adaptive immune cells, has a noteworthy role in atherosclerosis. It is well-known that atherosclerosis is linked to immune inflammation in blood vessels. In atherosclerotic lesions, there is a multitude of proinflammatory cytokines, adhesion molecules, and collagen, as well as smooth muscle cells, macrophages, and T lymphocytes, particularly the binding of CD40 and CD40L. Therefore, research on inhibiting the CD40-CD40L system to prevent atherosclerosis has been ongoing for more than 30 years. However, it's essential to note that long-term direct suppression of CD40 or CD40L could potentially result in immunosuppression, emphasizing the critical role of the CD40-CD40L system in atherosclerosis. Thus, specifically targeting the CD40-CD40L interaction on particular cell types or their downstream signaling pathways may be a robust strategy for mitigating atherosclerosis, reducing potential side effects. This review aims to summarize the potential utility of the CD40-CD40L system as a viable therapeutic target for atherosclerosis.

T-cell subsets and cytokines are indicative of neoadjuvant chemoimmunotherapy responses in NSCLC.

PURPOSE: Neoadjuvant PD-1 blockade combined with chemotherapy is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet the immunological mechanisms contributing to tumor regression and biomarkers corresponding to different pathological responses remain unclear. METHODS: Using dynamic and paired blood samples from NSCLC patients receiving neoadjuvant chemoimmunotherapy, we analyzed the frequencies of CD8 + T-cell and Treg subsets and their dynamic changes during neoadjuvant treatment through flow cytometry. Cytokine profiles and function-related gene expression of CD8 + T cells and Tregs were analyzed through flow cytometry and mRNA-seq. Infiltrating T-cell subsets in resected tissues from patients with different pathological responses were analyzed through multiplex immunofluorescence. RESULTS: Forty-two NSCLC patients receiving neoadjuvant chemoimmunotherapy were enrolled and then underwent surgical resection and pathological evaluation. Nineteen patients had pCR (45%), 7 patients had MPR (17%), and 16 patients had non-MPR (38%). In patients with pCR, the frequencies of CD137 + CD8 + T cells (P = 0.0475), PD-1 + Ki-67 + CD8 + T cells (P = 0.0261) and Tregs (P = 0.0317) were significantly different from those of non-pCR patients before treatment. pCR patients usually had low frequencies of CD137 + CD8 + T cells, PD-1 + Ki-67 + CD8 + T cells and Tregs, and their AUCs were higher than that of tissue PD-L1 expression. Neoadjuvant chemoimmunotherapy markedly improved CD8 + T-cell proliferation and activation, especially in pCR patients, as the frequencies of CD137 + CD8 + (P = 0.0136) and Ki-67 + CD8 + (P = 0.0391) T cells were significantly increased. The blood levels of cytokines such as IL-2 (P = 0.0391) and CXCL10 (P = 0.0195) were also significantly increased in the pCR group, which is consistent with the high density of activated cytotoxic T cells at the tumor site (P < 0.0001). CONCLUSION: Neoadjuvant chemoimmunotherapy drives CD8 + T cells toward a proliferative and active profile. The frequencies of CD137 + CD8 + T cells, PD-1 + Ki-67 + CD8 + T cells and Tregs at baseline might predict the response to neoadjuvant chemoimmunotherapy in NSCLC patients. The increase in IL-2 and CXCL10 might reflect the chemotaxis and enrichment of cytotoxic T cells at the tumor site and a better response to neoadjuvant chemoimmunotherapy.

Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics.

BACKGROUND: Despite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge in the field of immunotherapy; thus, more optimized translational models could strongly influence clinical trial development. To address this unmet need, we designed a preclinical model reflecting the heterogeneity in melanoma patients' clinical responses that can be used to evaluate novel immunotherapies and synergistic combinatorial treatment strategies. Using our all-autologous humanized melanoma mouse model, we examined the efficacy of a novel engineered interleukin 2 (IL-2)-based cytokine variant immunotherapy. METHODS: To study immune responses and antitumor efficacy for human melanoma tumors, we developed an all-autologous humanized melanoma mouse model using clinically annotated, matched patient tumor cells and peripheral blood mononuclear cells (PBMCs). After inoculating immunodeficient NSG mice with patient tumors and an adoptive cell transfer of autologous PBMCs, mice were treated with anti-PD-1, a novel investigational engineered IL-2-based cytokine (nemvaleukin), or recombinant human IL-2 (rhIL-2). The pharmacodynamic effects and antitumor efficacy of these treatments were then evaluated. We used tumor cells and autologous PBMCs from patients with varying immunotherapy responses to both model the diversity of immunotherapy efficacy observed in the clinical setting and to recapitulate the heterogeneous nature of melanoma. RESULTS: Our model exhibited long-term survival of engrafted human PBMCs without developing graft-versus-host disease. Administration of an anti-PD-1 or nemvaleukin elicited antitumor responses in our model that were patient-specific and were found to parallel clinical responsiveness to checkpoint inhibitors. An evaluation of nemvaleukin-treated mice demonstrated increased tumor-infiltrating CD4+ and CD8+ T cells, preferential expansion of non-regulatory T cell subsets in the spleen, and significant delays in tumor growth compared with vehicle-treated controls or mice treated with rhIL-2. CONCLUSIONS: Our model reproduces differential effects of immunotherapy in melanoma patients, capturing the inherent heterogeneity in clinical responses. Taken together, these data demonstrate our model's translatability for novel immunotherapies in melanoma patients. The data are also supportive for the continued clinical investigation of nemvaleukin as a novel immunotherapeutic for the treatment of melanoma.

STAT activation in regulatory CD4+ T cells of patients with primary sclerosing cholangitis.

INTRODUCTION: Regulatory CD4+ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK-STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines. METHODS: In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3+CD25+CD127lowCD4+ Tregs, their expression of ectonucleotidase CD39, and cytokine-induced phosphorylation of STAT1, 3, 5, and 6 using phospho-flow cytometry. In parallel, we measured cytokines IFN-gamma, interleukin (IL)-6, IL-2, and IL-4 in serum via bead-based immunoassays. RESULTS: In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN-gamma, IL-6, IL-2, and IL-4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL-6 induced increased phosphorylation of STAT3 in Tregs of PSC-patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts. CONCLUSIONS: In PSC, we observed enhanced STAT3 responsiveness of CD4+ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.

[Buzhong Yiqi Decoction ameliorates spleen deficiency syndrome by regulating gut microbiota].

This study aims to decipher the mechanism of Buzhong Yiqi Decoction(BZYQD) in the treatment of spleen deficiency syndrome via gut microbiota. The mouse models of spleen deficiency syndrome were established by fecal microbiota transplantation(FMT, from patients with spleen deficiency syndrome) and administration of Sennae Folium(SF, 10 g·kg~(-1)), respectively, and treated with BZYQD for 5 d. The pseudosterile mice(administrated with large doses of antibiotics) and the mice transplanted with fecal bacteria from healthy human were taken as the controls. The levels of IgA, interleukin(IL)-2, IL-1ß, interferon(IFN)-γ, tumor necrosis factor-alpha(TNF-α), and 5-hydroxytryptamine(5-HT) in the intestinal tissue of two models were measured by enzyme-linked immunosorbent assay, and the CD8~+/CD3~+ ratio was determined by flow cytometry. The composition and changes of the gut microbiota were determined by 16S rRNA high-throughput sequencing and qPCR. Furthermore, the correlation analysis was performed to study the mediating role of gut microbiota in the treatment. The results showed that BZYQD elevated the IgA level, lowered the IL-1ß, TNF-α, and 5-HT levels, and decreased the CD8~+/CD3~+ ratio in the intestinal tissue of the two models. Moreover, BZYQD had two-way regulatory effects on the levels of IL-2 and IFN-γ. BZYQD inhibited the overgrowth and reduced the richness of gut microbiota in the SF model, and improved the gut microbiota structure in the two models. Algoriphagus, Mycobacterium, and CL500＿29＿marine＿group were the common differential genera in the two models compared with the control. Acinetobacter, Parabacteroides, and Ruminococcus were the differential genera unique to the FMT model, and Sphingorhabdus, Lactobacillus, and Anaeroplasma were the unique differential genera in the SF model. BZYQD was capable of regulating all these genera. The qPCR results showed that BZYQD increased the relative abundance of Akkermansia muciniphila and decreased that of Bacteroides uniformis in the two models. The correlation analysis revealed that the levels of above intestinal cytokines were significantly correlated with characteristic gut microorganisms in different mo-dels. The IL-1ß level had a significantly positive correlation with Acinetobacter and CL500＿29＿marine＿group in the two models, while the different levels of IL-2 and IFN-γ in the two models may be related to its different gut microbiota structures. In conclusion, BZYQD could regulate the disordered gut microbiota structure in different animal models of spleen deficiency syndrome to improve the intestinal immune status, which might be one of the mechanisms of BZYQD in treating spleen deficiency syndrome.

Induction of regulatory T cells and efficacy of low-dose interleukin-2 in systemic sclerosis: interventional open-label phase 1-phase 2a study.

BACKGROUND: Systemic sclerosis (SSc) is a chronic autoimmune disease, with impaired immune response, increased fibrosis and endothelial dysfunction. Regulatory T cells (Tregs), which are essential to control inflammation, tissue repair and autoimmunity, have a decreased frequency and impaired function in SSc patients. Low-dose interleukin-2 (IL-2LD) can expand and activate Tregs and has, therefore, a therapeutic potential in SSc. OBJECTIVE: We aimed to assess the safety and biological efficacy of IL-2LD in patients with SSc. METHODS: As part of the TRANSREG open-label phase IIa basket trial in multiple autoimmune diseases, we studied nine patients with SSc without severe organ involvement. Patients received 1 million international units (MIU)/day of IL-2 for 5 days, followed by fortnightly injections for 6 months. Laboratory and clinical evaluations were performed between baseline and month 6. RESULTS: At day 8, the primary endpoint (Treg frequency) was reached with a 1.8±0.5-fold increase of Treg levels among CD4+ T lymphocytes (p=0.0015). There were no significant changes in effector T cells nor in B cells. IL-2LD was well tolerated, and no serious adverse events related to treatment occurred. There was a globally stable measurement in the modified Rodnan skin score and Valentini score at month 6. Disease activity and severity measures, the quality of life evaluated by EuroQL-5D-5L and pulmonary function test parameters remained stable during the study period. CONCLUSION: IL-2LD at a dosage of 1 MIU/day safely and selectively activates and expands Tregs. Clinical signs remain stable during the study period. This opens the door to properly powered phase II efficacy trials investigating IL-2LD therapeutic efficacy in SSc.

Vitamin D constrains inflammation by modulating the expression of key genes on Chr17q12-21.1.

Vitamin D possesses immunomodulatory functions and vitamin D deficiency has been associated with the rise in chronic inflammatory diseases, including asthma (Litonjua and Weiss, 2007). Vitamin D supplementation studies do not provide insight into the molecular genetic mechanisms of vitamin D-mediated immunoregulation. Here, we provide evidence for vitamin D regulation of two human chromosomal loci, Chr17q12-21.1 and Chr17q21.2, reliably associated with autoimmune and chronic inflammatory diseases. We demonstrate increased vitamin D receptor (Vdr) expression in mouse lung CD4+ Th2 cells, differential expression of Chr17q12-21.1 and Chr17q21.2 genes in Th2 cells based on vitamin D status and identify the IL-2/Stat5 pathway as a target of vitamin D signaling. Vitamin D deficiency caused severe lung inflammation after allergen challenge in mice that was prevented by long-term prenatal vitamin D supplementation. Mechanistically, vitamin D induced the expression of the Ikzf3-encoded protein Aiolos to suppress IL-2 signaling and ameliorate cytokine production in Th2 cells. These translational findings demonstrate mechanisms for the immune protective effect of vitamin D in allergic lung inflammation with a strong molecular genetic link to the regulation of both Chr17q12-21.1 and Chr17q21.2 genes and suggest further functional studies and interventional strategies for long-term prevention of asthma and other autoimmune disorders.

Omenn Syndrome in Two Infants with Different Hypomorphic Variants in Janus Kinase 3.

Biallelic null or hypomorphic variants in JAK3 cause SCID and less frequently Omenn syndrome. We investigated homozygous hypomorphic JAK3 mutations in two patients, and expression and function of a novel JAK3R431P variant in Omenn syndrome. Immunophenotyping of PBMC from the patient with the novel JAK3R431P variant was undertaken, by flow cytometry and Phosflow after stimulation with IL-2, IL-7, and IL-15. JAK3 expression was investigated by Western blotting. We report two patients with homozygous hypomorphic JAK3 variants and clinical features of Omenn syndrome. One patient had a previously described JAK3R775H variant, and the second had a novel JAK3R431P variant. One patient with a novel JAK3R431P variant had normal expression of JAK3 in immortalised EBV-LCL cells but reduced phosphorylation of STAT5 after stimulation with IL-2, IL-7, and IL-15 consistent with impaired kinase activity. These results suggest the JAK3R431P variant to be hypomorphic. Both patients are alive and well after allogeneic haematopoietic stem cell transplantation. They have full donor chimerism, restitution of thymopoiesis and development of appropriate antibody responses following vaccination. We expand the phenotype of hypomorphic JAK3 deficiency and demonstrate the importance of functional testing of novel variants in disease-causing genes.

Oncolytic herpes simplex virus expressing IL-2 controls glioblastoma growth and improves survival.

BACKGROUND: Glioblastoma (GBM), a highly immunosuppressive and often fatal primary brain tumor, lacks effective treatment options. GBMs contain a subpopulation of GBM stem-like cells (GSCs) that play a central role in tumor initiation, progression, and treatment resistance. Oncolytic viruses, especially oncolytic herpes simplex virus (oHSV), replicate selectively in cancer cells and trigger antitumor immunity-a phenomenon termed the "in situ vaccine" effect. Although talimogene laherparepvec (T-VEC), an oHSV armed with granulocyte macrophage-colony stimulating factor (GM-CSF), is Food and Drug Administration (FDA)-approved for melanoma, its use in patients with GBM has not been reported. Interleukin 2 (IL-2) is another established immunotherapy that stimulates T cell growth and orchestrates antitumor responses. IL-2 is FDA-approved for melanoma and renal cell carcinoma but has not been widely evaluated in GBM, and IL-2 treatment is limited by its short half-life, minimal tumor accumulation, and significant systemic toxicity. We hypothesize that local intratumoral expression of IL-2 by an oHSV would avoid the systemic IL-2-related therapeutic drawbacks while simultaneously producing beneficial antitumor immunity. METHODS: We developed G47Δ-mIL2 (an oHSV expressing IL-2) using the flip-flop HSV BAC system to deliver IL-2 locally within the tumor microenvironment (TME). We then tested its efficacy in orthotopic mouse GBM models (005 GSC, CT-2A, and GL261) and evaluated immune profiles in the treated tumors and spleens by flow cytometry and immunohistochemistry. RESULTS: G47Δ-mIL2 significantly prolonged median survival without any observable systemic IL-2-related toxicity in the 005 and CT-2A models but not in the GL261 model due to the non-permissive nature of GL261 cells to HSV infection. The therapeutic activity of G47Δ-mIL2 in the 005 GBM model was associated with increased intratumoral infiltration of CD8+ T cells, critically dependent on the release of IL-2 within the TME, and CD4+ T cells as their depletion completely abrogated therapeutic efficacy. The use of anti-PD-1 immune checkpoint blockade did not improve the therapeutic outcome of G47Δ-mIL2. CONCLUSIONS: Our findings illustrate that G47Δ-mIL2 is efficacious, stimulates antitumor immunity against orthotopic GBM, and may also target GSC. OHSV expressing IL-2 may represent an agent that merits further exploration in patients with GBM.

A novel dual mechanism-of-action bispecific PD-1-IL-2v armed by a "ßγ-only" interleukin-2 variant.

Introduction: Interleukin-2 (IL-2) is one of the first cytokines to be discovered as an immune agonist for cancer immunotherapy. Biased IL-2 variants had been discovered to eliminate Treg activation or enhance the tumor specific T cell cytotoxicity. However, all the biased IL-2 variants pose the risk to overstimulate immune response at a low-dose range. Here, we introduce a novel dual-MOA bispecific PD-1-IL-2v molecule with great anti-tumor efficacy in a high dosed manner. Methods: The novel IL-2 variant was designed by structural truncation and shuffling. The single armed bispecific PD-1-IL-2v molecule and IL-2v were studied by immune cell activations in vitro and in vivo and anti-tumor efficacy in mouse model. Results and discussion: The IL-2 variant in this bispecific antibody only binds to IL-2Rßγ complex in a fast-on/off manner without α, ß or γ single receptor binding. This IL-2v mildly activates T and NK cells without over stimulation, meanwhile it diminishes Treg activation compared to the wild type IL-2. This unique bispecific molecule with "ßγ-only" IL-2v can not only "in-cis" stimulate and expand CD8 T and NK cells moderately without Treg activation, but also block the PD-1/L1 interaction at a similar dose range with monoclonal antibody.

A phase I trial of autologous RAK cell immunotherapy in metastatic renal cell carcinoma.

BACKGROUND: Treatment of metastatic renal cell carcinoma (mRCC) remains a challenge worldwide. Here, we introduced a phase I trial of autologous RAK cell therapy in patients with mRCC whose cancers progressed after prior systemic therapy. Although RAK cells have been used in clinic for many years, there has been no dose-escalation study to demonstrate its safety and efficacy. METHODS: We conducted a phase I trial with a 3 + 3 dose-escalation design to investigate the dose-related safety and efficacy of RAK cells in patients with mRCC whose cancers have failed to response to systemic therapy (ChiCTR1900021334). RESULTS: Autologous RAK cells, primarily composed of CD8+ T and NKT cells, were infused intravenously to patients at a dose of 5 × 109, 1 × 1010 or 1.5 × 1010 cells every 28 days per cycle. Our study demonstrated general safety of RAK cells in a total of 12 patients. Four patients (33.3%) showed tumor shrinkage, two of them achieved durable partial responses. Peripheral blood analysis showed a significant increase in absolute counts of CD3+ and CD8+ T cells after infusion, with a greater fold change observed in naive CD8+ T cells (CD8+CD45RA+). Higher peak values of IL-2 and IFN-γ were observed in responders after RAK infusion. CONCLUSION: This study suggests that autologous RAK cell immunotherapy is safe and has clinical activity in previously treated mRCC patients. The improvement in peripheral blood immune profiling after RAK cell infusion highlights its potential as a cancer treatment. Further investigation is necessary to understand its clinical utility.

[Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/ß-catenin pathway].

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and ß-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, ß-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3ß(GSK-3ß) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and ß-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3ß and an increased protein content of Wnt3a, ß-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and ß-catenin was downregulated. The protein content of E-cadherin and GSK-3ß increased, while that of Wnt3a, ß-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/ß-catenin pathway.

IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Is There a Current Role for Combination Chemotherapy or High-Dose Interleukin 2 in Melanoma?

ABSTRACT: Immune checkpoint inhibition and targeted therapies have revolutionized the treatment of melanoma. However, chemotherapy and interleukin 2 (IL-2) therapy may still have a role in the later-line treatment of patients who do not have durable responses to other treatments. Chemotherapy can work transiently in patients whose disease has progressed on immune checkpoint inhibitors and for whom there are no appropriate targeted therapy options. High-dose IL-2 therapy can still be effective for a very small number of patients following progression on other therapies. In addition, modified IL-2 agents and IL-2 in combination with tumor-infiltrating lymphocyte therapy may play a role in future treatments for melanoma.

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

Predicting the mechanism of action of YQYYJD prescription in the treatment of non-small cell lung cancer using transcriptomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of the herbal formula Yiqi Yangyin Jiedu (YQYYJD) in the treatment of advanced lung cancer has been reported in clinical trials. However, the key anti-lung cancer herbs and molecular mechanisms underlying its inhibition of lung cancer are not well-understood. AIM OF THE STUDY: To identify the key anti-lung cancer herbs in the YQYYJD formula and investigate their therapeutic effect and potential mechanism of action in non-small cell lung cancer (NSCLC) using transcriptomics and bioinformatics techniques. MATERIALS AND METHODS: A mouse Lewis lung carcinoma (LLC) subcutaneous inhibitory tumor model was established with 6 mice in each group. Mice were treated with the YQYYJD split formula: Yiqi Formula (YQ), Yangyin Formula (YY), and Ruanjian Jiedu Formula (RJJD) for 14 days. The tumor volume and mouse weight were recorded, and the status of tumor occurrence was further observed by taking photos. The tumor was stained with hematoxylin-eosin to observe its histopathological changes. Immunohistochemistry was used to detect the expression of the proliferation marker Ki67 and the apoptotic marker Caspase-3 in tumor tissues. Flow cytometry was used to detect the number of CD4+ and CD8+ T cells and cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in the spleen and tumor tissues. The differential genes of key drugs against tumors were obtained by transcriptome sequencing of tumors. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed on differential genes to obtain pathways and biological processes where targets were aggregated. TIMER2.0 and TISIDB databases were used to evaluate the impact of drugs on immune cell infiltration and immune-related genes. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: YQ, YY, and RJJD inhibited the growth of subcutaneous transplanted tumors in LLC mice to varying degrees and achieved antitumor effects by inhibiting the expression of tumor cell proliferation, apoptosis, and metastasis-related proteins. Among the three disassembled prescriptions, YQ better inhibited the growth of subcutaneous transplanted tumors in LLC mice, significantly promoted tumor necrosis, significantly increased the expression of Caspase-3 protein in tumor tissue, and significantly decreased the expression of Ki-67 (P < 0.05), thereby increasing the infiltration of CD8+ T cells. YQ significantly increased the expression of CD4+ and CD8+ T cells in tumor and splenic tissues of tumor-bearing mice and up-regulated the expression of IL-2 and IFN-γ. Transcriptome sequencing and bioinformatics results showed that after YQ intervention, differentially expressed genes were enriched in more than one tumor-related pathway and multiple immune regulation-related biological functions. There were 12 key immune-related target genes. CONCLUSION: YQ was the key disassembled prescription of YQYYJD, exerting significant antitumor effects and immune regulation effects on NSCLC. It may have relieved T cell exhaustion and regulated the immune microenvironment to exert antitumor effects by changing lung cancer-related targets, pathways, and biological processes.

PRMT5 Promotes T follicular helper Cell Differentiation and Germinal Center Responses during Influenza Virus Infection.

Protein arginine methyltransferases (PRMTs) modify diverse protein targets and regulate numerous cellular processes; yet, their contributions to individual effector T cell responses during infections are incompletely understood. In this study, we identify PRMT5 as a critical regulator of CD4+ T follicular helper cell (Tfh) responses during influenza virus infection in mice. Conditional PRMT5 deletion in murine T cells results in an almost complete ablation of both Tfh and T follicular regulatory populations and, consequently, reduced B cell activation and influenza-specific Ab production. Supporting a potential mechanism, we observe elevated surface expression of IL-2Rα on non-T regulatory effector PRMT5-deficient T cells. Notably, IL-2 signaling is known to negatively impact Tfh differentiation. Collectively, our findings identify PRMT5 as a prominent regulator of Tfh programming, with potential causal links to IL-2 signaling.

Interleukin-2/anti-interleukin-2 immune complex attenuates cold ischemia-reperfusion injury after kidney transplantation by increasing renal regulatory T cells.

BACKGROUND: Cold ischemia-reperfusion injury (IRI) is an unavoidable complication of kidney transplantation. We investigated the role of regulatory T cells (Treg) in cold IRI and whether the interleukin (IL)-2/anti-IL-2 antibody complex (IL-2C) can ameliorate cold IRI. METHODS: We developed a cold IRI mouse model using kidney transplantation and analyzed the IL-2C impact on cold IRI in acute, subacute and chronic phases. RESULTS: Treg transfer attenuated cold IRI, while Treg depletion aggravated cold IRI. Next, IL-2C administration prior to IRI mitigated acute renal function decline, renal tissue damage and apoptosis and inhibited infiltration of effector cells into kidneys and pro-inflammatory cytokine expression on day 1 after IRI. On day 7 after IRI, IL-2C promoted renal regeneration and reduced subacute renal damage. Furthermore, on day 28 following IRI, IL-2C inhibited chronic fibrosis. IL-2C decreased reactive oxygen species-mediated injury and improved antioxidant function. When IL-2C was administered following IRI, it also increased renal regeneration with Treg infiltration and suppressed renal fibrosis. In contrast, Treg depletion in the presence of IL-2C eliminated the positive effects of IL-2C on IRI. CONCLUSION: Tregs protect kidneys from cold IRI and IL-2C inhibited cold IRI by increasing the renal Tregs, suggesting a potential of IL-2C in treating cold IRI. KEY POINTS: Interleukin (IL)-2/anti-IL-2 antibody complex attenuated acute renal injury, facilitated subacute renal regeneration and suppressed chronic renal fibrosis after cold ischemia-reperfusion injury (IRI) by increasing the renal Tregs. IL-2/anti-IL-2 antibody complex decreased reactive oxygen species-mediated injury and improved antioxidant function. This study suggests the therapeutic potential of the IL-2/anti-IL-2 antibody complex in kidney transplantation-associated cold IR.

Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models.

Cytokine release syndrome (CRS) is a significant side-effect of conventional chimeric antigen receptor (CAR) T-cell therapy. To facilitate patient accessibility, short-term (st) CAR T cells, which are administered to patients only 24 h after vector exposure, are in focus of current investigations. Their impact on the incidence and severity of CRS has been poorly explored. Here, we evaluated CD19-specific stCAR T cells in preclinical models. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and potent release of CRS-related cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered to NSG-SGM3 mice, stCAR T cells, but not conventional CAR T cells, induced severe acute adverse events within 24 h, including hypothermia and weight loss, as well as high body scores, independent of the presence of tumor target cells. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines, typical for severe CRS, were systemically elevated. Our data highlight potential safety risks of rapidly manufactured CAR T cells and suggest NSG-SGM3 mice as sensitive model for their preclinical safety evaluation.