Costimulatory Receptor CD28 Research Articles

CD28 co-stimulation: novel insights and applications in cancer immunotherapy.

Substantial progress in understanding T cell signalling, particularly with respect to T cell co-receptors such as the co-stimulatory receptor CD28, has been made in recent years. This knowledge has been instrumental in the development of innovative immunotherapies for patients with cancer, including immune checkpoint blockade antibodies, adoptive cell therapies, tumour-targeted immunostimulatory antibodies, and immunostimulatory small-molecule drugs that regulate T cell activation. Following the failed clinical trial of a CD28 superagonist antibody in 2006, targeted CD28 agonism has re-emerged as a technologically viable and clinically promising strategy for cancer immunotherapy. In this Review, we explore recentinsights into the molecular functions and regulation of CD28. We describe how CD28 is central to the success of current cancer immunotherapies and examine how new questions arising from studies of CD28 as a clinical target have enhanced our understanding of its biological role and may guide the development of future therapeutic strategies in oncology.

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

Differentiation of regulatory myeloid and T-cells from adult human hematopoietic stem cells after allogeneic stimulation.

Donor hematopoietic stem cell (DHSC) infusions are increasingly being studied in transplant patients for tolerance induction. To analyze the fate of infused DHSCs in patients, we developed an in vitro culture system utilizing CD34+DHSCs stimulated with irradiated allogeneic cells in cytokine supplemented medium long-term. Flow cytometric analyses revealed loss of the CD34 marker and an increase in CD33+ myeloid and CD3+ T-cell proportion by 10.4% and 72.7%, respectively, after 21 days in culture. T-cells primarily expressed TcR-αβ and were of both CD4+ and CD8+ subsets. Approximately 80% of CD3+ T cells lacked expression of the co-stimulatory receptor CD28. The CD4+ compartment was predominated by CD4+CD25+CD127-FOXP3+ Tregs (>50% CD4+CD127- compartment) with <1% of all leukocytes exhibiting a CD4+CD127+ phenotype. Molecular analyses for T-cell receptor excision circles showed recent and increased numbers of TcR rearrangements in generated T cells over time suggesting de novo differentiation from DHSCs. CD33+ myeloid cells mostly expressed HLA-DR, but lacked expression of co-stimulatory receptors CD80 and CD83. When studied as modulators in primary mixed lymphocyte reactions where the cells used to stimulate the DHSC were used as responders, the DHSC-lines and their purified CD8+, CD4+, CD33+ and linage negative subsets inhibited the responses in a dose-dependent and non-specific fashion. The CD8+ cell-mediated inhibition was due to direct lysis of responder cells. Extrapolation of these results into the clinical situation would suggest that DHSC infusions into transplant recipients may generate multiple subsets of donor "chimeric" cells and promote recipient Treg development that could regulate the anti-donor immune response in the periphery. These studies have also indicated that T cell maturation can occur in vitro in response to allogeneic stimulation without the pre-requisite of a thymic-like environment or NOTCH signaling stimulatory cell line.

DOP32 CD226+TIGITneg expression identifies pathogenic inflammatory CD4+ memory T cells in severe Crohn’s Disease patients, opening up opportunities for patient-tailored treatment strategies

Abstract Background CD4+ memory T cells (Tmem) drive chronicity of inflammation in Crohn’s disease (CD) and ulcerative colitis (UC). Treatments specifically targeting T cells effectively induce disease remission. However, due to disease heterogeneity and possibly variation in the Tmem response, not all patients achieve or maintain remission. As such, strategies are needed to identify and monitor the therapeutic target, i.e. the inflammatory Tmem response, in individual patients. Monitoring has been hampered by the large variety in both protective regulatory and pathogenic inflammatory Tmem phenotypes, thus, common denominators need to be identified to distinguish all inflammatory from all regulatory intestinal Tmem populations in individual patients. We have shown that the coinhibitory receptor T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is a common denominator identifying regulatory gut-homing Tmem. TIGIT shares its ligands with the costimulatory receptor CD226. While TIGIT ligation inhibits Tmem responses, CD226 engagement enhances Tmem activity. We hypothesize that TIGIT and CD226 differentiate the protective regulatory from the pathogenic inflammatory intestinal Tmem in individual IBD patients. Methods We studied CD38+CD62Lneg gut-homing Tmem in blood of therapy-naive pediatric IBD patients (CD n=59; UC n=32; IBD negative controls (IBDneg) n=24). Results In a subgroup of CD, but not UC patients, frequencies of regulatory TIGIT+ gut-homing Tmem at diagnosis were strongly reduced compared to IBDneg. Of these CD patients with low TIGIT frequencies, a significantly higher proportion had severe disease, defined as failing to achieve remission after 6 months or having a disease relapse within 1 year, compared to CD with normal TIGIT frequencies. As TIGIT is a common denominator for regulatory cells, we hypothesized that gut-homing Tmem without TIGIT (TIGITneg) contained inflammatory Tmem. Indeed, in CD, the TIGITneg Tmem population contained increased frequencies of pathogenic IFNg+IL-17+-double-producing cells compared to IBDneg. As TIGIT and CD226 frequencies inversely correlated (R=-0.78, p=4.8e-05) in CD patients, we assessed whether additional monitoring of CD226 could be used to more precisely identify and monitor inflammatory Tmem in CD. As anticipated, TIGITnegCD226+ gut-homing Tmem frequencies significantly increased in CD patients compared to UC patients and IBDneg. TIGITnegCD226+ Tmem were more proliferative and more activated compared to IBDneg, indicating increased inflammatory function. Conclusion These data establish that TIGIT and CD226 can be used to enumerate and differentiate the protective regulatory from the pathogenic inflammatory gut-homing Tmem in individual IBD patients, and to identify patients with severe disease.

Development of a c-MET x CD137 bispecific antibody for targeted immune agonism in cancer immunotherapy

BackgroundTargeting the costimulatory receptor CD137 has shown promise as a therapeutic approach for cancer immunotherapy, resulting in anti-tumor efficacy demonstrated in clinical trials. However, the initial CD137 agonistic antibodies, urelumab and utomilumab, faced challenges in clinical trials due to the liver toxicity or lack of efficacy, respectively. Concurrently, c-MET has been identified as a highly expressed tumor-associated antigen (TAA) in various solid and soft tumors. MethodsIn this study, we aimed to develop a bispecific antibody (BsAb) that targets both c-MET and CD137, optimizing the BsAb format and CD137 binder for efficient delivery of the CD137 agonist to the tumor microenvironment (TME). We employed a monovalent c-MET motif and a trimeric CD137 Variable Heavy domain of Heavy chain (VHH) for the BsAb design. ResultsOur results demonstrate that the c-MET x CD137 BsAb provides co-stimulation to T cells through cross-linking by c-MET-expressing tumor cells. Functional immune assays confirmed the enhanced efficacy and potency of the c-MET x CD137 BsAb, as indicated by activation of CD137 signaling, target cell killing, and cytokine release in various tumor cell lines. Furthermore, the combination of c-MET x CD137 BsAb with Pembrolizumab showed a dose-dependent enhancement of target-induced T cell cytokine release. ConclusionOverall, the c-MET x CD137 BsAb exhibits a promising developability profile as a tumor-targeted immune agonist by minimizing off-target effects while effectively delivering immune agonism. It has the potential to overcome resistance to anti-PD-(L)1 therapies.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

M9657 Is a Bispecific Tumor-Targeted Anti-CD137 Agonist That Induces MSLN-Dependent Antitumor Immunity without Liver Inflammation.

The costimulatory receptor CD137 (also known as TNFRSF9 or 4-1BB) sustains effective cytotoxic T-cell responses. Agonistic anti-CD137 cancer immunotherapies are being investigated in clinical trials. Development of the first-generation CD137-agonist monotherapies utomilumab and urelumab was unsuccessful due to low antitumor efficacy mediated by the epitope recognized on CD137 or hepatotoxicity mediated by Fcγ receptors (FcγR) ligand-dependent CD137 activation, respectively. M9657 was engineered as a tetravalent bispecific antibody (mAb2) in a human IgG1 backbone with LALA mutations to reduce binding to FCγRs. Here, we report that M9657 selectively binds to mesothelin (MSLN) and CD137 with similar affinity in humans and cynomolgus monkeys. In a cellular functional assay, M9657 enhanced CD8+ T cell-mediated cytotoxicity and cytokine release in the presence of tumor cells, which was dependent on both MSLN expression and T-cell receptor/CD3 activation. Both FS122m, a murine surrogate with the same protein structure as M9657, and chimeric M9657, a modified M9657 antibody with the Fab portion replaced with an anti-murine MSLN motif, demonstrated in vivo antitumor efficacy against various tumors in wild-type and human CD137 knock-in mice, and this was accompanied by activated CD8+ T-cell infiltration in the tumor microenvironment. The antitumor immunity of M9657 and FS122m depended on MSLN expression density and the mAb2 structure. Compared with 3H3, a murine surrogate of urelumab, FS122m and chimeric M9657 displayed significantly lower on-target/off-tumor toxicity. Taken together, M9657 exhibits a promising profile for development as a tumor-targeting immune agonist with potent anticancer activity without systemic immune activation and associated hepatotoxicity.

Apheresis for Production of Chimeric Antigen Receptor (CAR) T Cells in Patients with Multiple Myeloma - Clinical Factors, Cell Composition and T Cell Senescence

Chimeric antigen receptor (CAR) T cells have been proven as a highly promising immunotherapy for patients with multiple myeloma (MM). Apheresis of autologous CD3+ lymphocytes is the first pivotal step in the production process of chimeric antigen receptor (CAR) T cells. Repetitive activation of T lymphocytes can affect the qualitative properties, i.e. the fitness of CD3 cells leading to progressive loss of expression of the costimulatory receptors CD27 and CD28. CD3+27-28- cells are non-proliferating memory, senescent cells. In that regard, the factors influencing the manufacturing process of CAR T cells in patients with MM have not been sufficiently explored. We performed a retrospective analysis of clinical parameters influencing both collection of autologous lymphocytes and the subsequent manufacturing of idecabtagene-vicleucel with a special focus on T cell senescence, defined as loss of CD27 and CD28. Furthermore, we focused on cell-compositions of collections resulting in out of specification (OOS) events during CAR T cell manufacturing. Between January 2022 and January 2023, 36 lymphocyte collections of 32 patients with relapsed/refractory (r/r) MM were performed for subsequent CAR T cell therapy with idecabtagene-vicleucel. All collections were forwarded for further manufacturing without prior cryokonservation. 9 (25%) collections resulted in OOS; 7/9 resulting in insufficient proliferation or expansion of T cells, while 2/9 showed a microbial contamination during the manufacturing process after viral transduction for CAR generation. At apheresis, the patients were a median 63 (range 39-75) years old, 12 (38%) were female with median body weight 79 kg (49-103). The patients had a median of 7 previous treatment lines (range 3-13) and 15/36 collections were performed at partial remission or better. 8/36 collection had prior exposition to bispecific antibodies whereas in 13/36 collections the unspecific positivity for cytomegalovirus (CMV) in polymerase chain reaction (PCR) was detected. The collections were performed on a Spectra Optia cell-separator by processing median 3x total blood volume. CD3+ cells in peripheral blood (pB) were median 513.9 /µl (range 70.6-3616.0). The collections showed a median CD3+ yield with 29.8 x10 8 (range 4.9-171.6), with CD3+4+ and CD3+8+ yields 10.6 x10 8 (range 1-663.4) and 16.3 x10 8 (range 3.2-121.2) respectively, resulting in median CD4:CD8 ratio of 0.6 (range 0.1-2.6). The median yields for CD3+27+28+ and CD3+27-28- cells were 14.8 (range 2.3-88.8) x10 8 and 2.1 (range 0.1-59.8) x10 8 respectively. Noteworthy, OOS events were more common in patients with detectable cytomegalovirus replication at apheresis ( P=0.037) with a trend for prior exposition to bispecific antibodies ( P=0.086). In contrast, the collections with OOS did not differ regarding patients' age ( P=0.498), sex ( P=0.442), body weight ( P=0.364), number of previous treatment lines ( P=0.185) or remission status at apheresis ( P=0.443). In OOS collections, CD3+ cells in pB were significantly higher with median 991 /µl (range 558-2968) compared to 448.9 /µl (70.6-3616.0) ( P=0.002) and showed a trend for higher CD3+ yields with 38.9 x10 8 (range 23.9-62.2) and 28.2 x10 8 (range 4.9-171.6) respectively ( P=0.096). Interestingly, OOS products also had higher median yields of CD3+CD8+ cells with 28.1 x10 8 (range 12.2-52.3) compared to 15.3 x10 8 (range 3.2-121.2), ( P=0.046) and showed a trend for lower median CD3+CD4+ yields with 9.1 x10 8 (range 3.8-11.9) compared to 11.4 x10 8 (range 1.6-63.4), ( P=0.071). Median CD3+27+28+ yields in OOS and successful manufacturings were 9.7 x10 8 (4.7-26.0) and 17.0 x10 8(range 2.4-88.8) respectively ( P=0.030). OOS events yielded with higher median CD3+27-28- with 17.8 x10 8(range 0.2-46.1) compared to 1.4 x10 8 (0.1-59.8), ( P=0.031). We observed a negative prognostic role of CMV replication as well as of senescence of T lymphocytes on the manufacturing process. In contrast, a successful manufacturing was not influenced by patient age, sex, remission status at apheresis or number of previous treatment lines. Further analyses are required to determine measures appropriate for further optimization of autologous lymphocyte collections and improvement of collection outcomes in patients with MM.

High Response Rates Following Point-of-Care Anti-CD19 CAR T-Cell Therapy in Adults with Relapsed/Refractory Acute B Lymphoblastic Leukemia

Background Tisagenlecleucel and brexucabtagene autoleucel have demonstrated remarkable efficacy and safety results in adult patients with relapsed/ refractory acute B lymphoblastic leukemia (R/R B-ALL). Long term follow-up of the ZUMA-3 trial recently reported a median duration of response (DOR) of 14.6 months and an overall response rate of 71% (Hadjivassileva, EBMT 2023). Methods and patients We report results of a phase 1b/2 single center clinical trial (NCT02772198) involving point-of-care (POC) anti-CD19/CD28 costimulatory domain chimeric antigen receptor (CAR) T-cell therapy in adults with R/R B-ALL. Inclusion criteria were age ≥18 years, failure of at least 2 prior therapies, and a preserved organ function. Patients underwent a single leukapheresis procedure. Fresh CAR T products were delivered for immediate infusion. Lymphodepletion included fludarabine and cyclophosphamide. Cell dose was 1x10 6 CAR T cells/kg. Primary endpoints were 1-month disease response and safety. Secondary endpoints were the minimal residual disease (MRD) negativity rate, overall survival, progression-free survival (PFS), and production feasibility. Last follow-up was July 2023. Results Between 03/2017-05/2023, 28 patients enrolled. CAR T-cells were successfully produced in 27 patients (96%) who were included in the analysis. The median age was 33 years (range 19-77), and 7 (25%) patients had Karnofsky Performance Status &amp;lt;90%. Six patients (22%) had positive Philadelphia chromosome, and 14 patients (52%) had an extramedullary disease at leukapheresis. Nineteen patients (70%) had ≥3 prior lines of therapy, 18 patients (67%) underwent prior allogeneic hematopoietic stem-cell transplantation (allo-HCT), and 20 patients (74%) had prior exposure to inotuzumab-ozogamicin or blinatumomab. At leukapheresis, 16 patients (60%) had an active disease, while 11 patients (40%) were in complete morphological response (CR; MRD positive, n=7 [26%], MRD negative or not evaluated, n=4 [15%]). The median time between leukapheresis and cell infusion was 11 days (IQR 10-11). Only 3 patients (11%) received bridging chemotherapy. Grade 3-4 cytokine release syndrome and immune effector cell-associated neurotoxicity syndromes were observed in 30% of the patients, each. Six (22%) and 16 (59%) patients were treated with tocilizumab or corticosteroids, respectively. Severe neutropenia (&amp;lt;0.5k/μl) and thrombocytopenia (≤50 K/μl) occurred in 81% and 70% of the patients, respectively, and anemia requiring blood transfusion occurred in 71%. Three patients (11%) had grade 3 cardiovascular events, and 1 patient (4%) had grade 3 pleural effusion. Bloodstream bacterial infection occurred in 3 (11%) patients. Cellular therapy-related mortality was observed in 2 (7%) patients (both due to septic shock). The 1-month overall response rate was 88% (81% CR; MRD negative CR, 63%). MRD was evaluated by RT-PCR in most of the patients. The median follow-up was 19.7 months (IQR 4.7-43.2). Two-year overall survival and PFS were 62% (95% CI: 42-91), and 56% (95% CI: 38-84), respectively. The median DOR was not reached (95% CI: 6.1 months-not reached). PFS was not affected by prior exposure to inotuzumab-ozogamicin or blinatumomab (hazard ratio (HR) 0.7, 95% CI: 0.17-2.68, p=0.6). All eligible patients who achieved a response were offered consolidation with allo-HCT. Eight patients (30%) underwent allo-HCT (2 nd allo-HCT in 3 of them). The DOR was not affected by consolidation of CR with allo-HCT (HR 0.9, 95% CI: 0.2-4.1, p&amp;gt;0.9). Nevertheless, 2 patients (7%) died due to allo-HCT related complications while in CR. Conclusion Treatment with POC CAR T-cells, produced in an academic center, achieved high response and survival rates in adults with R/R B-ALL. These results are also comparable to the FDA approved CAR T-cells. The use of POC CAR T abrogates the need for cryopreservation and shipment of the cells, thus allowing a short vein-to-vein time without the need of bridging therapy in most patients.

CD19-CAR Cytokine Induced Killer Cells Armored with IL-18 Control Tumor Burden, Prolong Mouse Survival and Result in In Vivo Persistence of CAR-CIK Cells in a Model of B-Cell Acute Lymphoblastic Leukemia

Rationale: Cytokine induced killer cells (CIK) are a promising cancer immunotherapy. Recently CIK cells modified to express chimeric antigen receptors (CAR) target CD19 expressing hematological malignancies including B-cell acute lymphoblastic leukemia (B-ALL). Despite encouraging clinical trial results, insufficient CAR-CIK anti-tumor activity and persistence pose major obstacles towards improved efficacy in vivo. Therefore, we optimized our CAR-CIK platform by introducing two modifications to the CD19-CAR construct. The first modification “armored” CAR-CIK cells by inserting the IL-18 gene in a bicistronic DNA plasmid enabling simultaneous surface expression of the CAR protein and secretion of IL-18, which demonstrated improved CAR-T cell function. The second modification attenuated the CD28 cytoplasmic signaling domain of the CAR molecule to enhance anti-tumor activity and in vivo persistence since recent findings demonstrate that persistent or chronic CAR signaling can impair anti-tumor activity and in vivo persistence. Methods: CARCIK-1918 cells generated from donor-derived CIK cells are engineered to express CAR and IL-18 using the non-viral Sleeping Beauty (SB) transposon system. CIK cells were generated from donor PBMCs by the sequential addition of IFN-ϒ and stimulation with anti-CD3 antibody plus IL-2. After 2 days of stimulation CIK cells were co-electroporated with SB100X transposase mRNA and plasmid DNA containing a bicistronic transgene encoding both the anti-CD19 CAR and IL-18. This second-generation CAR contains a fusion of the CD28 transmembrane domain, intracellular CD28 and, TCR CD3ζ chain. In some instances, the cytoplasmic CD28 signaling domain is modified to attenuate CD28 signaling. This method of gene transfer results in stable surface expression of the CAR on CD3+ cells through a 17-day culture period. Day 17 cells are harvested, frozen and used for all subsequent studies. CARCIK-1918 cells are stimulated in vitro with CD19+ REH tumor cells and the phenotype of activated cells was characterized by multi-color flow cytometry. IL-18 secretion was determined by cytokine bead array analysis of supernatants collected post stimulation. CARCIK-1918 cells with or without the attenuated CD28 signaling domain were tested in vivo in a xenograft model of B-ALL, using NSG mice engrafted with CD19+ Raji tumor cells. Tumor burden was assessed weekly by bioluminescence imaging, the in vivo persistence of CARCIK-1918 cells was measured by flow cytometry, and survival was monitored throughout the study. Results: CARCIK-1918 cells express both IL-18 and CAR with or without attenuated CD28 signaling. Furthermore, stimulation of CARCIK-1918 cells with CD19+ REH tumor cells increases the frequency of CAR-CIK cells with a memory phenotype and concurrent upregulation of the high affinity IL-2 receptor, CD25, and the costimulatory receptor CD137. Expression of IL-18 leads to higher engraftment of CAR-CIK cells in tumor bearing NSG mice compared to unarmored controls. NSG mice engrafted with luciferase positive Raji cells demonstrated decreased bioluminescent signal following CARCIK-1918 treatment, consistent with CARCIK derived anti-leukemic activity. This anti-tumor activity corresponded to a statistically significant improvement in survival as mice treated with 2 nd generation CAR transgene CARCIK cells with or without attenuated CD28 signaling had a median survival of 37.5 (n=10; p=0.0001) and 38 (n=12; p =0.0001 days), respectively, compared to untreated mice that develop progressive highly disseminated tumor and succumb to disease by 3 weeks post tumor inoculation (n=11; median survival 18 days). Interestingly, mice treated with CARCIK-1918 cells with the CD28 attenuated signaling domain exhibited better tumor control and enhanced persistence in vivo, despite no significant improvement in survival compared to WT CD28 signaling CARCIK-1918 cells. Summary: This study demonstrates that administration of CAR-CIK cells armored with IL-18 results in a significant survival advantage for tumor challenged animals versus untreated animals. Furthermore, modifying the cytoplasmic CD28 domain to attenuate signaling, results in better control of tumor burden, and persistence of CARCIK-1918 cells in vivo. Our study is the first report of IL-18 armored CAR-CIK cells which may present a promising, novel strategy for the treatment of B-ALL.

Upregulation of the CD155-CD226 Axis Is Associated With Muscle Inflammation and Disease Severity in Idiopathic Inflammatory Myopathies.

The CD155-CD226/T-cell Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) pathway plays a critical role in regulating T-cell responses and is being targeted clinically. However, research on the role of this pathway in autoimmune diseases is limited. This study aimed to investigate the expression and tissue-specific roles of CD155-CD226/TIGIT pathway molecules in the inflamed muscles of patients with idiopathic inflammatory myopathies (IIMs). Immunohistochemistry, Western blot analysis, and polychromatic immunofluorescence staining were performed to examine the expression of CD155, CD226, and TIGIT in skeletal muscle biopsies from 30 patients with dermatomyositis (DM), 10 patients with amyopathic DM (ADM), 20 patients with immune-mediated necrotizing myopathy (IMNM), 5 patients with dysferlinopathy, and 4 healthy controls. Flow cytometry analysis was used to analyze the functions of T cells with different phenotypes. Strong expression of CD155 was observed in patients with DM and IMNM, while its expression was largely negative in those with ADM and dysferlinopathy and healthy controls. The costimulatory receptor CD226 was highly expressed on muscle-infiltrating cells, while the coinhibitory receptor TIGIT was expressed at low levels. These infiltrating CD226+ cells were mainly activated effector T cells that localized adjacent to CD155-expressing myofibers, but were faintly detectable within the muscle fascicles lacking CD155. A strong positive correlation between CD155 and CD226 expression scores was also observed. Polychromatic immunofluorescence staining revealed that CD155+ muscle cells coexpressed major histocompatibility complex classes I and II, and tumor necrosis factor alpha expression was detected in CD226+ T cells at their close sites with the myofibers. Furthermore, the expression levels of CD155 and CD226 showed a positive correlation with creatine kinase, lactate dehydrogenase, and the muscle histopathology damage scores and an inverse correlation with the Manual Muscle Testing-8 scores. In addition, CD155 and CD226 expressions were significantly decreased in representative patients who achieved remission posttreatment. These findings demonstrate that the CD155-CD226 axis is highly activated in inflamed muscle tissues of patients with IIM and is associated with muscle disease severity. Our data uncover the immunopathogenic role of the axis in the pathology of IIMs.

The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling

BackgroundThe inflammatory response is indispensable for protective immunity, yet microbial pathogens often trigger an excessive response, ‘cytokine storm’, harmful to the host. Full T-cell activation requires interaction of costimulatory receptors B7-1(CD80) and B7-2(CD86) expressed on antigen-presenting cells with CD28 expressed on the T cells. We created short peptide mimetics of the homodimer interfaces of the B7 and CD28 receptors and examined their ability to attenuate B7/CD28 coligand engagement and signaling through CD28 for inflammatory cytokine induction in human immune cells, and to protect from lethal toxic shock in vivo.MethodsShort B7 and CD28 receptor dimer interface mimetic peptides were synthesized and tested for their ability to attenuate the inflammatory cytokine response of human peripheral blood mononuclear cells, as well as for their ability to attenuate B7/CD28 intercellular receptor engagement. Mice were used to test the ability of such peptides to protect from lethal superantigen toxin challenge when administered in molar doses far below the toxin dose.ResultsB7 and CD28 homodimer interfaces are remote from the coligand binding sites, yet our finding is that by binding back into the receptor dimer interfaces, short dimer interface mimetic peptides inhibit intercellular B7-2/CD28 as well as the tighter B7-1/CD28 engagement, attenuating thereby pro-inflammatory signaling. B7 mimetic peptides exhibit tight selectivity for the cognate receptor in inhibiting intercellular receptor engagement with CD28, yet each diminishes signaling through CD28. In a prominent example of inflammatory cytokine storm, by attenuating formation of the B7/CD28 costimulatory axis, B7-1 and CD28 dimer interface mimetic peptides protect mice from lethal toxic shock induced by a bacterial superantigen even when administered in doses far submolar to the superantigen.ConclusionsOur results reveal that the B7 and CD28 homodimer interfaces each control B7/CD28 costimulatory receptor engagement and highlight the protective potential against cytokine storm of attenuating, yet not ablating, pro-inflammatory signaling via these receptor domains.

Cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on Tcells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ Tcells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ Tcell survival, migration, and cytokine production. In mouse tumor models, loss of Tcell-intrinsic cis-B7:CD28 interactions decreased intratumoral Tcells and accelerated tumor growth. Thus, B7 ligands on CD8+ Tcells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting Tcell functionality.

CD226 identifies functional CD8+T cells in the tumor microenvironment and predicts a better outcome for human gastric cancer.

It is well-known that CD226 serves as a critical activating receptor on various immune cells, such as lymphocytes and monocytes, and it is suggested to promote anti-tumor immunity in the tumor microenvironment (TME). Herein, we showed a crucial regulatory role of CD226 in CD8+T cell-mediated anti-tumor response in TME of human gastric cancer (GC). Specifically, the increased CD226 expression in cancer tissues was significantly associated with better clinical outcomes in GC patients. Moreover, the increased infiltrating CD226+CD8+T cells and the increased ratio of infiltrating CD226+CD8+T cells in CD8+T subpopulation within cancer tissues could also be valuable prognostic predictors for GC patients. Mechanically, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis revealed that the chromatin accessibility of CD226 in CD4+ and CD8+TILs was significantly higher than that in CD8+T cells in normal tissues. Further analysis showed that CD8+TILs highly expressed immune checkpoint molecules, such as TIGIT, LAG3, and HAVCR2, which means CD8+TILs are more exhausted. In addition, our multi-color immunohistochemical staining (mIHC) revealed that GC patients with higher frequency of IFN-γ+CD226+CD8+TILs showed poorer prognosis. Combined with the single-cell transcriptome sequencing (scRNA-seq) data analysis, we found that the expressions of IFN-γ and TIGIT in CD8+TILs were significantly and positively correlated. The expression of TIGIT in IFN-γ+CD226+CD8+TILs was higher, while that in IFN-γ-CD226+CD8+TILs was significantly lower. The correlation analysis showed that the expression of CD226 was positively correlated with the score of effector T cells but negatively correlated with that of immunosuppressive factors, such as Tregs and tumor-associated macrophages (TAMs). Collectively, we showed that the frequency of CD226+CD8+TILs was an excellent prognostic predictor for GC patients. Our findings provided insights into the interaction pattern between co-stimulatory receptor CD226 and tumor cells as well as the infiltrating immune cells in the TME in GC.

CD137 signaling aggravates myocardial ischemia-reperfusion injury by inhibiting mitophagy mediated NLRP3 inflammasome activation.

The inflammatory response caused by the NLRP3 is closely related to the formation of myocardial ischemia-reperfusion injury. Costimulatory receptor CD137 and its ligand play a crucial role in regulating the inflammatory immune response in atherosclerosis, which is the fundamental cause of cardiovascular diseases. However, the roles of CD137 signaling in the process of myocardial ischaemia-reperfusion (IR) injury remain unknown. Genetic ablation was used to determine the functional significance of CD137 in myocardial IR injury. Expression of CD137 was examined by Western-blot, quantitative real-time polymerase chain reaction, and immunohistochemistry in a murine IR model by coronary artery ligation. Even's blue-TTC staining and echocardiography to evaluate the severity of myocardial IR injury. Furthermore, HL-1 cardiomyocytes treated with agonist-CD137 recombinant protein were used to explore the underlying mechanism in CD137 signaling-induced NLRP3 inflammasome activation in response to hypoxia/reoxygenation or LPS/ATP. We demonstrated that CD137 knockout significantly improved cardiac function, accompanied by a markedly reduced NLRP3-mediated inflammatory response and IA/AAR which were reversed by mitophagy inhibitor Mdivi-1. Activating CD137 signaling significantly inhibited mitophagy and provoked NLRP3-mediated inflammatory response in H/R-injured or LPS-primed and ATP-stimulated HL-1 cardiomyocytes, the effects of which could be abolished by either anti-CD137 or mitophagy activator FCCP. Besides, mitochondrial ROS was augmented by activating CD137 signaling through the suppression of mitophagy. Our results reveal that activating CD137 signaling aggravates myocardial IR injury by upregulating NLRP3 inflammasome activation via suppressing mitophagy and promoting mtROS generation.

MUCOSAL IMMUNE SIGNATURES OF PERIANAL FISTULIZING CROHN’S DISEASE BY MASS CYTOMETRY

Abstract BACKGROUNDS Perianal fistulas occur in ~30-40% of patients associated with high morbidity and an impaired quality of life. The management of perianal fistulizing CD is a major clinical challenge, where its underlying etiology of is poorly understood. METHODS We recruited patients with a history of: 1) CD with perianal fistulas; 2) CD without perianal involvement; 3) idiopathic perianal fistulas. Biopsies were taken from the fistula tracts, external opening of the fistulas, and rectal mucosa during examination under anesthesia or colonoscopy. Isolated mucosal immune cells were analyzed using mass cytometry. Data was analyzed using viSNE for dimensionality reduction. RESULTS We have revealed previously unknown mucosal immune landscapes in perianal CD using mass cytometry. Key cell populations with significant difference in frequencies are summarize in Table 1 (data of external fistula opening not shown due to limited space): 1) CD45RO+ T cells including Tregs are elevated in CD fistulas compared to those from idiopathic fistulas, which is consistent with a prior histological study. The expression of co-inhibitory receptor TIGIT and co-stimulatory receptor CD226 are increased in Tregs in CD fistulas, although their function in IBD and especially perianal CD remains unclear. 2) Perianal CD is associated with increased Th17 cells in the fistula tracts and elevated IL-17-producing CD8 T cells (Tc17) in the rectum, which highlight the importance of IL-17 signaling in perianal CD. 3) CD fistulas show increased CD39 and decreased CD127 (IL-7Ra) in both CD4 and CD8 T cells. CD39 and CD127 are markers of T cell exhaustion, which may control the pathogenesis of perianal CD by regulating cytokine production. 4) Total B cells are elevated in CD fistulas compared to idiopathic fistulas. In contrast, regulatory B cells (Bregs), with immunomodulatory function in the gut, are dramatically diminished in CD fistulas compared to idiopathic fistulas. 5) Besides adaptive cells, CD3-CD19- innate immune cells including innate lymphoid cells (ILCs), macrophages, and NK cells are also skewed in perianal CD. Fistula tracts, external fistula opening (data not shown), and rectum of perianal CD all show dramatically elevated CD172+TREM1+ macrophages, which were previously found to promote chronic inflammation in luminal CD. CONCLUSIONS The immune responses in perianal CD demonstrate unique features that are not present in luminal CD (summarized in Figure 1). This indicates that perianal CD represents an immune identity distinct from luminal CD in the intestine. Importantly, altered T cell exhaustion markers (CD39 and CD127) and increased CD172+TREM1+ pathogenic macrophages are present in all three locations (fistula tracts, external opening, and rectum) of perianal CD, suggesting their roles in the pathogenesis of perianal CD and may become novel therapeutic targets.

Stratification of PD-1 blockade response in melanoma using pre- and post-treatment immunophenotyping of peripheral blood.

Efficacy of checkpoint inhibitor therapies in cancer varies greatly, with some patients showing complete responses while others do not respond and experience progressive disease. We aimed to identify correlates of response and progression following PD-1-directed therapy by immunophenotyping peripheral blood samples from 20 patients with advanced malignant melanoma before and after treatment with the PD-1 blocking antibody pembrolizumab. Our data reveal that individuals responding to PD-1 blockade were characterised by increased CD8 T cell proliferation following treatment, while progression was associated with an increase in CTLA-4-expressing Treg. Remarkably, unsupervised clustering analysis of pre-treatment T cell subsets revealed differences in individuals that went on to respond to PD-1 blockade compared to individuals that did not. These differences mapped to expression of the proliferation marker Ki67 and the costimulatory receptor CD28 as well as the inhibitory molecules 2B4 and KLRG1. While these results require validation in larger patient cohorts, they suggest that flow cytometric analysis of a relatively small number of T cell markers in peripheral blood could potentially allow stratification of PD-1 blockade treatment response prior to therapy initiation.

CAR T Cells and Their Immune Environment Synergize to Shape Distinct Immune Profiles in Response Versus Toxicity in B Cell Lymphoma Patients

Genetically engineered CD19-redirected chimeric antigen receptor T (CAR T) cells represent a breakthrough immunotherapy for B cell malignancies. However, response rates are variable and many patients face life-threatening side effects such as cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). The impact of the patient's immune landscape as a whole - consisting of CAR T cells and non-CAR T immune cells - on patients' outcome is largely unknown. Furthermore, reliable biomarkers to predict response and toxicity are lacking. To decode the crosstalk of CAR T cells and their circulating immune environment, we used longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided computational analysis of 47 peripheral blood samples from 19 lymphoma patients, together with 6 healthy controls. Patient samples were harvested one day before (day -1) up to 104 days after CAR T infusion. The generated immune map, based on 69 parameters and 2078 extracted immune features, was enriched with numerous clinical parameters from the lymphoma patients, including response and toxicity data of a follow-up period ≥ 315 days. When comparing healthy controls and B cell lymphoma patient samples at baseline (day -1 and day 0, pre-CAR T infusion), the two cohorts demonstrated distinct immune profiles, including a strong lymphodepletion and proportional expansion of the myeloid/ dendritic cell fraction in lymphoma patients. Furthermore, this translational proteomics approach revealed a lymphoma patient-specific, heterogeneous CAR T cell population (Figure 1). However, most of the identified 13 CAR T subsets, such as γδ T cells and CD4 T regulatory cells next to naive/effector/memory T cell populations, appeared in each sample. The temporal composition of the CAR+ T population varied, presenting an inverse relationship over time with a bias of CD8 CAR T cells at early timepoints and dominating CD4 CAR T subsets later on. When stratifying the overall immune profile at the time of CAR T cell expansion and toxicities (day 9 to 21), immune alterations associated with response to CAR T therapy (best overall response complete remission (CR)) were primarily found in the non-CAR T immune compartments, while CRS- and ICANS-associated signatures affected predominantly the CAR T population (Figure 2; deregulated immune features: p < 0.5 and wilcoxon effect size > 0.6). In particular, higher expression of TCF1 in CD8 CAR T cells and EOMES in CD8 T stem cell memory (Tscm) cells - suggestive for stem-like features that allow expansion/long-term persistence - and CD223 (LAG3) in CD4 CAR T cells marked the immune profile of CR patients. The interplay of CAR T and non-CAR T immune alterations was even more striking in toxicity: both CRS and ICANS were linked to a dysbalance in the CD226/TIGIT-axis with higher expression of the co-stimulatory receptor CD226 together with its regulator EOMES in CD4 and CD8 CAR T cells, whereas the co-inhibitory and competing receptor TIGIT demonstrated reduced levels in several CAR T subsets as well as CD8 T central memory and Tscm cells. In line with the latter result, lower expression of the immune checkpoint CTLA-4 was detected in both CAR positive and negative unconventional T cells, indicating immunopathological signatures in CRS and ICANS are based on imbalanced activating/inhibitory receptor profiles. Supportive for a pivotal role of the immune environment in CAR T treated patients, stratifying immune signatures associated with patient outcomes were already detected pre-CAR-T-infusion (Figure 2). Towards clinical translation, these altered signatures affecting the T, NK and myeloid/dendritic cell compartment are further investigated and could serve as potential biomarkers predicting patient outcome. In-depth immune profiling of CAR T treated lymphoma patients allowed us to ultimately define the CAR T specific immune landscape and uncovered a synergy of CAR T and surrounding immune cells in stratifying immune profiles. These results could serve to delineate CAR T-based therapeutic strategies targeting the immunopathologic cascade, aimed to enhance anti-cancer activity and confine toxicity. . Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

297 (PB077) - CAR T cells in marginal zone lymphoma (MZL) models with acquired resistance to PI3K and BTK inhibitors

Background. MZLs are characterized by an indolent course with median survival over 10 years. However, relapses/progressions are common and if they occur during the during the first 2 years, they heavily impact the median survival. Among the 23 relapsed/refractoryMZL patients treated with the anti-CD19 CAR-T cells axicabtagene ciloleucel (Axi-Cel), the overall response rate was 83% with 73% of complete responses (Jacobson, Lancet Onc 2022; Neelapu, ASH2021). We previously presented MZL-derived models with secondary resistance to BTK/PI3K inhibitors, observing, in some of the resistant cells, an increase in CD19 levels and higher sensitivity to the anti-CD19 loncastuximab tesirine antibody drug conjugate (Arribas, ASH2019, ENA2019, Haematologica 2022). Here, we tested CAR-T cells with costimulatory receptors CD28 or 4-1BB (28z CAR-T and BBz CAR-T), representative of Axi-Cel and tisagenlecleucel (Tisa-Cel), respectively, in these models. Methods: CD19-specific 28z CAR-T and BBz CAR-T produced from 4 healthy donors with retroviral vectors. Transduction efficacy evaluated by protein L staining. MZL models of secondary resistance to PI3K/BTK inhibitors and their parental cells cocultured for 48 h with CAR-T cells (1:1 E: T ratio) after adjusting for transduction efficacy. CAR-T cell-mediated cytotoxicity (CX) calculated as percentage of cell death compared to tumor cells cultured alone. CD19 expression determined by flow cytometry (protein) and RNA-Seq (RNA). Results. We first tested 28z CAR-T and BBz CAR-T against the VL51 cell line and in the 2 derivatives, resistant to various PI3K inhibitors and ibrutinib, respectively. Median CX against parental was 24% and 29% with 28z or BBz CAR-T, respectively. CX was significantly increased against PI3K resistant cells, with a median CX of 62% after coculture with 28z CAR-T and 55% with BBz CAR T cells. Ibrutinib resistant VL51 cells displayed an even higher sensitivity to CAR-T cell killing, with a median CX of 68% after co-culture with 28z CAR-T and 79% with BBz CAR-T cells. Differently from VL51, both parental Karpas1718 and PI3K resistant derivative were equally sensitive to CAR-T cells, with a median 28z CAR-T CX of 72% against parental and 72% against PI3K resistant Karpas1718. Similar results were observed after coculture with BBz CAR-T cells (median CX 67% against parental and 66% against PI3K resistant Karpas1718). The results agreed with CD19 levels, higher in Karpas1718 (parental and derivative) and in the resistant VL-51 derivatives than in parental VL51. Conclusions: Anti-CD19 28z and BBz CAR-T showed maintained or even increased in vitro antitumor activity in MZL models after acquisition of secondary resistance to PI3K/BTK inhibitors. The data support exploring CD19 directed therapeutic modalities for MZL patients. FS, FB: co-senior authors. Work supported by Barletta and Henriette Meyer Foundations. Conflict of interest: Advisory Board: Gilead, AbbVie, Janssen, AstraZeneca, MSD, BMS/ Celgene, Roche, Mei Pharma, Astra Zeneca, Celltrion Healthcare, Incyte, Kite/Gilead. Corporate-sponsored Research: Celgene, Roche, Janssen, Acerta, ADC Therapeutics, Bayer AG, Cellestia, CTI Life Sciences, EMD Serono, Helsinn, ImmunoGen, Menarini Ricerche, NEOMED Therapeutics 1, Nordic Nanovector ASA, Oncology Therapeutic Development, PIQUR Therapeutics AG, Gilead, AbbVie, Janssen.