Murine CD4 Research Articles

Murine CD4+ T cells exhibit sexually dimorphic responses to estrogen signaling

Abstract Many autoimmune diseases exhibit sexual dimorphism. 17β-estradiol (estrogen, “E2”), a steroid sex hormone primarily known for its reproductive roles, has also been shown to modulate CD4+ T cells. E2 signals through two nuclear receptors, ERα and ERβ, which regulate gene transcription through direct DNA binding and other mechanisms. We previously showed that in the SAMP/YitFC model of Crohn’s-like ileitis, loss of ERβ enhanced inflammation selectively in female mice. We also found that loss of ERβ resulted in decreased expression of Foxp3 in CD4+ T cells, suggesting that ERβ is required for differentiation of Foxp3+ Tregs. These prior findings suggest a pro-inflammatory role for ERα and anti-inflammatory role for ERβ, potentially in a sex-specific manner. In the current study, we investigated mechanisms of E2 signaling in CD4+ T cells isolated from males vs. females. Using standard in vitro assays, we found that the proliferation of TGFβ-induced regulatory T cells (Tregs) was enhanced in female cells co-treated with E2 compared to male cells, suggesting that E2 enhances Treg differentiation more strongly in females. Interestingly, using the CD45RB T cell transfer model of colitis, we found that recipients of female donor cells exhibited more severe disease compared to recipients of male cells, suggesting that female-derived CD45RB highT cells are more colitogenic than male cells. Future studies will determine the effect of E2 signaling on both effector and regulatory CD4+ T cell populations, identifying the contributions of ERα vs. ERβ-specific signaling in these T cell subsets and the functional impact of these signaling pathways in males versus females. Supported by grants from NIH (R01DK128143, R03DK123579, T32AI089474)

Prohibitin is Upregulated on Donor T cells in Murine Acute Graft-versus-Host Disease and Promotes Th17 Response

Abstract Introduction: Acute graft-versus-host disease (GVHD) is a T cell-mediated disorder and the main cause of non-relapse mortality in allogeneic hematopoietic cell transplantation recipients. Here, we investigate PHB in Th17-mediated inflammation and as a therapeutic target for acute GVHD. Methods: Irradiated mice received T cell depleted bone marrow cells and allogeneic or syngeneic splenocytes. At day 25 post-transplant, blood, spleen, liver, and colon were harvested and analyzed for surface PHB, IFNγ, and IL-17 on donor T cells by flow cytometry. Splenocytes were cultured in the presence of PHB pharmacological inhibitor rocaglamide (RocA) and analyzed for PHB and IL-17 expression in donor T cells. Naïve murine CD4 T cells were cultured in Th17 polarizing conditions with RocA. CRISPR/Cas9-mediated knockout of PHB was performed followed by evaluation of Il17a, Il17f, and Il22 gene expression by quantitative PCR. Results: Surface expression of PHB is increased in murine allogeneic donor T cells in liver, spleen, colon, and peripheral blood in acute GVHD. PHB-expressing splenic T cells secrete IL-17, which is reduced with treatment with a PHB inhibitor in ex vivo experiments. PHB inhibition of murine CD4 T cells in vitro impairs differentiation into the Th17 subtype resulting in reduced IL-17 secretion and downregulation of Il17a, Il17f, and Il22 genes. Knockout of PHB reduces transcription of Il17 and Il22 genes. Conclusions: These findings show relevancy of PHB in Th17-mediated inflammation and suggest a potential role for PHB in acute GVHD. To our knowledge, this study is the first to investigate PHB in acute GVHD, and our findings will offer greater support for clinical use of PHB inhibition to treat this fatal disease. Supported by grants from AAI (KS, PR), ACS (PR; RSG-22-053-01-IBCD), and NIH NCI (PR; R01 CA252469).

Inflammasome activation occurs in CD4 +and CD8 +T cells during graft-versus-host disease

Abstract A severe complication of hematopoietic stem cell transplantation is graft-versus-host disease (GvHD), a reaction that occurs following the transfer of donor immune cells (the graft) into an allogeneic host. Transplanted cells recognize host alloantigens as foreign, resulting in the activation of donor T cells and migration of these pathological cells into host tissues. In this study, we found that caspase-1 is active in alloreactive murine and human CD4 +and CD8 +T cells early during acute GvHD (aGvHD) development. The presence of inflammasome-bound, active caspase-1 (p33) and ASC speck formation confirmed inflammasome activation in these cells. Isolated T cells with high levels of active caspase-1 had a strong inflammatory transcriptional signature and a metabolic phenotype similar to inflammatory myeloid cells, including the upregulation of proinflammatory cytokines and metabolic switch from oxidative phosphorylation to aerobic glycolysis. We also observed oxidative stress and mitochondrial dysfunction, in alloreactive T cells. For the first time, this study characterizes caspase-1 activation in transplanted T cells during aGvHD, using mouse and human models, adding to a body of literature supporting inflammasome function in cells of the adaptive immune system. NIH

Unfolded Protein Response Factor ATF6 Augments T Helper Cell Responses and Promotes Mixed Granulocytic Airway Inflammation

Abstract The unfolded protein response (UPR) plays a critical role in many diseases, including airway inflammation. Several recent studies demonstrated a pathogenic role of Activating transcription factor 6a (ATF6a or ATF6), one of the essential arms of UPR, in airway structural cells. However, its role in T helper (TH) cells has not been well examined. In this study, we found that ATF6 was selectively induced by STAT6 and STAT3 in TH2 and TH17 cells, respectively. ATF6 upregulated UPR genes and promoted the differentiation and cytokine production of TH2 and TH17 cells. T cell-specific Atf6-deficiency impaired TH2 and TH17 responses in vitro and in vivo and attenuated mixed granulocytic experimental asthma. ATF6 inhibitor Ceapin A7 suppressed ATF6 downstream gene expression and TH cell cytokine expression in both murine and human memory CD4 +T cells. Administration of Ceapin A7 lessened both TH2 and TH17 responses in vivo, leading to alleviation of both airway neutrophilia and eosinophilia. Thus, our results demonstrate a novel role of ATF6 in TH2 and TH17 cell-driven mixed granulocytic airway disease, suggesting a novel option to combat steroid-resistant mixed and even T2-low endotypes of asthma through targeting ATF6. This work was supported in part by NIH grants HL148337 and AI142200 (X.O.Y.), and DK110439 and DK132643 (M.L.). We acknowledge the University of New Mexico Comprehensive Cancer Center Flow Cytometry Core supported by NIH CA118100 and the Autophagy, Inflammation, and Metabolism in Disease Center Inflammation and Metabolism Core supported by NIH GM121176

Metformin improves cancer immunotherapy by directly rescuing tumor-infiltrating CD8 T lymphocytes from hypoxia-induced immunosuppression

BackgroundDespite their revolutionary success in cancer treatment over the last decades, immunotherapies encounter limitations in certain tumor types and patients. The efficacy of immunotherapies depends on tumor antigen-specific CD8 T-cell...

Reducing PI3Kδ signaling in a dose-dependent manner promotes human T cell stemness

Abstract Adoptive T Cell Therapy (ACT) is a promising type of highly personalized immunotherapy against cancer. However, during the clinical production of T cell products for ACT, the cells experience cues that lead to early senescence and poor quality. Thus, ways to improve T cell quality for ACT is warranted. Previous mouse data indicate that inhibiting the PI3Kδ pathway in T cells using Idelalisib, an FDA-approved selective inhibitor of the δ subunit of PI3K, produces less differentiated T cells with enhanced efficacy against cancer. We hypothesized that Idelalisib treatment in human T cells would recapitulate a dose-dependent acquisition of stemness features. Mouse T cells, bulk donor human peripheral blood mononuclear cells (PBMCs), and isolated human CD3+ T cells were cultured in vitrofor seven days with dose escalation of Idelalisib. We counted the number of T cells to track expansion and assessed the phenotype through flow cytometry. Reducing PI3Kδ signaling produced a less differentiated phenotype in both murine and human CD3+ T cells. Human CD3+ T cells had lower expression of TCF-1 and LEF-1, transcription factors regulating stemness, due to Idelalisib. However, other markers (CD62L and CD45RA) still suggested dose escalation up to 25 μM led to less differentiation. PBMCs and CD3+ T cells treated with dosages of Idelalisib above 10 μM had limited expansion and decreased viability compared to mouse T cells. Altogether, our data demonstrate that blocking PI3Kδ with Idelalisib in human T cells supports acquisition of stemness features.

Mitochondrial translation inhibition in CD4+T cells selectively sensitizes the Th2 subset to increased cell death

Abstract Upon stimulation with antigen, resting T cells undergo extensive proliferation followed by differentiation into effector cells and long-lived memory cells. Upregulation of mitochondrial oxidative phosphorylation (OXPHOS) is essential for the activation and proliferation of resting T cells. Mitochondrial DNA encodes 13 proteins, which are integral parts of the complexes that form the mitochondrial respiratory chain. Since mitochondrial ribosomes are evolutionarily similar to bacterial ribosomes, antibiotics targeting bacterial ribosomes inhibit mitochondrial protein synthesis as well. But how these antibiotics affect T cell proliferation and differentiation is not well understood. To address this, we used chloramphenicol to inhibit mitochondrial translation during the activation of naive murine CD4+T cells in Th1 or Th2 polarizing conditions. Cell proliferation and effector functions were reduced in both Th1 and Th2 cells when activated in presence of chloramphenicol. Moreover, chloramphenicol significantly altered metabolic reprogramming in Th1 and Th2 cells indicated by a lower rate of OXPHOS and upregulation of glycolysis to maximum levels. Interestingly, inhibition of mitochondrial translation resulted in increased apoptosis following activation specifically in the Th2 effector subset. Th2 cells displayed a significant increase in caspase 3/7 activity upon mitochondrial translation inhibition. RNA sequencing analysis identified the upregulation of Bim and Puma in Th2 cells, two pro-apoptotic proteins that are known to be upregulated in response to metabolic stress. These results show that Th2 cells specifically undergo apoptosis in response to antibiotics that target mitochondrial translation. Support from ICMR, NII-DBT

The Tox Gene Encodes Two Proteins with Distinct and Shared Roles in Gene Regulation.

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection-associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

Thelper1 and T helper2 cytokine production patterns in stressed beta2-adrenergic receptor-deficient mice during chlamydia genital infection.

Murine CD4+ T cells have two distinct cytokine secretion patterns to play different functions. We have demonstrated that cold-induced stress (CIS) suppresses the immune system leading to increased intensity of Chlamydia muridaum in mice. We have reported that beta2-adrenergic receptor (b2-AR) knockout (KO) mice resist chlamydia genital infection. However, the cytokine profile of CD4+ T cells is not well explored. This study aimed to determine the cytokine production of Th1 and Th2 types in stressed and non-stressed b2-AR KO mice. We investigated the cytokine production levels of stressed and non-stressed mice during C. muridarum genital infection. Significantly increased production of cytokines was observed in plates pre-coated with anti-CD3/CD28 or in the presence of Con A or LPS. A decrease in the production of IFN-g and IL-2, whereas an increase in the secretion of IL-10, IL-13, and IL-23 in CD4+ T cells of stressed wild-type mice was obtained; however, the secretion of IFN-g and IL-2 was restored in T cells of b2-AR KO mice. Moreover, in vitro proliferation of CD4 T cells in the presence of b2-AR antagonists, ICI 118, 551 stimulated the production of Th1 cytokines, whereas b2-AR agonist, Fenoterol, decreased the production of Th1-type cytokines. Overall, Th1 cytokine responses are reduced in stressed mice suggesting that the cytokine status was polarized toward a Th2 immune response that can be restored by removing b2-AR from immune cells.

CD4+ T cell memory is impaired by species-specific cytotoxic differentiation, but not by TCF-1 loss.

CD4+ T cells are typically considered as 'helper' or 'regulatory' populations that support and orchestrate the responses of other lymphocytes. However, they can also develop potent granzyme (Gzm)-mediated cytotoxic activity and CD4+ cytotoxic T cells (CTLs) have been amply documented both in humans and in mice, particularly in the context of human chronic infection and cancer. Despite the established description of CD4+ CTLs, as well as of the critical cytotoxic activity they exert against MHC class II-expressing targets, their developmental and memory maintenance requirements remain elusive. This is at least in part owing to the lack of a murine experimental system where CD4+ CTLs are stably induced. Here, we show that viral and bacterial vectors encoding the same epitope induce distinct CD4+ CTL responses in challenged mice, all of which are nevertheless transient in nature and lack recall properties. Consistent with prior reports, CD4+ CTL differentiation is accompanied by loss of TCF-1 expression, a transcription factor considered essential for memory T cell survival. Using genetic ablation of Tcf7, which encodes TCF-1, at the time of CD4+ T cell activation, we further show that, contrary to observations in CD8+ T cells, continued expression of TCF-1 is not required for CD4+ T cell memory survival. Whilst Tcf7-deficient CD4+ T cells persisted normally following retroviral infection, the CD4+ CTL subset still declined, precluding conclusive determination of the requirement for TCF-1 for murine CD4+ CTL survival. Using xenotransplantation of human CD4+ T cells into murine recipients, we demonstrate that human CD4+ CTLs develop and persist in the same experimental conditions where murine CD4+ CTLs fail to persist. These observations uncover a species-specific defect in murine CD4+ CTL persistence with implications for their use as a model system.

Abstract 2737: A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice

Abstract In the United States, colorectal cancer (CRC) is the second leading cause of cancer-related death in both sexes. Immune checkpoint blockade therapy (ICBT) has emerged as a powerful new tool for cancer therapy. However, only CRCs associated with microsatellite instability (MSI) or DNA mismatch repair gene defects (dMMR, ~15% of all CRCs) are sensitive to this therapy. This poor sensitivity to ICBT is either due to poor tumor infiltration of functional T cells or T cell exhaustion. To overcome this, we investigated a combination treatment with an oncolytic virus and an immune checkpoint inhibitor (ICI). Because tumor cell oncolysis generates neoantigens and increases tumor immunogenicity, T cell infiltration into the tumor tissue will be increased and the efficacy of ICBT will be enhanced. Accordingly, local immune checkpoint inhibition coupled with oncolysis may be an ideal ICBT. In the present study, a novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. Fourteen candidate peptide sequences were generated using the PinaColada algorithm and the x-ray structure of the PD-1:PD-L1 complex and screened in molecular dynamics simulations on the microsecond timescale. Two were chosen for experimental testing, wherein the peptide denoted PD-L1ip3 showed a binding affinity for PD-L1 in the micromolar range. In cell culture studies, treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by antigens from CT26 cells. In immunocompetent mice, growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells. This tumor growth attenuation was further enhanced by cotreatment with the peptide form of PD-L1ip3. The present study suggests that this peptide can stimulate host antitumor immunity via a blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth. This research was supported by Kansas State University Johnson Cancer Research center (MT), K-INBRE Scholar Award (MB), National Cancer Institute (MT, JC) and the National Science Foundation (JC). Citation Format: Susumu Ishiguro, Deepa Upreti, Molly Bassette, E. R. Azhagiya Singam, Ravindra Thakkar, Mayme Loyd, Makoto Inui, Jeffrey Comer, Masaaki Tamura. A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice [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 2737.

Abstract 341: Critical role of defensins in respiratory viral infection induced breast cancer progression

Abstract Respiratory viral infections (RVI) such as influenza and COVID19 impact the host systemic immune system along with causing deleterious chronic inflammatory responses and respiratory distress. While the role of chronic inflammation in cancer is well-established, the role of RVI on tumorigenesis is poorly defined. To study the role of RVI on breast cancer, we first infected murine respiratory epithelial cells (mRES) with murine sendai virus (mSV), an analog for human parainfluenza virus. These infected mRES were co-cultured with 4T1 murine breast cancer cells in 1:1 dilution on a single 2D plate and also in trans-well format. Both in co-culture and transwell culture we saw a 40-80% (p<0.05) increased proliferation of breast cancer cells. Similarly, when 4T1 cells were treated with the supernatant collected from infected mRES cells in 1:5 dilution, also demonstrated a 2.3 fold increased breast cancer cell proliferation. The cytokine analysis from the supernatant collected from infected mRES cells demonstrated a 17-23 fold enhanced secretion of α/β-defensins. Direct treatment of α-defensin (cyptidin-4, 10 pg/mL) and β-defensin-3 (mBD3, 20 pg/mL) on 4T1 cells demonstrated enhanced expression of chemokine metastatic receptor, CXCR4 (4.3 fold), angiogenic factor, VEGF (12.8 fold) and cell division favoring factor, CDK2 (8.1 fold). Further, analysis of infected mRES cells demonstrated upregulation of toll-like receptor 2 (TLR2) and NOD-like receptor protein 3 (NLRP3) expression. Interesting, co-cultured of infected mRES with syngeneic murine CD4 T cells induced exhaustion phenotype (PD1+ and CTLA4+) differentiation of CD4 T cells. Taken together, these data suggest that respiratory viral infections through induction of cancer cell proliferation and inhibiting anti-tumor adaptive immune responses promote breast cancer proliferation. Citation Format: Umer Ali, Venkataswarup Tiriveedhi. Critical role of defensins in respiratory viral infection induced breast cancer progression [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 341.

IL-12 and IL-27 Promote CD39 Expression on CD8+ T Cells and Differentially Regulate the CD39+CD8+ T Cell Phenotype.

Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells' effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using invitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12- and IL-27-induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27-induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12-mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

The IgV domain of the poliovirus receptor alone is immunosuppressive and binds to its receptors with comparable affinity

PVR (poliovirus receptor) functions as a ligand that signals through TIGIT and CD96 to induce suppression of T-cell and NK-cell responses. Alternatively, PVR binds to CD226, resulting in a co-stimulatory signal. To date, TIGIT antibody antagonists have been developed to restore immune functions and allow PVR to signal though CD226 in the context of cancer immunotherapy. Due to PVR receptor heterogeneity, agonizing either of these pathways with a recombinant form of the PVR extracellular domain represents a therapeutic strategy for either immunosuppression or activation. Here, we developed a minimal murine PVR-Fc fusion construct, consisting of only the IgV domain of PVR (vdPVR-Fc), and assessed its ability to dampen inflammatory responses in a murine model of psoriasis. vdPVR-Fc and PVR-Fc containing the full-length extracellular domain bound to TIGIT, CD96 and CD226 with similar low nanomolar affinities as defined by surface plasmon resonance. vdPVR-Fc was also able to suppress the in-vitro proliferation of murine CD4+ and CD8+ T-cells in mixed splenocyte cultures. Importantly, vdPVR-Fc delayed the onset, and reduced inflammatory responses (scaling and thickness) in a murine model of psoriasis. Collectively, our results suggest that the minimal IgV domain of PVR is sufficient to dampen immune responses in-vitro and attenuate symptoms of psoriasis in-vivo.

T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.

Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.

A Synthetic SARS-CoV-2-Derived T-Cell and B-Cell Peptide Cocktail Elicits Full Protection against Lethal Omicron BA.1 Infection in H11-K18-hACE2 Mice.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developing the capacity for immune evasion and resistance to existing vaccines and drugs. To address this, development of vaccines against coronavirus disease 2019 (COVID-19) has focused on universality, strong T cell immunity, and rapid production. Synthetic peptide vaccines, which are inexpensive and quick to produce, show low toxicity, and can be selected from the conserved SARS-CoV-2 proteome, are promising candidates. In this study, we evaluated the effectiveness of a synthetic peptide cocktail containing three murine CD4+ T-cell epitopes from the SARS-CoV-2 nonspike proteome and one B-cell epitope from the Omicron BA.1 receptor-binding domain (RBD), along with aluminum phosphate (Al) adjuvant and 5' cytosine-phosphate-guanine 3' oligodeoxynucleotide (CpG-ODN) adjuvant in mice. The peptide cocktail induced good Th1-biased T-cell responses and effective neutralizing-antibody titers against the Omicron BA.1 variant. Additionally, H11-K18-hACE2 transgenic mice were fully protected against lethal challenge with the BA.1 strain, with a 100% survival rate and reduced pulmonary viral load and pathological lesions. Subcutaneous administration was found to be the superior route for synthetic peptide vaccine delivery. Our findings demonstrate the effectiveness of the peptide cocktail in mice, suggesting the feasibility of synthetic peptide vaccines for humans. IMPORTANCE Current vaccines based on production of neutralizing antibodies fail to prevent the infection and transmission of SARS-CoV-2 Omicron and its subvariants. Understanding the critical factors and avoiding the disadvantages of vaccine strategies are essential for developing a safe and effective COVID-19 vaccine, which would include a more effective and durable cellular response, minimal effects of viral mutations, rapid production against emerging variants, and good safety. Peptide-based vaccines are an excellent alternative because they are inexpensive, quick to produce, and very safe. In addition, human leukocyte antigen T-cell epitopes could be targeted at robust T-cell immunity and selected in the conserved region of the SARS-CoV-2 variants. Our study showed that a synthetic SARS-CoV-2-derived peptide cocktail induced full protection against lethal infection with Omicron BA.1 in H11-K18-hACE2 mice for the first time. This could have implications for the development of effective COVID-19 peptide vaccines for humans.

Modulating the proliferative and cytotoxic properties of patient-derived TIL by a synthetic immune niche of immobilized CCL21 and ICAM1.

A major challenge in developing an effective adoptive cancer immunotherapy is the ex-vivo generation of tumor-reactive cells in sufficient numbers and with enhanced cytotoxic potential. It was recently demonstrated that culturing of activated murine CD8+ T-cells on a "Synthetic Immune Niche" (SIN), consisting of immobilized CCL21 and ICAM-1, enhances T-cell expansion, increases their cytotoxicity against cultured cancer cells and suppresses tumor growth in vivo. In the study reported here, we have tested the effect of the CCL21+ICAM1 SIN, on the expansion and cytotoxic phenotype of Tumor Infiltrating Lymphocytes (TIL) from melanoma patients, following activation with immobilized anti-CD3/CD28 stimulation, or commercial activation beads. The majority of TIL tested, displayed higher expansion when cultured on the coated SIN compared to cells incubated on uncoated substrate and a lower frequency of TIM-3+CD8+ cells after stimulation with anti-CD3/CD28 beads. Comparable enhancement of TIL proliferation was obtained by the CCL21+ICAM1 SIN, in a clinical setting that included a 14-day rapid expansion procedure (REP). Co-incubation of post-REP TIL with matching target cancerous cells demonstrated increased IFNγ secretion beyond baseline in most of the TIL cultures, as well as a significant increase in granzyme B levels following activation on SIN. The SIN did not significantly alter the relative frequency of CD8/CD4 populations, as well as the expression of CD28, CD25, several exhaustion markers and the differentiation status of the expanded cells. These results demonstrate the potential capacity of the CCL21+ICAM1 SIN to reinforce TIL-based immunotherapy for cancer patients.

Hyperlipidaemia elicits an atypical, T helper 1-like CD4+ T-cell response: a key role for very low-density lipoprotein.

Hyperlipidemia and T cell driven inflammation are important drivers of atherosclerosis, the main underlying cause of cardiovascular disease. Here, we detailed the effects of hyperlipidemia on T cells. In vitro, exposure of human and murine CD4+ T cells to very low-density lipoprotein (VLDL), but not to low-density lipoprotein (LDL) resulted in upregulation of Th1 associated pathways. VLDL was taken up via a CD36-dependent pathway and resulted in membrane stiffening and a reduction in lipid rafts. To further detail this response in vivo, T cells of mice lacking the LDL receptor (LDLr), which develop a strong increase in VLDL cholesterol and triglyceride levels upon high cholesterol feeding were investigated. CD4+ T cells of hyperlipidemic Ldlr-/- mice exhibited an increased expression of the C-X-C-chemokine receptor 3 (CXCR3) and produced more interferon-γ (IFN-γ). Gene set enrichment analysis identified IFN-γ-mediated signaling as the most upregulated pathway in hyperlipidemic T cells. However, the classical Th1 associated transcription factor profile with strong upregulation of Tbet and Il12rb2 was not observed. Hyperlipidemia did not affect levels of the CD4+ T cell's metabolites involved in glycolysis or other canonical metabolic pathways but enhanced amino acids levels. However, CD4+ T cells of hyperlipidemic mice showed increased cholesterol accumulation and an increased arachidonic acid (AA) to docosahexaenoic acid (DHA) ratio, which was associated with inflammatory T cell activation. Hyperlipidemia, and especially its VLDL component induces an atypical Th1 response in CD4+ T cells. Underlying mechanisms include CD36 mediated uptake of VLDL, and an altered AA/DHA ratio.

Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells.

CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis. To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate. The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma. Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.

Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells invivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRβ-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRβ-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRβ-sufficient Tfh cells. Loss of LXRβ confers inactivation of GSK3β induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/β-catenin pathway, leading to elevated TCF-1 expression in CD4+ Tcells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ Tcells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3β-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.