Primary CD4 Research Articles

Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3

Abstract Although prior research implicated c-Jun N-terminal kinase (JNK) in helper CD4 +T cell differentiation, mechanisms governing JNK signaling and function in human lymphocytes remain nebulous. CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor (AgR) engagement to transcriptional programs necessary for effector cell survival, proliferation, and differentiation. In addition to NF-κB, CARD11 is also required for AgR-induced mTORC1 and JNK2 signaling. We sought to define the impact of dominant interfering (DI) CARD11 variants on JNK signaling and elucidate transcriptomic changes governed by CARD11-dependent JNK activation in T cells. We transfected WT and CARD11 knockout (KO) Jurkat T cells with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling and performed RNA sequencing on mitogen-activated WT and CARD11 KO Jurkat T cells ± JNK inhibition. Our results revealed that AgR-dependent JNK1 andJNK2 activation are CARD11-dependent. Additionally, heterozygous DI CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, mirroring the extent of impaired NF-κB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4 +T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. Our novel findings suggest that defects in CARD11-dependent JNK signaling in CD4 +T cells may contribute to severe allergic disease noted in CADINS patients, unveiling a new potential therapeutic target. Supported by funding from the Jeffrey Modell Specific Defect Research Program (ALS) and the Henry M. Jackson Foundation for the Advancement of Military Medicine (BMB).

Yi-Qi-Jian-Pi formula ameliorates immune function in acute-on-chronic liver failure by upregulating autophagy and mitochondrial biogenesis in CD8+ T lymphocytes.

A key event in the pathogenesis of acute-on-chronic liver failure (ACLF) is the imbalance in the systemic immune response; immunosuppression in patients with ACLF contributes to poor prognosis. The Yi-Qi-Jian-Pi formula (YQJPF) may improve T lymphocyte immune function in patients with ACLF. To investigate the immune mechanism of YQJPF in vivo and in vitro. An ACLF rat model was established by injection of CCl4, lipopolysaccharide, and D-galactosamine. We examined the effect of different doses of YQJPF (6.43, 12.87, 25.74g/kg) on liver injury and immune function in the ACLF rat model. Magnetic-activated cell sorting was used to sort the CD8+ T lymphocytes in the spleen for lymphocyte function detection. In primary CD8+ T lymphocytes and Jurkat cell lines, the expression of mitochondrial function and biogenesis and autophagy related markers were detected using molecular biological methods and flow cytometry analysis. YQJPF improved the peripheral blood lymphocyte count and proportion of CD8+ T lymphocytes in ACLF rats, increased pro-inflammatory factors (IL-2, IFN-λ, and TNF-α), and reduced anti-inflammatory factors (IL-10 and TGF β1). YQJPF also improved metabolism and mitochondrial homeostasis in CD8+ T lymphocytes, alleviated lymphocyte immune dysfunction by promoting autophagy, upregulated mitochondrial biogenesis by promoting PGC-1α, NRF-1, and TFAM expression, and regulated the relationship between autophagy and mitochondrial biogenesis via PGC-1α. Our results suggest that YQJPF could improve immune function in a rat model of ACLF, possibly via affecting the homeostasis of lymphatic mitochondria in CD8+ T lymphocytes. YQJPF may enhance lymphocyte mitochondrial biosynthesis and promote lymphocyte autophagy. PGC-1α is a possible upstream regulatory target of YQJPF.

QRICH1 is a negative regulator of T cell activation via the CARD11 signalosome

Abstract Controlled lymphocyte activation is crucial for defending against pathogens while preventing diseases such as autoimmunity and lymphoma. In T lymphocytes, the scaffold protein and signaling hub CARD11 mediates NF-κB, JNK, and mTOR pathway activation downstream of T cell receptor (TCR) engagement. We recently identified QRICH1 as a previously unrecognized CARD11 interactor recruited to the CARD11 complex downstream of TCR engagement and discovered that QRICH1 protein levels are upregulated in wild-type activated mouse primary CD8+ T cells. Overexpression of QRICH1 in 293T and Jurkat T cells dose-dependently inhibited NF-κB activation by wild-type CARD11 as well as by constitutively active and oncogenic CARD11 mutants. QRICH1-deficient Jurkat T cells had increased NF-κB activation and IL-2 secretion downstream of TCR stimulation. To study the in vivo role of QRICH1 in immunity, we generated a novel QRICH1 conditional KO mouse model. Compared to control mice, QRICH1 T cell KO mice had decreased splenic CD4+ and CD8+ T cells, despite no differences in thymic CD4+, CD8+, or CD4+CD8+ T cell populations. The CD8+ T cell population in QRICH1 T cell KO mice was skewed towards an effector memory phenotype. QRICH1-deficient primary CD8+ T cells had increased proliferation and production of pro-inflammatory cytokines IFN-γ and TNF-α in response to ex vivo stimulation with anti-CD3/CD28, and QRICH1-deficient OT-1 CD8+ T cells also had increased cytokine production in response to antigen-specific stimulation with Ova peptide. Our findings reveal that QRICH1 negatively regulates T cell activation by modulating CARD11’s signaling output. This work was supported by NIH grants R01AI148143, F31CA254167, and T32GM007445.

Abstract PR08: TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development

Abstract Background: KRAS mutations drive tumorigenesis in adenocarcinomas of the lung (30-40%), colon (40-50%) and pancreas (>90%). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with KRAS-mutated advanced solid tumors. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. Here, we describe the pre-clinical development of TCR1020, a human TCRab specific for KRAS G12V restricted to HLA-A*11:01. Methods: In vitro stimulation assays were used to generate CD8+ T cell responses specific for a decamer epitope of KRAS G12V (VVVGAVGVGK) using blood specimens collected from A*11:01+ healthy donors. TCR1020 was identified by TCRab RNA sequencing of flow cytometrically sorted peptide/HLA multimer+ CD8 T cells. Transgenic expression of TCR1020 was attained via lentiviral transduction of reporter cell lines and gene-edited (TCRabnull) primary T cells. TCR1020 antigen specificity, affinity and cross-reactivity was assessed using in vitro coculture, limiting peptide dilution, and combinatorial peptide scanning assays. TCR1020 reactivity to G12V/A*11:01+ cell lines was assessed using in vitro cytokine release and cytotoxicity assays, as well as mouse xenograft tumor models. Results: TCR1020 exhibits specificity to the cognate KRAS G12V epitope with cross-reactivity to G12C but not wild-type KRAS. TCR1020 has a measured functional avidity (EC50) of 262 pM. TCR1020-redirected primary CD8+ T cells promote cytolysis of A*11:01+ tumor cell lines of lung, colonic or pancreatic origin harboring endogenous KRAS G12V mutations. The adoptive transfer of TCR1020-engineered primary CD8+ T cells exhibits potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. TCR1020 demonstrates CD8 co-receptor independence, and CD4+ T cells expressing TCR1020 express cytokines and lyse G12V/A*11:01+ tumor cell lines. In-depth characterization of the TCR1020 peptide recognition motif using alanine/glycine scanning libraries reveals a unique antigen binding motif with 5 critical amino acid residues excluding peptide/HLA anchor residues. Combinatorial peptide library scanning identifies five A*11:01-restricted peptide candidates within the human proteome with potential cross-reactivity to TCR1020, none of which activate TCR1020-engineered T cells over a wide range of concentrations. TCR1020 exhibits no evidence of reactivity against cell lines derived from healthy human tissues nor alloreactivity against a panel of lymphoblastoid cell lines encompassing a wide array of HLA class I alleles. Conclusion: TCR1020 exhibits high potency and exquisite antigen cognate specificity, and it is under clinical development for the treatment of A*11:01+ patients with KRAS G12V+ advanced solid tumors. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR08.

Antigen loaded monocytes prime CD4+ T cell responses through Notch2 dependent cDC2

Abstract Cellular vaccines, primarily monocyte-derived dendritic cells (MoDC), have recently been realized as therapeutic cancer vaccines with modest clinical efficacy. Understanding the mechanism by which these vaccines lead to antigen presentation is key to improving their potency. A significant advancement was the discovery that MoDC vaccines do not directly present antigen, but instead rely on endogenous dendritic cells for their antigen presenting cell (APC) function. Subsequent research has also demonstrated that primary monocytes can be loaded with antigen and used as cellular vaccines, outperforming MoDC vaccines in mouse models of melanoma and glioblastoma. In this study, we characterized how monocyte vaccines generate CD4+ T cell responses. We utilized the CD11c-Cre RBPJfl/flmouse model, which lack a subset of Notch2 dependent cDC2 characterized by expression of Endothelial cell-selective adhesion molecule (ESAM hi). We demonstrate that the ESAM hicDC2 subset is required for effective CD4+ OT-II T cell priming after vaccination with ovalbumin (OVA) loaded monocytes. Interestingly, depletion of the ESAM hiDC population does not affect monocyte vaccine-induced CD8+ T cell priming, a process previously shown to be dependent on cDC1. This indicates that monocyte vaccines generate T cell responses by independently transferring antigen to both ESAM hiand CD8+ DCs, leveraging their APC function to prime CD4+ and CD8+ T cell responses, respectively. This work furthers the mechanistic understanding of monocyte vaccines, and will inform the design of future monocyte vaccines as they enter clinical trials as therapeutic cancer vaccines. Supported by NCI P50 CA 190991 and funding from the Stead Scholarship Program of the Duke University Department of Medicine.

Reciprocity between IL-4 and opioid immune regulation: IL-4 primes human CD4+ T cells for opioid receptor driven MAP kinase signaling

Abstract Opioid receptor (OR) signaling induces transcriptomic and functional changes in human T cell responses at both a molecular and cellular level, yet the signaling pathways initiated by OR engagement in human T cells are poorly characterized. ORs are G-protein coupled receptors with three subtypes: MuOR, KappaOR, and DeltaOR. Using the human lymphoma Jurkat T cell line and negatively selected human peripheral blood CD4 +T cells, the effects of opioid exposure on the activation kinetics of the MAP kinases ERK 1/2, JNK, and p38, scored as their level of phosphorylation, was followed by immunoblot. Opioid-driven phosphorylation of the MAPKs was dependent on IL-4 pretreatment of Jurkat cells, but not blood-derived CD4+ T cells. After Jurkat cells were primed with a 3-day exposure to IL-4, a series of opioid agonists selective for each OR subtype induced the phosphorylation of all three MAPKs (ERK, JNK, p38) within 2 min. Phosphorylation of the MAPKs was independent of OR subtype and opioid agonist. Notably, the kinetics were different than that observed in neurons. When primary human CD4 +T cells were treated with opioids, only p38 MAPK, not ERK or JNK, was activated with similar kinetics and OR subtype independence as that seen in Jurkat cells. This difference between primary cells and Jurkat cells suggests an additional level of regulation of opioid signaling in primary cells not found in Jurkat cells. The requirement for IL-4 pretreatment suggests that opioids may play a role in Th2 biology. Preliminary data indicate that morphine modestly increases the expression of IL-4 mRNA. Overall, these results reveal that p38 functions as a primary effector of opioid receptor signal transduction in human T cells. Supported by grants from NIH / NIDA (R01 DA 043253, )

Abstract A009: AFNT-111: A novel TCR-engineered T cell therapy targeting the oncogenic driver KRAS G12V

Abstract Mutations in the RAS family are highly prevalent in human cancers, including up to 35% of non-small cell lung, 45% of colorectal, and 95% of pancreatic cancers. Kirsten rat sarcoma viral oncogene homologue (KRAS) is the most frequently mutated RAS oncogene and patients with KRAS mutations have poor responses to standard treatment regimens. Therefore, successful KRAS-targeted therapies represent a high unmet need. The AFNT-111 cellular therapy consists of autologous CD8+ and CD4+ T cells expressing a high affinity TCR specific for the prevalent KRASG12V mutation presented by HLA-A*11:01, one of the most common HLA alleles worldwide. AFNT-111 is engineered to express the CD8α/β coreceptor, enabling a coordinated CD4+/CD8+ tumor response to induce robust T cell activity and persistence while minimizing T cell exhaustion. Lentiviral vector was used to transduce primary healthy human CD4+ and CD8+ T cells with the KRASG12V-specific human TCR and CD8α/β coreceptor. Engineered T cells were assessed against exogenous KRASG12V peptide as well as a panel of tumor cell lines endogenously expressing KRASG12V and probed for in vitro activation, proliferation, cytotoxicity and cytokine secretion. In parallel, in vitro safety studies were performed to evaluate autoantigen cross-reactivity and alloreactivity. In vivo efficacy studies were conducted using established human KRASG12V xenografts in NSG mice. AFNT-111 demonstrated potent functional avidity towards the KRASG12V peptide with no reactivity against KRASWT. Several naturally expressing KRASG12V human tumor cell lines led to significant AFNT-111 T cell activation and proliferation, as well as potent cytotoxicity. The addition of the CD8α/β coreceptor revealed a coordinated and synergistic cytotoxic CD4+ and CD8+ T cell response, enhancing activation of the AFNT-111 cell product. Robust cytotoxicity was associated with increased T cell secretion of IFNγ and TNFα. Durable in vivo anti-tumor efficacy was observed in three established mouse xenograft tumor models. XScan studies using amino acid substitutions of the index KRASG12V peptide revealed a restrictive TCR recognition motif. Further, potentially cross-reactive human self-peptides matching this motif were tested and no significant cross-reactivities were identified. To test alloreactivity, a large lymphoblastoid cell line library was assessed with no alloreactive responses detected. A streamlined manufacturing process has also been developed in which CD4+/CD8+ T cell ratios are controlled, with the final AFNT-111 product retaining a high degree of stem-like properties. AFNT-111 preclinical data demonstrate a highly potent and specific TCR-engineered T cell product that is cytotoxic to KRASG12V-expressing tumor cells both in vitro and in vivo. Cross-reactivity and alloreactivity assessments establish a potentially favorable safety profile of AFNT-111, supporting clinical translation. First-in-human clinical studies will focus on advanced or metastatic pancreatic, colorectal, and lung cancer indications. Citation Format: Michele Hoffmann, Xingyue He, Cheryl Black, Joshua Francis, James Parsons, Christian Roy, Jinsheng Liang, Hongjing Qu, Martin Campbell, Tomasz Sewastianik, Jessica Webb, Aude Chapuis, Thomas Schmitt, Philip Greenberg, Damien Hallet, Markus Vallaster, Piotr Pierog, Gary Shapiro, Hubert Lam, Loic Vincent. AFNT-111: A novel TCR-engineered T cell therapy targeting the oncogenic driver KRAS G12V [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A009.

Boosting NAD blunts T H17 inflammation via arginine biosynthesis and redox regulatory control in healthy control and psoriasis human subjects

Abstract Although NAD +levels modulate metabolism, how this regulates immunometabolism/inflammation remains unclear. Employing Nicotinamide Riboside (NR) to boost NAD, we explored T H17-linked inflammatory in Psoriasis. Primary Psoriasis CD4 +T cells exposed to NR reduced IL-17 secretion and RNA-seq/pathway analysis implicated NR in modulating sequestosome 1 (SQSTM1/p62)-coupled oxidative stress. In parallel, NR reduced CD4 +T cell reactive oxygen species (ROS) levels and activated the NRF2 transcription factor. Depletion of NRF2 or SQSTM1 diminished NR effects on ROS and T H17 activation. Metabolomic analysis implicated NR in arginine biosynthesis and L-arginine replicated NR CD4 +T cell effects. Genetic disruption of the argininosuccinate lyase synthesis enzyme ameliorated NR antiinflammatory effects, supporting that NR, via arginine biosynthesis orchestrates NRF2 activation, CD4 +T cell antioxidant defenses and blunted T H17 responsiveness. A pilot in-vivo human study similarly showed that NR increased arginine, antioxidant enzyme gene expression and blunted T H17 regulation, implicating NAD metabolism in CD4 +T cell-linked inflammation. NHLBI Division of Intramural Research (MNS – ZIA-HL005102), NIH Bench to Bedside award (MNS and RT, HL-129510-04S1) and the NIH Office of Dietary Supplementation (JT) and the UK MRC (JLG – MR/P011705/2; UKDRI-5002; MAP UK).

CRISPR-Cas9 screen identifies potential novel regulators of CD8 +T cell dysfunction

Abstract CD8 +T cells directly target and kill cancer cells and are required for effective antitumor immunity and response to immunotherapy. However, CD8 +T cells may become dysfunctional and exhausted when exposed to chronic antigen stimulation during cancer. Dysfunction is characterized by increased expression of inhibitory receptors as well as impaired effector function, yet the genetic mechanisms controlling dysfunction remain poorly understood. To address this gap, we have developed an ex vivo restimulation model that recapitulates features of CD8 +T cell dysfunction seen within the tumor microenvironment. By coupling this system with genome-wide CRISPR-Cas9 loss of function screening, we have identified genetic factors which may play a novel role in the progression of CD8 +T cells to a dysfunctional state. Deletion of select factors restored CD8 +T cell survival and effector function during chronic restimulation conditions in vitro, including tumor cell killing capability. Future studies will focus on further characterizing the phenotypic and functional roles of these regulators, as well as translating this model to primary human CD8 +T cells. In summary, we have uncovered novel potential regulators of CD8 +T cell dysfunction and targeting these genes may improve antitumor immunity and immunotherapy efficacy.

P152 Allele-associated chromatin interactions in primary immune cells uncover mechanisms of gene regulation

Abstract Background/Aims Genome-wide association studies studies have identified many genetic variants associated with the risk of developing common rheumatological conditions. These variants primarily affect regulatory elements of the genome which can affect the expression of genes located far away from their genomic location through chromatin interaction mechanisms. Furthermore, common genetic variants have been associated with specific changes in chromatin interactions in cell lines. Studying chromatin interactions can improve our understanding of these diseases. Here, we aim to study chromatin interactions and how they are affected by genetic variation in primary immune cells that are relevant for rheumatological conditions. Methods We generated promoter Capture Hi-C (pCHi-C) data for primary CD4+ T cells and CD14+ monocytes isolated from peripheral blood of 10 systemic sclerosis patients and 5 healthy controls. We developed a new computational pipeline to explore how genetic variation affects chromatin interactions. Briefly, genotypes were called from the pCHi-C data using a modified GLIMPSE pipeline. Genotypes were phased using the integrated phasing pipeline which combined population haplotype information with the pCHi-C data. Reads were then assigned to the correct allele using SNPsplit and assigned to chromatin loops (CHiCAGO score > 5). A test for allelic imbalance was carried out using a binomial test, and results were consolidated using Fisher’s method. Results We identified 171 and 139 allele-associated loops (FDR < 0.05) in CD4+ T cells and CD14+ monocytes respectively. These interactions are cell-type specific and highly associated with expression quantitative trait loci (eQTLs). We found that 80% of the variants involved in allele-associated loops were eQTLs for at least one gene. These interactions link to the promoters of 62 and 57 distal genes in CD4+ T cells and CD14+ monocytes respectively. Of these, 38 and 31 respectively were also eQTL for those genes. Many of these allele-associated interactions affect genomic regions that are highly significant for the function of these cell populations. For example, a region with variants affecting T cell-specific chromatin interactions is the TRAF3IP3/IRF6/UTP25 locus. In this region there are separate groups of variants which affect gene expression with a complex mechanism. A group of variants overlapping the UTP25 promoter increases interaction strength with the TRAF3IP3 and IRF6 gene bodies. Interestingly, these are also associated with an increase in UTP25 expression but a decrease in TRAF3IP3 and IRF6 expression. Another group of variants located near the IRF6 promoter is associated with increased interactions with the UTP25 promoter. In contrast, these variants are associated with a reduction of UTP25 expression but an increase in TRAF3IP3 and IRF6 expression. Conclusion We created a new pipeline that allows the identification of allele-associated chromatin interactions in primary cells. These new data will be helpful in uncovering mechanisms of gene regulation in loci highly significant to autoimmune conditions. Disclosure D. González-Serna: None. C. Shi: None. M. Kerick: None. J. Hankinson: None. J. Ding: None. A. McGovern: None. M. Tutino: None. G. Villanueva Martin: None. N. Ortego-Centeno: None. J. Luis Callejas: None. J. Martin: None. G. Orozco: None.

Lysophosphatidylcholine facilitates the pathogenesis of psoriasis through activating keratinocytes and T cells differentiation via glycolysis.

Although abnormal metabolism plays a critical role in the pathogenesis of psoriasis, the details are unclear. Here, we identified to explore the role and mechanism of lysophosphatidylcholine (LPC) on the pathogenesis of psoriasis. The level of LPC in plasma and skin lesions and the expression of G2A on skin lesions of psoriasis patients were detected by enzyme-linked immunosorbent assay, liquid chromatography-tandem mass spectrometry, or immunohistochemistry, respectively. The glycolysis in the skin lesions of imiquimod (IMQ)-induced psoriasis-like mouse model was detected by extracellular acidification rate. LPC was subcutaneously injected into IMQ-treated mouse ears, and the phenotype as well as the glycolysis were evaluated. Exploring the effects and mechanism of LPC on keratinocytes and CD4+ T cells by culturing primary keratinocytes and CD4+ T in vitro. We found that LPC was significantly increased both in the plasma and skin lesions of psoriatic patients, while G2A, exerting an essential role in LPC-inducing biological functions, was increased in psoriatic lesions. The abundance of LPC was positively correlated with glycolytic activity in the psoriasis-like mouse model. LPC treatment facilitated psoriasis-like inflammation and glycolytic activity in skin lesions. Mechanistically, the LPC/G2A axis significantly triggered glycolytic activity and produced inflammatory factors in keratinocytes, and blockade of glycolysis abrogated LPC-induced expression of inflammatory mediators in keratinocytes. LPC activated STAT1, resulting in recognition and binding to the promoters of GCK and PKLR, which are glycolytic rate-limiting enzymes. Furthermore, the LPC/G2A axis directly benefited Th1 differentiation, which was dependent on LPC-induced glycolytic activity. Notably, LPC indirectly facilitated Th17 differentiation by inducing the secretion of IL-1β in keratinocytes-T cells coculture system. Taken together, our findings revealed the role of the LPC/G2A axis in the pathogenesis of psoriasis; targeting LPC/G2A is a potential strategy for psoriasis therapy.

Abstract 1825: A bacterial consortium engineered from feces of patients responding to anti-PD1 treatment mediates tumor response through IFNγ in non-small cell lung cancer

Abstract Introduction: We previously showed that non-small cell lung cancer (NSCLC) patients that respond (R) to immune checkpoint blockade (ICB) have a different microbial community structure than non-responders (NR) pre-treatment (Genome Med 2022). Importantly, we showed that pooled R microbiota transplantation into gnotobiotic xenograft mice decreased tumor growth compared to NR colonized mice following anti-PD1 therapy, a phenomenon associated with enrichment of the Bacteroides genus. However, the bacterial strains driving this synergistic effect with anti-PD1 therapy and mechanism of action are unknown. Method: Feces collected from R mice were used as source material for high-throughput microbial isolation performed with the GALT Prospector technology. Bacterial identification was performed with MALDI-TOF Biotyper and Sanger sequencing. The cell free supernatants and under 3 kDa small molecules of 183 Bacteroides isolates were screened for their ability to stimulate IFNγ production by primary CD8+ T cells by flow cytometry. A consortium composed of 6 IFNγ-stimulating isolates or NR feces was transplanted into a gnotobiotic mouse model of lung cancer treated with anti-PD1, with or without anti-IFNγ monoclonal antibody depletion. Tumors were harvested at endpoint for flow cytometric analysis, and blood serum for IFNγ ELISA. Bioactivity-guided fractionation coupled with LC-MS analysis was used to identify small molecules from the cell free supernatant of a single stimulating Bacteroides isolate. Potential bioactive small molecule characterization was performed using LC-MS/MS supported by computational pipelines for structural prediction followed by NMR for structural confirmation. Results: We cultured and identified 679 isolates from 30 unique species. The cell free supernatant from 6 out of 183 Bacteroides isolates stimulated IFNγ production from primary CD8+ T cells. Small molecules from the six stimulatory isolates’ supernatants significantly induced IFNγ production compared to 6 taxonomy-matched non-stimulatory isolates (P=0.039). A defined consortium composed of the six stimulatory isolates (R-consortium) was able to colonize germ free mice, and decreased tumor growth compared to NR feces-colonized mice (P=0.041). IFNγ depletion of R-consortium mice significantly increased tumor growth (P=0.012) compared to non-depleted mice. Intratumor IFNγ+ CD8+ T cell frequency and circulating serum IFNγ was elevated only in R-consortium mice. Bioassay guided fractionation revealed 5 small molecule metabolites produced by Bacteroides and their structure and bioactivity are currently being investigated. Conclusion: A microbial consortium engineered from R patients’ feces synergizes with anti-PD1 therapy to reduce lung cancer growth through an IFNγ-dependent mechanism which may be mediated by small molecule metabolites. Citation Format: Rachel C. Newsome, Joonseok Oh, Wenya Jian, Raad Z. Gharaibeh, Jason Crawford, Christian Jobin. A bacterial consortium engineered from feces of patients responding to anti-PD1 treatment mediates tumor response through IFNγ in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1825.

Abstract 6543: Comparative analysis of immune landscape according to EGFR mutation and tumor PD-L1 proportion scores in non-small cell lung cancer at single cell resolution

Abstract Introduction: In EGFR-mutant non-small cell lung cancer (NSCLC), the efficacy of immune checkpoint blockade (ICB) is limited with higher incidence of toxicity profiles. EGFR-mutant NSCLC is associated with immune suppressive microenvironment, including lower programmed death-ligand 1 (PD-L1), tumor mutational burden (TMB), and tumor-infiltrating CD8+ T lymphocytes. However, tumors expressing high levels of PD-L1 show improved and durable clinical benefit in immunotherapy. In addition, Understanding the dynamics of tumor microenvironment may elucidate immune response in terms of immune composition, and help overcome resistance to immune checkpoint inhibitors. Here, we demonstrate that conventional type 1 dendritic cells (cDC1s) is one of the key tumor microenvironment, resulting in priming of CD8+ T cells and production of chemokines, such as CXCL9/CXCL10 and cytokines that augment anti-tumor immunity. Methods: A total of 31 surgical samples of NSCLC, including EGFR wild and mutant NSCLC of stages I-III, and 16 normal lung tissue samples were collected. PD-L1 expression in EGFR wild type was categorized into tumor proportion score (TPS) of < 50% and ≥ 50% using immunohistochemistry (IHC). Single cell RNA sequencing was performed with the Scanpy toolkit on the retrospective cohorts of normal (n=16), EGFR mutant (n=14, L858R=6, E19Del=8), EGFR wild TPS ≥ 50% (n=10), and TPS < 50% (n=7). Results: We observed that an overall tumor specific immune response occurred in EGFR wild-type TPS ≥ 50% compared with others. XCL1+ innate-like T cells, which act as an orchestra of tumor specific immune response, were found. These cells had high expression of FCER1G and IL2RB, a receptor for IL-15, However, expression of CD8A/B, CD4 and FCGR3A was low. Interestingly, in these innate-like T cells, the expression of ITGAE, which interacts with E-cadherin in cancer cells, and CD81, which interacts with CD3 and enhances immunological synapse, was observed in EGFR wild TPS ≥ 50%. According to these results, XCR1+ cDC1s were enriched in EGFR wild-type TPS ≥ 50%. CXCL9/CXCL10, which recruits CXCR3+ effector T cells which are primed at the lymph node, was highly expressed in activated DCs at EGFR wild TPS ≥ 50%. CD8+ T cells were significantly enriched in EGFR wild-type TPS ≥ 50%. These cells also expressed IFNG and FLT3LG, which maintain the function of intra-tumoral dendritic cells (DCs), and were also up-regulated in EGFR wild-type TPS ≥ 50%. Among them, exhausted CD8+ T cells expressing PDCD1 were observed in EGFR wild TPS ≥ 50%, and these cells also expressed ITGAE and CD81. In addition, these cells also showed high expression of CXCR3, a receptor for CXCL9/CXCL10 which were expressed in the activated DCs. Conclusion: cDC1s-mediated tumor specific immune response occurred in patients with EGFR wild TPS ≥ 50%, which could be an important mechanism to predict anti-PD-1 response. Citation Format: YoungJoon Park, Su-Jin Choi, Jii Bum Lee, Dong Kwon Kim, Sun Min Lim, Seong Gu Heo, Ke Jia Qi, Byoung Chul Cho, Kyoung-Ho Pyo. Comparative analysis of immune landscape according to EGFR mutation and tumor PD-L1 proportion scores in non-small cell lung cancer at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6543.

Abstract 1624: eIF4E allosteric regulators cause rapid commitment to apoptosis in cancer cells while sparing immune cells

Abstract Advanced or metastatic breast cancers represents a large patient population. Targeted therapies following hormonal or chemotherapeutic approaches offer some benefit. Subsequently, molecular profiling has identified over 100 mutations that render inhibitor resistance, creating challenges for physicians, patients, and the healthcare system broadly. While advances in therapeutic options have improved treatment outcomes by prolonging PFS with better QOL, long-term solutions remain a challenge for the majority of patients. The aim of our work is to drive strong mechanistic responses in this large patient population by developing targeted therapies to a key fundamental mechanism at the convergence of many oncogenic signaling pathways through which resistance arises. Our work in established cell lines and primary patient derived organoids demonstrated potent and complete responses in breast cancers that represent patient populations where unmet needs remain high including ER positive, TNBC, PTEN and RB null molecular profiles. The regulation of CAP dependent protein translation is a central element in oncogenesis. One of a family of CAP binding proteins, eIF4E serves to anchor the m7GTP CAP of RNA into several protein complexes and regulates multiple aspects of mRNA processing such as nuclear export and ribosomal translation. Previous communications revealed our eIF4E regulators potently cause cell death in primary breast cancer organoid models differentially to models derived from healthy tissue. We have expanded our efforts following our observations of both rapid apoptosis and differential sensitivity of healthy cells. In vitro, in breast cancer cells we observed stable responses in mechanistic markers of pharmacodynamic response, including phospho-Rb, with short term drug incubation. In vivo, in a ZR-75-1 breast CDX model, intermittent dosing (QW) of our lead compound resulted in strong tumor growth inhibition (>95%). Consistent with growth impacts, we observed stable pharmacodynamic marker (p-Rb) changes in tumor tissue at 24 and 48 hours. To understand the impact on immune cells, we evaluated our regulators against primary cells including PBMCs, CD4+ T cells, and bone marrow mononuclear cells. Our eIF4E regulators did not impair survival of these cells up to a maximum tested dose of 25 micromolar. Primary CD4+ T cells were also functional in the presence of eIF4E regulators up to 5 micromolar, which included assessments of CD3/CD28 stimulated cytokine production and proliferative responses. Our findings suggest that allosteric modulation of eIF4E provides a unique and efficient way to address multiple resistance mechanisms while sparing normal immune cellular function. Thus, our allosteric eIF4E regulators represent a potential beneficial therapeutic approach to address multiple resistant cancer patient populations and fulfill the promise of this elusive target. Citation Format: Lisa-Marie Sturla, Xuemei Zhang, Katherine Bowdish, Susan E. Alters, Christopher VanDeusen. eIF4E allosteric regulators cause rapid commitment to apoptosis in cancer cells while sparing immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1624.

Abstract 5833: Crosstalk between cancer-associated fibroblasts and CD8 T cells leads to improved T cell activation in head and neck cancer

Abstract Cancer-associated fibroblasts (CAFs) have been shown to modulate immune responses in the tumor microenvironment (TME), however, the extensive heterogeneity of these cells has precluded rigorous understanding of their immunoregulatory role in the head and neck TME. To gain a better understanding of the relationship between CAFs and the immune environment in head and neck cancer, we leveraged protein activity inference tools to analyze single-cell RNA sequencing of advanced-stage head and neck squamous cell carcinoma (HNSCC) patients pre- and post-treatment with the αPD-1 therapy, nivolumab. From this analysis, we identified a novel CAF population predictive of patient response to αPD-1 therapy. Utilizing in vitro co-culture assays with primary human CD8 T cells, we discovered that this novel CAF population was able to elicit features associated with enhanced CD8 T cell function, namely the reduction of PD-1+TIM-3+ exhaustive phenotypes and induction of CD103+NKG2A+ tissue-resident memory phenotypes and cytotoxicity. Due to their unique ability to stimulate T cells in this manner, we termed this novel CAF population T cell-stimulating CAFs (tsCAFs). In efforts to elucidate the mechanism behind this pro-inflammatory function, we determined that tsCAFs secrete a heat-sensitive small molecule/s in order to alter CD8 T cell behavior. However, tsCAFs will only secrete this factor in the presence of activated T cells demonstrating crosstalk between T cells and CAFs in the head and neck TME. This pro-inflammatory crosstalk between CAFs and T cells represents a novel finding that further contributes to the idea of immunoregulatory pleiotropy among CAFs. Accordingly, our findings highlight the functional importance of distinct CAF subsets in modulating the immunoregulatory milieu of human HNSCC and warrant more extensive characterization of CAF-T cell interactions in human squamous cell carcinoma. Citation Format: Diana Graves, Aleksandar Obradovic, Henry Schares, Michael Korrer, Brian Bachmann, Young Kim, Ben Park, Paula Hurley. Crosstalk between cancer-associated fibroblasts and CD8 T cells leads to improved T cell activation in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5833.

Abstract 4787: The cancer targeting synergy of tertbutylhydroquinone and a manganese porphyrin

Abstract Cancer cells utilize high basal levels of reactive oxygen species (ROS) to create conditions that facilitate rapid cell proliferation. This creates an Achilles heel, making cancer cells more susceptible than normal cells to cell death caused by a bolus dose of ROS. The common food preservative, tert-butylhydroquinone (tBHQ), generates ROS intracellularly via redox cycling. Herein, we tested the effect of combining tBHQ with a manganese porphyrin, compounds originally designed as super oxide dismutase mimetics which can also generate ROS intracellularly in cancer cells and have radioprotective effects under clinical investigation. We find that the combination treatment of tBHQ and the commercially available Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) is much more toxic to leukemic Jurkat lymphocytes than either compound alone, e.g., lowering the LC50 of tBHQ by two orders of magnitude to 1.0 μM. MnTMPyP acts as a catalyst to oxidize tBHQ, shown by UV-Vis spectroscopy. Flow cytometry analysis revealed that while 5 μM tBHQ decreases cell viability by less than 10% in 4 hours, the combination of 5 μM tBHQ and 12 μM MnTMPyP decreases viability by 60%, primarily by apoptosis. A clinically tested manganese porphyrin, Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (Mn2BuOE), was as effective and more potent than MnTMPyP. We evaluated the cancer-targeting effect of tBHQ and Mn2BuOE in both Jurkats and a matched primary CD4 T cell line. The combination treatment induced significantly more cell death in cancerous Jurkats than in the normal CD4 cells. With the promise Mn2BuOE shows in pre-clinical studies and early clinical trials as adjunctive to radiotherapy, and the wide use of tBHQ in prepared food, the results shown here in CD4 cells suggest their combination may be an effective therapy to target cancer cells. Citation Format: Joseph P. LaMorte, Sandra Tamarin, Margueritte Lalo, David J. Sokoloski, Jared J. Paul, Aimee L. Eggler. The cancer targeting synergy of tertbutylhydroquinone and a manganese porphyrin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4787.

Tumor Suppressor Adenomatous Polyposis Coli Sustains Dendritic Cell Tolerance through IL-10 in a β-Catenin-Dependent Manner.

Dendritic cells (DC) play important roles in balancing immunity and tolerance, in which β-catenin signaling plays an important role, yet the underlying mechanisms remain elusive. In this study, we investigated the functions of the tumor suppressor adenomatous polyposis coli (APC), also a key component of the β-catenin upstream destruction complex in DC. APC depletion in DC does not alter DC and T cell homeostasis under resting conditions. However, APC deficiency in DC leads to attenuated antitumor immunity in mice, which exhibit fewer CD8+ T cells and more Foxp3+ regulatory T cells in tumor and draining lymph nodes. Loss of APC in DC does not affect the expression levels of costimulatory molecules. However, APC-deficient DC produce more IL-10 and exhibit a higher ability of inducing regulatory T cells but a lower ability of priming CD8+ T cells, both of which can be reversed by IL-10 inhibition. Lastly, β-catenin depletion in APC-deficient DC rescues their antitumor immunity and reverses elevated IL-10 production. Taken together, our results identify that APC drives DC tolerance via the β-catenin/IL-10 axis.

Chemotherapy-Sensitized In Situ Vaccination for Malignant Osteosarcoma Enabled by Bioinspired Calcium Phosphonate Nanoagents.

How to effectively treat malignant osteosarcoma remains clinically challenging. Programmed delivery of chemotherapeutic agents and immunostimulants may offer a universal strategy for killing osteosarcoma cells while simultaneously eliciting in situ antitumor immunity. However, targeted chemoimmunotherapy lacks a reliable delivery system. To address this issue, we herein developed a bioinspired calcium phosphonate nanoagent that was synthesized by chemical reactions between Ca2+ and phosphonate residue from zoledronic acid using bovine serum albumin as a scaffold. In addition, methotrexate combination with a phosphorothioate CpG immunomodulator was also loaded for pH-responsive delivery to enable synergistic chemoimmunotherapy of osteosarcoma. The calcium phosphonate nanoagents were found to effectively accumulate in osteosarcoma for nearly 1 week, which is favorable for exerting the vaccination effects in situ by maturing dendritic cells and priming CD8+ T cells to suppress the osteosarcoma progression and pulmonary metastasis through controlled release of the three loaded agents in the acidic tumor microenvironment. The current study may thus offer a reliable delivery platform for achieving targeted chemotherapy-induced in situ antitumor immunity.

The transcriptional regulator Sin3A balances IL‐17A and Foxp3 expression in primary CD4 T cells

The Sin3 transcriptional regulator homolog A (Sin3A) is the core member of a multiprotein chromatin‐modifying complex. Its inactivation at the CD4/CD8 double‐negative stage halts further thymocyte development. Among various functions, Sin3A regulates STAT3 transcriptional activity, central to the differentiation of Th17 cells active in inflammatory disorders and opportunistic infections. To further investigate the consequences of conditional Sin3A inactivation in more mature precursors and post‐thymic T cell, we have generated CD4‐Cre and CD4‐CreERT2 Sin3AF/F mice. Sin3A inactivation in vivo hinders both thymocyte development and peripheral T‐cell survival. In vitro, in Th17 skewing conditions, Sin3A‐deficient cells proliferate and acquire memory markers and yet fail to properly upregulate Il17a, Il23r, and Il22. Instead, IL‐2+ and FOXP3+ are mostly enriched for, and their inhibition partially rescues IL‐17A+ T cells. Notably, Sin3A deletion also causes an enrichment of genes implicated in the mTORC1 signaling pathway, overt STAT3 activation, and aberrant cytoplasmic RORγt accumulation. Thus, together our data unveil a previously unappreciated role for Sin3A in shaping critical signaling events central to the acquisition of immunoregulatory T‐cell phenotypes.

Analysis of clinical features in primary cutaneous T cell lymphoma

Background: Primary cutaneous T cell lymphoma is a diverse and rare group of diseases with clinical manifestations that, if not biopsied, are easily confused with benign skin diseases. Objective: To determine some clinical features of primary cutaneous T cell lymphomas and classify them according to the 2018 update of World Health Organization and European Organisation for Research and Treatment of Cancer classification. Methods: a case series study with 39 cases of primary cutaneous T cell lymphomas diagnosed at Pathology Department of University Medical Center at Ho Chi Minh city from 01/2018 to 02/2022. Results: Median age at diagnosis is 59 years old. Male:Female ratio is 1:2.3. In order of frequency, diagnoses were: subcutaneous panniculitis-like T cell lymphoma (35.8%), mycosis fungoides (28.2%), primary cutaneous anaplastic large cell lymphoma (15.4%), peripheral T cell lymphoma not otherwise specified (7.7%), extranodal NK/T cell lymphoma, nasal type (7.7%), primary cutaneous CD4+ small/medium T cell lymphoproliferative disorder (2.6%) and primary cutaneous CD8+ aggressive epidermotropic T- cell lymphoma (2.6%). Subcutaneous panniculitis-like T cell lymphoma always presents as subcutaneous nodule-tumor (100%); the most common site is lower extremities (71.4%). Mycosis fungoides has various appearance of skin lesions (36.4%) including patch-plaque (100%), and usually involves in trunk (90.9%). Extranodal NK/T cell lymphoma, nasal type always has skin ulcer (100%). Conclusion: The study documented the frequence and clinical characteristics of each subtype of primary cutaneous T cell lymphoma. The most common were subcutaneous panniculitis-like T cell lymphoma and mycosis fungoides. Although the morphology and location of skin lesions in the subtypes overlap, they still aid in the diagnosis of skin lymphoma. Key words: T-cell lymphoma, primary, cutaneous.