TCR Expression Research Articles

Food for thought: Nutrient metabolism controlling early T cell development.

T cells develop in the thymus by expressing a diverse repertoire of either αβ- or γδ-T cell receptors (TCR). While many studies have elucidated how TCR signaling and gene expression control T cell ontogeny, the role of nutrient metabolism is just emerging. Here, we discuss how metabolic reprogramming and nutrient availability impact the fate of developing thymic T cells. We focus on how the PI3K/mTOR signaling mediates various extracellular inputs and how this signaling pathway controls metabolic rewiring during highly proliferative and anabolic developmental stages. We highlight the role of the hexosamine biosynthetic pathway that generates metabolites that are utilized for N- and O-linked glycosylation of proteins and how it impacts TCR expression during T cell ontogeny. We consider the dichotomy in metabolic needs during αβ- versus γδ-T cell lineage commitment as well as how metabolism is also coupled to molecular signaling that controls cell fate.

Cbl-b inhibition promotes less differentiated phenotypes of T cells with enhanced cytokine production

For adoptive therapy with T cell receptor engineered T (TCR-T) cells, the quantity and quality of the final cell product directly affect their anti-tumor efficacy. The post-transfer efficacy window of TCR-T cells is keen to optimizing attempts during the manufacturing process. Cbl-b is a E3 ubiquitin ligase previously shown with critical negative impact in T cell functions. This study investigated whether strategic inclusion of a commercially available small inhibitor targeting Cbl-b (Cbl-b-IN-1) prior to T cell activation could enhance the quality of the final TCR-T cell product. Examination with both PBMCs and TCR-T cells revealed that Cbl-b-IN-1 treatment promoted TCR expression efficiency, T cell proliferation potential and, specifically, cell survival capability post antigenic stimulation. Cbl-b-IN-1 exposure facilitated T cells in maintaining less differentiated states with enhanced cytokine production. Further, we found that Cbl-b-IN-1 effectively augmented the activation of TCR signaling, shown by increased phosphorylation levels of Zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase c-γ1 (PLCγ1). In conclusion, our results evidence that the inclusion of Cbl-b inhibitor immediately prior to TCR-T cell activation may enhance their proliferation, survival, and function potentials, presenting an applicable optimization strategy for immunotherapy with adoptive cell transfer.

A synthetic cytotoxic T cell platform for rapidly prototyping TCR function

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented a granzyme-activatable sensor of T cell cytotoxicity in a universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24 h exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate knowledge regarding the patterns of T cell receptor recognition, and optimize therapeutic T cell receptors.

HIV infection is associated with compromised tumor microenvironment adaptive immune reactivity in Hodgkin lymphoma

AbstractThe impact of HIV infection on the tumor microenvironment (TME) of classic Hodgkin lymphoma (cHL), one of the most common comorbidities after HIV infection, is not well understood. Here, we have used multiplexed immunofluorescence and spatial transcriptomic analysis to dissect the impact of viral infections (Epstein-Barr virus [EBV] and HIV/EBV) on cHL TME. HIV−EBV+ cHL TME was characterized by higher cell densities of CD8high T cells coexpressing inhibitory receptors (PD-1 and TIGIT), macrophage subsets, and an in situ inflammatory molecular profile associated with increased expression of T-cell receptor (TCR) and B-cell receptor cell signaling pathways than HIV−EBV− cHL TME. Compared with HIV−EBV+, HIV+EBV+ cHL TME was characterized by significantly less CD8high T cells coexpressing PD-1 and TIGIT, a profile concomitant with significantly increased cell densities of CD155high neoplastic cells. Significant downregulation of in situ TCR signaling and upregulation of extracellular matrix reorganization pathways were found in HIV+EBV+ cHL TME, in line with an altered topological organization of CXCL13 and heparan sulfate, an extracellular matrix glycosaminoglycan. Our data reveal the complexity of the cellular and molecular composition of cHL TME in the presence of viral infections, with possible implications for combinatorial immunotherapies. Furthermore, the data suggest specific molecular targets and pathways for further investigation that could improve our understanding of possible mechanistic links between HIV and lymphomagenesis.

A pan-cancer gamma delta T cell repertoire.

This report presents the largest collection of gamma-delta T cell receptor (γδ TCR) reads in human cancer to date, analyzing about 11,000 patient tumor samples across 33 cancer types using the TRUST4 algorithm. Despite γδ T cells being a small fraction of the T cell population, they play a key role in both innate and adaptive immunity. Our comprehensive analysis reveals their significant presence across all cancer types, specifically highlighting the diverse spectrum and clonality patterns of their γδ receptors. This research highlights the complex roles of γδ T cells in tumor tissues and their potential as prognostic biomarkers. We also demonstrate the utility of T cell receptor gamma (TRG) and delta (TRD) gene expression values from standard RNA-seq data. Ultimately, our work establishes a fundamental resource for future tumor-infiltrating γδ T cell research and may facilitate the development of novel γδ-T-cell-based therapeutic strategies. Together, we demonstrate the strong diversity and prognostic potential of γδ T cells in multiple cancer types.

Nonsense CD247 mutations show dominant-negative features in T-cell receptor expression and function

BackgroundThe invariant TCRζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its three immunoreceptor tyrosine‐based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. ObjectiveTo analyze and model the potential impact on TCR expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. MethodsJurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with wild-type CD247 or mutations ablating one (Q142X), two (Q101X), or three (Q70X) ITAMs. ResultsThree patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2+3, respectively. Both mutations were associated with low surface CD3 expression, normal intracellular CD247 levels using a transmembrane-specific antibody but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO could not restore normal surface CD3 expression (only 60%, 22% and 10%, respectively), whereas in WT they reduced it (to 39%, 19% and 9% of normal levels), and both effects were ITAM number dependent. All six transfectants showed reduced CD69 induction (25-50%), indicating that they were unable to signal downstream properly neither isolated nor associated with wild-type CD247. ConclusionOur results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

Oleic acid enhances proliferation and calcium mobilization of CD3/CD28 activated CD4+ T cells through incorporation into membrane lipids.

Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function.

γδ intraepithelial lymphocytes acquire the ability to produce IFN-γ in a different time course than αβ intraepithelial lymphocytes.

An age-dependent increase in IFN-γ expression by intestinal intraepithelial lymphocytes (IELs) contributes to the acquisition of resistance to infection by pathogens. However, how IELs acquire the ability to produce IFN-γ remains to be elucidated. Here, we report that IELs in the small intestine acquire the ability to rapidly produce IFN-γ at two distinct life stages. TCRαβ+ IELs (αβIELs) started producing IFN-γ at 4 weeks of age, within 1 week after weaning. In contrast, TCRγδ+ IELs (γδIELs) started producing IFN-γ at 7 weeks of age. In mice lacking Eγ4, an enhancer of the TCRγ locus (Eγ4-/- mice), Thy-1+ Vγ5+ γδIELs, a major subpopulation of γδIELs, were specifically reduced and their ability to produce IFN-γ was severely impaired, whereas Vγ2+ γδIELs normally produced IFN-γ. In Eγ4-/- mice, TCR expression levels were reduced in Vγ5+ γδIEL precursors in the thymus but unchanged in the Vγ5+ IELs. Nevertheless, TCR responsiveness in Vγ5+ γδIELs was impaired in Eγ4-/- mice, suggesting that the TCR signal received in the thymus may determine TCR responsiveness and the ability to produce IFN-γ in the gut. These results suggest that αβIELs and γδIELs start producing IFN-γ at different life stages and that the ability of Vγ5+ γδIELs to produce IFN-γ in the gut may be predetermined by TCR signaling in IEL precursors in the thymus.

A CRISPR-edited allogeneic anti-CD19 CAR-T cell therapy with a PD-1 knockout (CB-010) in patients with relapsed/refractory B cell non-Hodgkin lymphoma (r/r B-NHL): Updated phase 1 results from the ANTLER trial.

7025 Background: CB-010 is an allogeneic anti-CD19 CAR-T cell therapy derived from healthy donor T cells using CRISPR hybrid RNA-DNA (chRDNA) technology. This technology is used to introduce 3 genome edits: (1) knockout of TRACto eliminate TCR expression and reduce risk of GvHD, (2) insertion of a CD19-specific CAR (scFv FMC63) into the TRAC locus, and (3) knockout of PD-1 to prevent premature CAR-T cell exhaustion and potentially enhance antitumor activity. Methods: ANTLER is a Phase 1 clinical trial (NCT04637763) with a 3+3 dose escalation phase and a dose expansion phase designed to evaluate safety, tolerability, and antitumor activity of CB-010 in patients (pts) with r/r B-NHL and determine RP2D. In dose escalation, pts must have received ≥2 prior lines of chemoimmunotherapy or had primary refractory disease to 1L therapy. Pts received lymphodepletion with sequential cyclophosphamide (60 mg/kg/day x 2 days) and fludarabine (25 mg/m2/day x 5 days) followed by a single CB-010 infusion. Results: 16 pts with r/r B-NHL (10 LBCL, 3 MCL, 2 FL with POD24, 1 MZL) received CB-010 at 40 x 106 CAR-T cells (dose level 1; N=8), 80 x 106 CAR-T cells (dose level 2; N=5), or 120 x 106 CAR-T cells (dose level 3; N=3) during dose escalation. Median age was 66 years (range 55-82). Median time since first diagnosis was 2.4 years (range 0.2-16.4). Median prior lines of therapy was 2 (range 1-8). CB-010 was generally well tolerated. No GvHD was seen. CRS occurred in 7/16 (44%) pts (no CRS grade ≥3). Median time to CRS onset was 3.5 days and median duration was 3 days. ICANS occurred in 4/16 (25%) pts (13% grade ≥3). Median time to ICANS onset was 7.5 days and median duration was 2 days. The 3 most common TEAEs grade ≥3 were thrombocytopenia (11/16; 69%), neutropenia (9/16; 56%), and anemia (8/16; 50%). One grade 3 infection (antecubital cellulitis) occurred unrelated to CB-010. After a single CB-010 infusion, 15/16 (94%) pts achieved an overall response, 11/16 (69%) achieved a CR, and 7/16 (44%) achieved a CR at ≥6 months. Median time to CR was 28 days. Among LBCL pts (n=10), 9 (90%) achieved an overall response, 7 (70%) achieved a CR, and 5 (50%) achieved a CR at ≥6 months. To date,2 pts have completed the 24-month study period with ongoing CR. Peak expansion of CB-010 occurred at days 7-10 post-infusion. T and NK cells recovered rapidly in peripheral blood (<3 weeks) after lymphodepletion, and B cells remained below the limit of quantification beyond 3 months, supporting specific targeting of B cells by CB-010. Conclusions: CB-010 showed a manageable safety profile and promising efficacy for treatment of pts with r/r B-NHL, including aggressive subtypes. The dose escalation phase is complete. Enrollment of 2L LBCL pts in dose expansion is ongoing. Initial dose expansion data at the CB-010 RP2D and translational data will be presented for the first time at the meeting. Clinical trial information: NCT04637763 .

Type B thymomas in patients with myasthenia gravis display a distinctive pattern of αβ TCR and IL-7 receptor α expression on CD4+CD8+ thymocytes

Thymoma is closely associated with myasthenia gravis (MG). However, due to the heterogeneity of thymoma and the intricate pathogenesis of MG, it remains unclear why some patients with thymoma develop MG and others do not. In this study, we conducted a comparative phenotype analysis of thymocytes in type B thymomas in patients with MG (MG (+) thymomas) and without MG (MG (−) thymomas) via fluorescence-activated cell sorting (FACS). Our results show that the developmental stages defined by the expression of CD3, CD4, and CD8 were largely maintained in both MG (+) and MG (−) thymomas, with CD4+CD8+ cells constituting the majority of thymocytes in type B thymoma, and no significant difference between this cell population was observed in MG (+) and MG (−) thymomas.We discovered that CD4+CD8+ thymocytes in MG (+) thymomas expressed low levels of αβ TCR and high levels of IL-7 receptor α (IL-7Rα), whereas in MG (−) thymomas, CD4+CD8+ thymocytes exhibited the opposite pattern of αβ TCR and IL-7Rα expression. These results suggest that the positive and negative selection processes of CD4+CD8+ thymocytes might differ between MG (+) thymomas and MG (−) thymomas. The expression of the Helios transcription factor is induced during negative selection and marks a group of T cells that have undergone negative selection and are likely to be deleted due to strong TCR binding with self-peptides/MHC ligands. We observed that the percentage of Helios-positive CD4SP T cells was greater in MG (−) than in MG (+) thymomas. Thus, the differentially regulated selection process of CD4+CD8+ thymocytes, which involves TCR and IL-7/IL-7Rα signaling, is associated with the presence of MG in type B thymomas.

Abstract 27: A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity

Abstract Background: Genetically engineered T cells with T cell receptor (TCR-T) cell therapy have shown great potential for cancer treatment by targeting tumor-associated antigens. Despite its success in treating various tumor types, such as synovial sarcoma, head and neck tumors, and cervical cancer, its application in liver cancer is limited. Alpha-fetoprotein (AFP), a highly expressed tumor-associated antigen in liver cancer, is an ideal target for TCR-T cell therapy. Although early human trials have confirmed the safety of AFP-targeting TCR-T products, their efficacy is still modest, highlighting the need for further research and development. Methods: We developed a robust strategy combining ex vivo stimulation and single-cell paired TCR sequencing to identify AFP-recognizing TCRs in liver cancer patients. To optimize TCR expression and minimize mispairing, we employed codon optimization and replaced the constant regions with murine counterparts. Furthermore, structural analysis guided the affinity optimization of the CDR3 region to enhance the antitumor function of the selected TCR. The anti-tumor efficacy of TCR-T cells was rigorously evaluated through in vitro cytotoxicity assays using the Real-Time Cell Analyzer (RTCA) system and in vivo antitumor assessment in immunodeficient mouse xenograft models. Antigen specificity, HLA restriction, and other potential toxicity were carefully evaluated in both in vitro and in vivo models. Results: We successfully cloned a highly effective TCR that recognizes the AFP158-166 epitope presented by HLA-A*0203. After sequence optimization, we further changed a single amino acid on the TCR vβ chain based on AI prediction. The expression and functionality of this TCR was significantly improved. When introduced into human T cells, the single amino acid mutated TCR demonstrated robust and specific anti-tumor activity in various models, including cultured tumor cells, patient-derived organoids, and mouse xenografts models. Notably, this TCR also redirected CD4 T cells to recognize tumor cells, induced the secretion of multiple cytokines, and maintained long-lasting anti-tumor effects. Our rigorous screening assays confirmed that the TCR showed no cross-reactivity with the human proteome or major HLA subtypes. Additionally, extensive preclinical safety assessments in tumor-bearing animals treated with ST08A01-AM14 TCR-T cells showed no significant safety concerns. Conclusion: In this study, we have developed an HLA-A*0203 restrict AFP158-166 specific TCR-T therapy, which exhibits exceptional anti-tumor effects while ensuring a favorable safety profile. The promising results support the further investigation against AFP-positive solid tumors in clinic. Citation Format: Chen-Song Huang, shudan Ou, Jun Li, Junjun Chu, Yanyan Han. A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 27.

Effects of therapeutic hypercapnia on the expression and function of γδT cells in transplanted lungs in rats.

To investigate the effect of therapeutic hypercapnia on the expression and function of gamma delta T (γδ T) cells during ischemia-reperfusion injury (IRI) after lung transplantation. We randomly divided male Wistar rats into three groups (n = 6 in each group), the control group (group N), the IRI group (group I), and the therapeutic hypercapnia group (group H). We then assessed pulmonary edema, neutrophil infiltration, wet-to-dry (W/D) weight ratio, and microscopic histopathology and separately measured the levels of γδT cell surface antigen (TCR) and Interleukin-17 (IL-17) using flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The infiltration of neutrophils and the expression of TCR and IL-17 were significantly increased in the I group compared to the control, and the biopsy edema in group I was more severe. Arterial partial pressure of oxygen (PaO2) was decreased after reperfusion in group I compared with the control group. W/D weight ratio, neutrophil infiltration, and the expression of TCR and IL-17 decreased drastically in the H group compared to the I group. Our findings suggest that γδ T lymphocytes were directly involved in lung injury. In addition, therapeutic hypercapnia effectively reduced the expression of γδ T cells and IL-17, and this has the potential to become a treatment strategy for IRI and an intervention to improve lung function.

The Efficacy of Nile Tilapia Vaccination by Formalin killed Streptococcus agalactiae Encapsulated in Alginate Microcapsules

Nile tilapia is one of the major farmed fish that is often infected with Streptococcus. Oral vaccination is the most preferable technique, but the antigen is usually degraded by gastric juice. Encapsulation by alginate could be effective in protecting the antigen. The aim of this study was to assess the effectiveness of vaccinating Nile tilapia using formalin-killed Streptococcus agalactiae encapsulated in alginate. The average diameter of fabricated microcapsules was 500±20 μm, with 86% encapsulation efficacy. Fish were then divided into four groups (30 fish per group): group A: fish vaccinated by alginate microcapsules covered by formalin-killed cells (FKC), group B: fish vaccinated by non-covered FKC, group C: fish fed with empty alginate microcapsules and group D: fish feed with commercial pellets as control group. All groups were treated for 14 days and then fed with commercial pellets. Blood samples were collected on days 7, 14, 21, and 28 days post-vaccination. Bactericidal activity, lysozyme activity and serum antibody titer were significantly elevated in group A (p<0.001) compared to other treated groups. Then, gene expression analysis was performed at 29 days post-vaccination to evaluate the expression of main immune contributors such IgM, IgT, TCR β, CD4, CD8α, IL 1β, IL 8, IFN 1, and TNFα. The analysis showed significant upregulation of all tested genes in group A compared to other treated groups (p<0.001). Lastly, a challenge test was done on day 30 post-vaccination by injecting 4.6×106 CFU mL−1 of virulent S. agalactiae. The relative percent of survival (RPS) were 92± 2%, 48± 5% and 4± 3% for groups A, B and C respectively. The obtained result indicated that alginate encapsulation provided antigen protection due to its higher immunogenicity compared to non-covered vaccine. Hence, the fabricated vaccine could be incorporated with food and orally administered to Nile tilapia to prevent S. agalactiae infection.

Exploitation of CD3ζ to enhance TCR expression levels and antigen-specific T cell function.

The expression levels of TCRs on the surface of human T cells define the avidity of TCR-HLA/peptide interactions. In this study, we have explored which components of the TCR-CD3 complex are involved in determining the surface expression levels of TCRs in primary human T cells. The results show that there is a surplus of endogenous TCR α/β chains that can be mobilised by providing T cells with additional CD3γ,δ,ε,ζ chains, which leads to a 5-fold increase in TCR α/β surface expression. The analysis of individual CD3 chains revealed that provision of additional ζ chain alone was sufficient to achieve a 3-fold increase in endogenous TCR expression. Similarly, CD3ζ also limits the expression levels of exogenous TCRs transduced into primary human T cells. Interestingly, transduction with TCR plus CD3ζ not only increased surface expression of the introduced TCR, but it also reduced mispairing with endogenous TCR chains, resulting in improved antigen-specific function. TCR reconstitution experiments in HEK293T cells that do not express endogenous TCR or CD3 showed that TCRα/β and all four CD3 chains were required for optimal surface expression, while in the absence of CD3ζ the TCR expression was reduced by 50%. Together, the data show that CD3ζ is a key regulator of TCR expression levels in human T cells, and that gene transfer of exogenous TCR plus CD3ζ improved TCR surface expression, reduced TCR mispairing and increased antigen-specific function.

Comparative analysis of the effects of cyclophosphamide and dexamethasone on intestinal immunity and microbiota in delayed hypersensitivity mice.

The T cell-mediated delayed-type hypersensitivity (DTH) response is critical for elucidating cellular immune mechanisms, especially the role of memory T cells upon antigen re-exposure. This study aimed to investigate the specific effects of the immunosuppressive drugs Cyclophosphamide (CY) and Dexamethasone (DEX) on intestinal immunity and microbiota in a DTH mouse model, contributing to a more nuanced understanding of their immunomodulatory mechanisms. Female BALB/c mice were sensitized to 2,4-dinitrofluorobenzene (DNFB) and randomly allocated into control, CY, and DEX groups. The impact of CY and DEX on immune function was assessed through measurement of thymus and spleen indices, lymphocyte proliferation in mesenteric lymph nodes (MLNs) using MTT assay, and flow cytometric analysis of T cell subsets and TCR expression. Intestinal secretory IgA (sIgA) was quantified by ELISA, and gut microbiota diversity was evaluated using 16S rRNA gene sequencing. CY and DEX significantly reduced the immune function in DNFB-induced sensitized mice, as indicated by decreased thymus and spleen indices, MLN enlargement, intestinal sIgA content, and ear swelling degree. Flow cytometry revealed that CY increased the proportion of total CD3+ T cells but reduced CD3+CD69+ activated T cells and CD3+TCRγ/δ+ T cells, while DEX increased CD3+CD4+ helper T cells. Both drugs induced distinct changes in gut microbiota diversity and structure, with CY enhancing α diversity and DEX reducing it. The study demonstrates that CY and DEX have distinct regulatory effects on the immune organ index, distribution of T cell subsets, and diversity and structure of gut microbiota on DTH-induced immune responses mice, suggesting their differential influence on intestinal mucosal immunity. These findings have implications for the development of targeted immunotherapies and understanding the interplay between immunosuppressive drugs and gut microbiota.

Alternatively Spliced Variants of Murine CD247 Influence T Cell Development and Activation, Revealing the Importance of the CD3ζ C-Terminal Region.

CD247, also known as CD3ζ, is a crucial signaling molecule that transduces signals delivered by TCR through its three ITAMs. CD3ζ is required for successful thymocyte development. Three additional alternatively spliced variants of murine CD247 have been described, that is, CD3ι, CD3θ, and CD3η, that differ from CD3ζ in the C terminus such that the third ITAM is lost. Previous studies demonstrated defects in T cell development in mice expressing CD3η, but the TCR signaling pathways affected by CD3η and the impacts of the CD3ι and CD3θ on T cell development were not explored. In this study, we used a retrovirus-mediated gene transfer technique to express these three isoforms individually and examined the roles of them on T cell development and activation. Rag1-/- mice reconstituted with CD3θ-expressing bone marrow failed to develop mature T cells. CD3ι-expressing T cells exhibited similar development and activation as cells expressing CD3ζ. In contrast, thymic development was severely impaired in CD3η-reconstituted mice. Single-positive but not double-positive CD3η-expressing thymocytes had reduced TCR expression, and CD5 expression was decreased at the double-positive stage, suggesting a defect in positive selection. Peripheral CD3η-expressing T cells had expanded CD44hi populations and upregulation of exhaustion markers seen by flow cytometry and RNA sequencing analysis. Analysis of early signaling events demonstrated significantly reduced activation of both the PLCγ1 and Akt/mTOR signaling pathways. There was also a reduction in the frequency of activation of CD3η-expressing T cells. These studies reveal the importance of the CD3ζ C-terminal region in T cell development and activation.

The mitochondrial fission protein DRP1 influences memory CD8+ T cell formation and function.

Pharmacological methods for promoting mitochondrial elongation suggest that effector T cells can be altered to support a memory T cell-like metabolic state. Such mitochondrial elongation approaches may enhance the development of immunological memory. Therefore, we hypothesized that deletion of the mitochondrial fission protein dynamin-related protein 1 (DRP1) would lead to mitochondrial elongation and generate a large memory T cell population, an approach that could be exploited to enhance vaccination protocols. We find that, as expected, while deletion of DRP1 from T cells in dLckCre × Drp1flfl does compromise the magnitude and functionality of primary effector CD8+ T cells, a disproportionately large pool of memory CD8+ T cells does form. In contrast to primary effector CD8+ T cells, DRP1-deficient memory dLckCre × Drp1flfl CD8+ T cells mount a secondary response comparable to control memory T cells with respect to kinetics, magnitude, and effector capabilities. Interestingly, the relative propensity to form memory cells in the absence of DRP1 was associated with neither differentiation toward more memory precursor CD8+ T cells nor decreased cellular death of effector T cells. Instead, the tendency to form memory CD8+ T cells in the absence of DRP1 is associated with decreased T cell receptor expression. Remarkably, in a competitive environment with DRP1-replete CD8+ T cells, the absence of DRP1 from CD8+ T cells compromised the generation of primary, memory, and secondary responses, indicating that approaches targeting DRP1 need to be carefully tailored.

Durable Multiplex Epigenetic Editing for Generation of Allogeneic CAR T without Chromosomal Rearrangements

CAR T therapies have provided significant breakthroughs for patients with CD19+ malignancies and multiple myeloma, but a major limitation is the need for individualized manufacturing of autologous T cells, increasing the cost and reducing the availability of these life-saving drugs. An allogeneic CAR T product, available off-the-shelf, could greatly reduce manufacturing burden and improve access to therapy. To date, allogeneic CAR T clinical trials have shown limited durable responses and have reduced only TCR expression to avoid a graft versus host response. Further editing to reduce MHC class I and II expression may produce a cell resistant to allogeneic CD4+ and CD8+ T cell rejection to potentially improve allogeneic CAR T persistence. Conventional genome editing technologies that rely on double or single stranded DNA breaks have the potential to introduce genomic changes including translocations, especially when multiple edits are introduced. Epigenetic editing, in contrast, enables durable modulation of gene expression without cutting or nicking the DNA sequence and is extremely well-suited for simultaneous targeting of multiple genes to enable generation of persistent allogeneic CAR T cells. In this study, we evaluated the use of a dCas9-based epigenetic editor to silence TCR and MHC class I and II expression in primary T cells to produce allogeneic CAR T transduced for expression of CAR and HLA-E. Epigenetic editor mRNA and sgRNAs targeting the genes of interest were delivered via nucleofection to activated T cells. Edited T cells were then maintained in proliferative culture, with CD3/CD28 or PMA/ionomycin restimulation, and expression of the target genes was assessed by flow cytometry. Expression of TCR and MHC class I and II was durably suppressed in both single and multiple gene target silencing, demonstrating the robustness of epigenetic editing for ex vivo cell therapies. In addition, genomic integrity assessments demonstrated that epigenetic editing did not induce genomic changes as detected by either a quantitative sequencing-based assay or a fluorescence-based technique with single-cell resolution. In primary T cells, multiplex silencing did not cause genomic rearrangements, including translocations and truncations, above the level observed in controls, whereas Cas9 multiplex editing did. Mixed lymphocyte reaction (MLR), specific killing, and degranulation assays were used to demonstrate allogeneic function of silencing edits and the overexpression of an NK shield in vitro. In these assays, epigenetically-edited cells performed similarly to control gene edited cells in which the same gene targets were knocked out. TCR-silenced T cells did not proliferate or produce IFN-γ in response to allogeneic PBMCs in an MLR assay, and MHC class I- and II-silenced T cells did not evoke a proliferative response or IFN-γ production from allogeneic CD4+ or CD8+ T cells. Furthermore, overexpression of the HLA-E NK shield protected HLA-E expressing cells relative to B2M KO cells in a specific killing assay and reduced the degranulation response of NKG2A+ NK cells without a corresponding increase in NKG2C+ NK cell degranulation observed with other single chain HLA-E constructs tested. Finally, multiplex epigenetically-edited allogeneic CAR T were produced by transduction for expression of an anti-BCMA CAR, followed by simultaneous editing to silence target genes. These cells displayed potent anti-tumor activity against the BCMA+ MM.1S cell line, comparable to activity of unedited CAR T cells. These data support multiplex epigenetic editing to produce functional allogeneic CAR T cells that (1) do not exhibit a GVHD response; (2) resist CD8+ and CD4+ T cell alloresponses; and (3) resist the NK missing-self response normally consequent of reduced MHC class I expression without introduction of genotoxic events.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (<1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (>90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells

NK cells armed with chimeric antigen receptors (CAR) enable NK cells specifically to target cancer cells by recognizing tumor associated antigens. Previous studies have proven that human iPSC-derived CAR-NK cells have enhanced anti-tumor activity. Targeted B-cell maturation antigen (BCMA) therapy for relapsed refractory MM (rrMM) has been widely used with antibody-drug conjugate (ADC), bispecific antibodies, and CART cells, which has produced a rapid response, but relapse is common. Recent work has revealed GPRC5D as an alternative target in MM for immunotherapy and patients who have previously received anti-BCMA therapy have responded to GPRC5D CART cells. To assess target GPRC5D therapy with CAR-NK cells, which may in future serve as allogeneic immunotherapy, we transduced anti-GPRC5D CAR into an iPSC line derived from a healthy donor using a piggybac transposon system. More than 97% of CAR inserted-iPSCs (CAR-iPSCs) expressed anti-GPRC5D scFV with a similar expression level identified in CAR-iPSC-derived-NK cells (CAR-iNK, 92.7%). The anti-GPRC5D CAR-iNK demonstrated high purity (>99.9% for CD45 + and CD56 +) and expressed a high level of CD16 (63.6%), the NK cell activating receptor NKG2D (96.3%) and NKp30 (98.7%), and the co-stimulatory receptors CD244 (99.6%) and CD226 (97.8%). Nonetheless expression of TCR⍺β and TCRƳ- was relatively low (<2%). Cytotoxicity assay revealed that anti-GPRC5D CAR-iNK had similar cytotoxicity against K562 cells (No GPRC5D antigen) to cord blood-derived NK cells (CB-NK), wide type (WT) iNK and anti-BCMA CAR-iNK. Cytotoxicity was dose-dependent indicating that these NK cells had similar innate anti-cancer cytotoxicity. When incubated at a ratio of 4:1 with NCI-H929 cells (MM) for 4 hours, which express a high level of both BCMA and GPRC5D, anti-GPRC5D CAR-iNK demonstrated an overwhelming advantage (approximately 90% killing rate) over the CB-NK (no killing) and WT iNK (around 10% killing rate). Furthermore, the anti-GPRC5D CAR-iNK also showed superior cytotoxicity to anti-BCMA CAR-iNK (Figure 1), indicating that GPRC5D may be a more efficient target for CAR-NK-based immunotherapy in MM. We further confirmed that anti-GPRC5D CAR-NK have acceptable cytotoxicity to NCI-H929 and OPM-2 cells, even when transported over a long distance (more than 2000 km) and following cryopreservation. The lower expression by OPM-2 cells of BCMA and GPRC5D compared with NCI-H929 indicates that both fresh and cryopreserved anti-GPRC5D CAR-iNK can specifically target MM with a higher and lower expression of GPRC5D. In addition, cryopreserved anti-GPRC5D CAR-iNK cells maintain comparable antigen-specific cytotoxicity in vitro, and demonstrate the ability to reduce tumor burden in an antigen-specific manner in an OPM-2 xenograft model. Our results demonstrate that genome-engineered human iPSCs can provide an unlimited source for manufacturing scalable, off-the-shelf, and cost-effective CAR-NK cells, and GPRC5D is a potent immunotherapeutic target in multiple myeloma.