Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

BACKGROUND: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). METHODS: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. RESULTS: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. CONCLUSIONS: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Pre-Clinical Assessment of SAR442257, a CD38/CD3xCD28 Trispecific T Cell Engager in Treatment of Relapsed/Refractory Multiple Myeloma.

Current treatment strategies for multiple myeloma (MM) are highly effective, but most patients develop relapsed/refractory disease (RRMM). The anti-CD38/CD3xCD28 trispecific antibody SAR442257 targets CD38 and CD28 on MM cells and co-stimulates CD3 and CD28 on T cells (TCs). We evaluated different key aspects such as MM cells and T cells avidity interaction, tumor killing, and biomarkers for drug potency in three distinct cohorts of RRMM patients. We found that a significantly higher proportion of RRMM patients (86%) exhibited aberrant co-expression of CD28 compared to newly diagnosed MM (NDMM) patients (19%). Furthermore, SAR442257 mediated significantly higher TC activation, resulting in enhanced MM killing compared to bispecific functional knockout controls for all relapse cohorts (Pearson's r = 0.7). Finally, patients refractory to anti-CD38 therapy had higher levels of TGF-ß (up to 20-fold) compared to other cohorts. This can limit the activity of SAR442257. Vactoserib, a TGF-ß inhibitor, was able to mitigate this effect and restore sensitivity to SAR442257 in these experiments. In conclusion, SAR442257 has high potential for enhancing TC cytotoxicity by co-targeting CD38 and CD28 on MM and CD3/CD28 on T cells.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

PD-1-CD28-enhanced receptor and CD19 CAR-modified tumor-infiltrating T lymphocytes produce potential anti-tumor ability in solid tumors.

The limited expansion ability and functional inactivation of T cells within the solid tumor microenvironment are major problems faced during in the application of using tumor-infiltrating lymphocytes (TILs) in vivo. We sought to determine whether TILs carrying a PD-1-CD28-enhanced receptor and CD19 CAR could overcome this limitation and mediate tumor regression. First, anti-tumor effects of PD-1-CD28-enhanced receptor or CD19 CAR modified NY-ESO-1-TCR-T cells to mimic the TILs function (hereafter "PD-1-CD28-TCR-T" or "CD19 CAR-TCR-T" cells, respectively) were tested using the NY-ESO-1 over-expressed tumor cell line in vitro and in a tumor-bearing model. Furthermore, the safety and anti-tumor ability of S-TILs (TILs modified through transduction with a plasmid encoding the PD-1-CD28-T2A-CD19 CAR) were evaluated in vivo. PD-1-CD28-TCR-T cells showed a formidable anti-tumor ability that was not subject to PD-1/PD-L1 signaling in vivo. CD19 CAR-TCR-T cells stimulated with CD19+ B cells exhibited powerful expansion and anti-tumor abilities both in vitro and in vivo. Three patients with refractory solid tumors received S-TILs infusion. No treatment-related mortality was observed, and none of the patients experienced serious side effects. One patient with melanoma achieved a partial response, and two patients with colon or kidney cancer achieved long-term stable disease following S-TILs therapy. To the best of our knowledge, this is the first study describing the safety and efficacy of the adoptive transfer of autologous S-TILs to control disease in patients with advanced cancers, suggesting that S-TILs may be a promising alternative therapy for cancer.

TOP CAR with TMIGD2 as a safe and effective costimulatory domain in CAR cells treating human solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy shows impressive efficacy treating hematologic malignancies but requires further optimization in solid tumors. Here, we developed a TMIGD2 optimized potent/persistent (TOP) CAR that incorporated the costimulatory domain of TMIGD2, a T and NK cell costimulator, and monoclonal antibodies targeting the IgV domain of B7-H3, an immune checkpoint expressed on solid tumors and tumor vasculature. Comparing second- and third-generation B7-H3 CARs containing TMIGD2, CD28, and/or 4-1BB costimulatory domains revealed superior antitumor responses in B7-H3.TMIGD2 and B7-H3.CD28.4-1BB CAR-T cells in vitro. Comparing these two constructs using in vivo orthotopic human cancer models demonstrated that B7-H3.TMIGD2 CAR-T cells had equivalent or superior antitumor activity, survival, expansion, and persistence. Mechanistically, B7-H3.TMIGD2 CAR-T cells maintained mitochondrial metabolism; produced less cytokines; and established fewer exhausted cells, more central memory cells, and a larger CD8/CD4 T cell ratio. These studies demonstrate that the TOP CAR with TMIGD2 costimulation offered distinct benefits from CD28.41BB costimulation and is effective against solid tumors.

CD28-expressing δ T cells are increased in perivascular adipose tissue of hypertensive mice and in subcutaneous adipose tissue of obese humans.

OBJECTIVES: γδ T-lymphocytes play a role in angiotensin II (AngII)-induced hypertension, vascular injury and T-cell infiltration in perivascular adipose tissue (PVAT) in mice. Mesenteric arteries of hypertensive mice and subcutaneous arteries from obese humans present similar remodeling. We hypothesized that γδ T-cell subtypes in mesenteric vessels with PVAT (MV/PVAT) from hypertensive mice and subcutaneous adipose tissue (SAT) from obese humans, who are prone to develop hypertension, would be similar. METHODS: Mice were infused with AngII for 14 days. MV/PVAT T-cells were used for single-cell RNA-sequencing (scRNA-seq). scRNA-seq data (GSE155960) of SAT CD45 + cells from three lean and three obese women were downloaded from the Gene Expression Omnibus database. RESULTS: δ T-cell subclustering identified six δ T-cell subtypes. AngII increased T-cell receptor δ variable 4 ( Trdv4 ) + γδ T-effector memory cells and Cd28high δ T EM -cells, changes confirmed by flow cytometry. δ T-cell subclustering identified nine δ T-cell subtypes in human SAT. CD28 expressing δ T-cell subclustering demonstrated similar δ T-cell subpopulations in murine MV/PVAT and human SAT. Cd28+ γδ NKT EM and Cd28high δ T EM -cells increased in MV/PVAT from hypertensive mice and CD28high δ T EM -cells in SAT from obese women compared to the lean women. CONCLUSION: Similar CD28 + δ T-cells were identified in murine MV/PVAT and human SAT. CD28 high δ T EM -cells increased in MV/PVAT in hypertensive mice and in SAT from humans with obesity, a prehypertensive condition. CD28 + δ T-lymphocytes could have a pathogenic role in human hypertension associated with obesity, and could be a potential target for therapy.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

Non-coding autoimmune risk variant defines role for ICOS in T peripheral helper cell development.

Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4+ T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.

Differential Impact of CD43 and CD28 on T-Cell Differentiation Depending on the Order of Engagement with the TCR.

The combination of signals from the T-cell receptor (TCR) and co-stimulatory molecules triggers transcriptional programs that lead to proliferation, cytokine secretion, and effector functions. We compared the impact of engaging the TCR with CD28 and/or CD43 at different time points relative to TCR engagement on T-cell function. TCR and CD43 simultaneous engagement resulted in higher CD69 and PD-1 expression levels than in TCR and CD28-stimulated cells, with a cytokine signature of mostly effector, inflammatory, and regulatory cytokines, while TCR and CD28-activated cells secreted all categories of cytokines, including stimulatory cytokines. Furthermore, the timing of CD43 engagement relative to TCR ligation, and to a lesser degree that of CD28, resulted in distinct patterns of expression of cytokines, chemokines, and growth factors. Complete cell activation was observed when CD28 or CD43 were engaged simultaneously with or before the TCR, but ligating the TCR before CD43 or CD28 failed to complete a cell activation program regarding cytokine secretion. As the order in which CD43 or CD28 and the TCR were engaged resulted in different combinations of cytokines that shape distinct T-cell immune programs, we analyzed their upstream sequences to assess whether the combinations of cytokines were associated with different sets of regulatory elements. We found that the order in which the TCR and CD28 or CD43 are engaged predicts the recruitment of specific sets of chromatin remodelers and TFSS, which ultimately regulate T-cell polarization and plasticity. Our data underscore that the combination of co-stimulatory molecules and the time when they are engaged relative to the TCR can change the cell differentiation program.

CD28null T cells in aging and diseases: From biology to assessment and intervention.

CD28null T cells, an atypical subset characterized by the loss of CD28 costimulatory molecule expression, exhibit functional variants and progressively expand with age. Moreover, T cells with these phenotypes are found in both typical and atypical humoral immune responses. Consequently, they accumulate during infectious diseases, autoimmune disorders, cardiovascular conditions, and neurodegenerative ailments. To provide an in-depth review of the current knowledge regarding CD28null T cells, we specifically focus on their phenotypic and functional characteristics as well as their physiological roles in aging and diseases. While uncertainties regarding the clinical utility remains, we will review the following two crucial research perspectives to explore clinical translational applications of the research on this specific T cell subset: 1) addressing the potential utility of CD28null T cells as immunological markers for prognosis and adverse outcomes in both aging and disease, and 2) speculating on the potential of targeting CD28null T cells as an interventional strategy for preventing or delaying immune aging processes and disease progression.

Persistence of peripheral CD8 + CD28- T cells indicates a favourable outcome and tumour immunity in first-line HER2-positive metastatic breast cancer.

BACKGROUND: The contradictory role of CD8 + CD28- T cells in tumour immunity has been reported, while their biological and clinical significance in HER2-positive metastatic breast cancer (MBC) is still unknown. METHODS: HER2-positive MBC patients with no prior therapy in the metastatic setting were retrospectively recruited at two medical centres. Peripheral CD8 + CD28- T cells (pTCD8+CD28-) were detected at baseline and following therapeutic intervals. Progression-free survival (PFS) was compared according to pTCD8+CD28- levels. The molecular features of pTCD8+CD28- and its correlation with tumour immunity were also investigated. RESULTS: A total of 252 patients were enrolled, and the median follow-up time was 29.6 months. pTCD8+CD28- high at baseline has prolonged PFS compared to pTCD8+CD28- low (P = 0.001). Patients who maintained pTCD8+CD28- high had a longer PFS than those who kept pTCD8+CD28- low (P < 0.001). The enhanced pTCD8+CD28- level also indicates a longer PFS compared to pTCD8+CD28- low (P = 0.025). Here, pTCD8+CD28- was demonstrated as an antigen-experienced effector T cell. Higher IL-2 level (P = 0.034) and lower TGF-ß level (P = 0.016) in the serum and highly infiltrated CD8 + CD28- T cells (P = 0.037) were also connected to pTCD8+CD28- high. CONCLUSIONS: High pTCD8+CD28- level is associated with a favourable tumour immunity and a better PFS of HER2-targeting therapy in MBC patients.

NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

The B7:CD28 family and friends: Unraveling coinhibitory interactions.

Immune responses must be tightly regulated to ensure both optimal protective immunity and tolerance. Costimulatory pathways within the B7:CD28 family provide essential signals for optimal T cell activation and clonal expansion. They provide crucial inhibitory signals that maintain immune homeostasis, control resolution of inflammation, regulate host defense, and promote tolerance to prevent autoimmunity. Tumors and chronic pathogens can exploit these pathways to evade eradication by the immune system. Advances in understanding B7:CD28 pathways have ushered in a new era of immunotherapy with effective drugs to treat cancer, autoimmune diseases, infectious diseases, and transplant rejection. Here, we discuss current understanding of the mechanisms underlying the coinhibitory functions of CTLA-4, PD-1, PD-L1:B7-1 and PD-L2:RGMb interactions and less studied B7 family members, including HHLA2, VISTA, BTNL2, and BTN3A1, as well as their overlapping and unique roles in regulating immune responses, and the therapeutic potential of these insights.

Co-stimulatory pathway competitive assay development using Liquid chromatography-tandem mass spectrometry (LC-MS/MS).

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Costimulatory receptors in the channel catfish: CD28 family members and their ligands.

The CD28-B7 interaction is required to deliver a second signal necessary for T-cell activation. Additional membrane receptors of the CD28 and B7 families are also involved in immune checkpoints that positively or negatively regulate leukocyte activation, in particular T lymphocytes. BTLA is an inhibitory receptor that belongs to a third receptor family. Fish orthologs exist only for some of these genes, and the potential interactions between the corresponding ligands remain mostly unclear. In this work, we focused on the channel catfish (Ictalurus punctatus), a long-standing model for fish immunology, to analyze these co-stimulatory and co-inhibitory receptors. We identified one copy of cd28, ctla4, cd80/86, b7h1/dc, b7h3, b7h4, b7h5, two btla, and four b7h7 genes. Catfish CD28 contains the highly conserved mammalian cytoplasmic motif for PI3K and GRB2 recruitment, however this motif is absent in cyprinids. Fish CTLA4 share a C-terminal putative GRB2-binding site but lacks the mammalian PI3K/GRB2-binding motif. While critical V-domain residues for human CD80 or CD86 binding to CD28/CTLA4 show low conservation in fish CD80/86, C-domain residues are highly conserved, underscoring their significance. Catfish B7H1/DC had a long intracytoplasmic domain with a P-loop-NTPase domain that is absent in mammalian sequences, while the lack of NLS motif in fish B7H4 suggests this protein may not regulate cell growth when expressed intracellularly. Finally, there is a notable expansion of fish B7H7s, which likely play diverse roles in leukocyte regulation. Overall, our work contributes to a better understanding of fish leukocyte co-stimulatory and co-inhibitory receptors.