Population Of CD4 Research Articles

Abstract 1975: Potential novel Immuno-oncology mechanism revealed during translational phase I Immuno-blood profiling of experimental ADC medicine OBT076 in a gastric cancer patient

Abstract CD205 is a type I transmembrane glycoprotein, with unique characteristics that make it an ideal target for Antibody Drug Conjugate (ADC) therapy. Here we report on a potential novel immuno-oncology mechanism revealed during the Translational Phase I (NCT04064359) immuno-blood profiling of a chemo-refractory patient treated with OBT076, an experimental CD205-directed ADC. A chemo-refractory advanced gastric cancer patient with 60% CD205 expression in the primary tumor via IHC and having previously undergone 2 lines of chemotherapy treatment (Docetaxel/cisplatin/5FU and Ramucirumab/Paclitaxel), received five 21-day cycles of OBT076 (one at 2.5mg/kg and four cycles at 2.0 mg/kg) followed by 1 cycle of Pembrolizumab (PZ; 200mg) ~4 weeks later. Clinical response was evaluated and immunological markers (CD45, CD205, CD4, CD8, and PD1) in peripheral blood cells were quantified using flow cytometry. After 2 OBT076 cycles at 2.0 mg/kg, there was an ~40% shrinkage in the primary gastric tumor size and resolution of ascites and lymph node metastases were observed. Following 2 further cycles and PZ, complete response was achieved for the primary tumor. Flow cytometry showed (1) an initial decrease in the absolute numbers of dendritic cells by day 8, followed by a 2-fold increase in numbers by day 21 after treatment; (2) a near total decrease in the population of CD8+ CD205+ cells by day 8, no recovery in levels were observed; (3) a 3-fold increase in CD4+ and CD8+ T-cell numbers between days 8 and 21 and (4) an initial decrease in CD4+ PD1+ and CD8+ PD1+ T-cell numbers followed by an ~4-fold increase between days 8 and 21. In summary our results show that increases in PD1+ T-cells, T-cell induction, and decreases in immuno-suppressive CD4+ CD205+ and CD8+ CD205+ cells occur simultaneously; coinciding with rapid resolution of the primary tumor, lymph node metastases and ascites. These findings suggest that OBT076 activates the patient’s immune response against the tumor through a potentially novel mechanism: drug-induced depletion of CD8+ CD205+ immuno-suppressive cells and subsequent T-cell activation. Additionally, our data support the use of immune checkpoint inhibitors in conjunction with OBT076 to achieve favorable clinical outcomes. Citation Format: Christian Rohlff, Solmaz Sahebjam, Alain Mita, Rosen Lee, Chander Sekhar Peddaboina, Arnima Bisht, Lindsey Hudson, Wolf Fridman, Abderrahim Fandi, Olivier Rixe. Potential novel Immuno-oncology mechanism revealed during translational phase I Immuno-blood profiling of experimental ADC medicine OBT076 in a gastric cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1975.

Abstract 6129: Investigate the mechanism and treatment efficacy of regorafenib combined with anti-PD-L1 on bladder cancer

Abstract Bladder cancer is one of the most common cancer in urinary system in Taiwan. According to Ministry of Health and Welfare’s research, bladder cancer plays 15th leading causes of death in Taiwan in 2020. The patients who suffer from bladder cancer after undergoing resection surgery about 60% will relapse, and 80% of them will relapse in less than a year, 15% of the relapsing patients will aggravate and become invasive carcinoma spread all over the body. PD-L1 (Programmed death-ligand 1), also known as B7-H1 and CD274 is a transmembrane glycoprotein, which interacts with PD-1 (Programmed death-1) receptor on T cells to elicit the immune checkpoint response. Overexpression of PD-L1 suppresses the function of CD8+ T cells and facilitate tumor progression. Targeting tumor PD-L1 may be a possible treatment option; however, PD-L1 expression was only detected in small amount of population. Regorafenib is a multiple tyrosine kinase inhibitor, which has been approved by the US Food and Drug Administration to treat advanced HCC. In our research, we found that regorafenib may upregulation the surface PD-L1 expression in both mice and human cell line. Based on this finding, we suggested that regorafenib may show potential to enhance treatment efficacy anti-PD-L1. Therefore, we established MB-49 bearing animal model to identify the immune regulation and treatment efficacy of regorafenib combined with anti-PD-L1. In ours animal data we found that combination therapy effectively increased the tumor infiltrating population of NK cells and CD8+ lymphocytes which were determined by flow cytometry assay. Not only anti-tumoral leukocytes, we also detected the population of immune suppressor cells including regulatory T cells, MDSCs and M2 macrophage was suppressed by combination therapy. We also detected the tumor microenvironment related markers (VEGF, IDO-1, PD-L1, CD8, CD31, CD49b, FOXP3, granzyme B) to evaluate combination therapy effect by using immunohistochemistry staining. In conclusion, regorafenib may effectively enhance anti-PD-L1 efficacy via triggering positive immune regulation and increasing the interaction of anti-PD-L1 with tumor. Citation Format: Kai-Jen Pai, Yuan Chang, I-Tsang Chiang, Fei-Ting Hsu. Investigate the mechanism and treatment efficacy of regorafenib combined with anti-PD-L1 on bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6129.

Abstract 2070: Differentiating PD-1+TCF-1+ stem-like resource CD8 T cells towards a distinct effector Tcell population with enhanced anti-tumor potential by delivering an engineered IL-2 variant to PD-1 via cis-targeting

Abstract PD1-IL2v is a novel immunocytokine constituted by a high affinity bivalent blocking PD-1 antibody and a heterodimeric silent Fc-part fused to a monomeric IL-2 variant (IL2v). In contrast to wildtype IL2, IL2v does not bind to CD25 (IL2Ra) and, therefore, the preferential targeting of T regulatory cells (Tregs) and endothelial cells through CD25 is abolished. The PD-1 antibody, having ca. 50-fold higher affinity than IL2v, dictates the preferential cis-delivery of IL2v to PD-1 positive T cells, such as antigen-experienced tumor-reactive T cells. In addition, due to the reduced expression levels of PD-1 on Tregs compared to CD8 and conventional T cells, PD1-IL2v preferentially binds to the latter in vitro and ex vivo, rescuing them from Treg-mediated suppression. To assess the effects of the cis-delivery of IL2v to PD-1+ T cells in vivo, we applied subcutaneous B16F10-OVA and Panc02-H7-Fluc tumor models in syngeneic mice. PD1-IL2v lead to significantly improved survival of the B16F10-OVA tumor bearing and tumor regression in the Panc02-H7-Fluc tumor bearing mice. The OVA-specific CD8 T cells expressed the highest amount of PD-1 in the tumor microenvironment, and PD1-IL2v not only increased the total number of CD8 tumor infiltrating lymphocytes (TILs), but, more importantly, it expanded the OVA-specific T cells. The recently discovered PD-1+ TCF1+ stem-like or resource T cells responsible for the proliferative burst following anti-PD-1 therapy, correlate with clinical responses and better prognosis. However, stem-like T cells ultimately differentiate into terminally exhausted T cells resulting in relapses and tumor growth. Here, we show that PD1-IL2v targets OVA-specific stem-like T cells, and leads to their expansion and differentiation into a unique population of effector CD8 T cells, so-called “fresh and better effectors”. These cells have a distinct phenotypic and molecular signature in addition to diverse TCR clonality in comparison to the terminally exhausted CD8 TILs generated by anti-PD1 therapy alone or in combination with a non-cis targeted FAP-IL2v immunocytokine. Thus, PD1-IL2v is a differentiated immune cell-targeted IL2v, which promotes an effective and long-term anti-tumor immune-response by delivering IL-2v to PD-1+ tumor-specific T cells, like stem-like T cells, and by generating a distinct effector T cell population. Citation Format: Maria Karagianni, Eleni Maria Varypataki, Valeria Nicolini, Christian Münz, Pablo Umana, Christian Klein, Laura Codarri Deak. Differentiating PD-1+TCF-1+ stem-like resource CD8 T cells towards a distinct effector Tcell population with enhanced anti-tumor potential by delivering an engineered IL-2 variant to PD-1 via cis-targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2070.

Abstract 5419: A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features

Abstract Introduction: Mismatch repair (MMR) protein expression in colorectal cancer (CRC) is usually homogeneously retained or lost. However, rare CRC lesions may show a heterogeneous pattern of MMR protein expression. We aimed to evaluate the molecular effect of a heterogeneous expression of the MMR proteins in a complex context of concomitant loss of one of the other effectors of the DNA repair mechanism. Experimental procedures: We evaluated MMR protein expression (by immunohistochemistry -IHC- for MLH1, MSH2, MSH6 and PMS2) in 200 CRCs, identifying 3 groups with proficient (MMRp), deficient (MMRd) and heterogeneous (MMRh) MMR. MMRh tumors were micro-dissected based on the expression of the heterogeneous marker, DNA was extracted and subjected to targeted sequencing (TSO500 panel, Illumina). RNA was purified from bulk tumors of all MMRh cases and in a control series of 10 MMRp and 10 MMRd CRCs and analyzed using the IO360 panel (NanoString). The degree of tumor infiltrating lymphocytes (TILs) was calculated on H&E slides and on IHC sections stained with antibodies raised against CD8. Results: Twenty-nine out of 200 cases were MMRd (14.5%). Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, resulting in a microsatellite instable (MSI) phenotype. Five out of the 9 cases were suitable for separate mutational analysis of IHC-positive and IHC-(double)negative components of the tumor. Both components of all the lesions exhibited a high tumor mutation burden (mean TMB: 59 mut/Mb) in line with the MSI status, nevertheless a significant increase in TMB in the double-negative components was observed (mean TMB: neg. 67 mut/Mb vs pos. 53 mut/Mb), due to a higher number of sub-clonal, non-synonymous variants compared to the positive component. Comparative gene expression analyses between MMRd, MMRp and MMRh CRCs highlighted differential gene expression patterns, with MMRh tumors displaying a strong activation of angiogenesis, MAPK and PI3K-AKT axes. Moreover, these tumors showed the highest number of TILs, characterized by a substantial population of exhausted CD8+ lymphocytes. Conclusions: We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins, in a background of concomitant loss of one of the other markers. This heterogeneity is associated with i) an increase of TMB and of unstable loci in the negative component, with additional markedly sub-clonal variants, ii) a high number of TILs, mostly exhausted CD8+ lymphocytes, and iii) a differential activation of signaling pathways. Taken together these data provide indirect evidence that heterogeneous loss of MMR proteins may impact on the biology of CRCs. Whether this pattern may influence response to immune checkpoint inhibition remains to be determined. Citation Format: Enrico Berrino, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, Anna Sapino, Silvia Marsoni, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Emanuela Bonoldi, Caterina Marchio. A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5419.

Abstract 1347: Novel lung cancer mouse model to study the effects of cigarette smoke on immune therapy

Abstract Background: In 2021, 235,760 new cases of lung cancer will be diagnosed in the United States. The treatment regimen of non-small cell lung cancer (NSCLC) has drastically changed owing to the superior anti-cancer effects generated by the immune-checkpoint inhibitor (ICI). However, only a subset of patients experience benefit after receiving ICI. Tumor Mutation Burden (TMB) and PD-L1 expression in tumor cells are known to be potential biomarkers in predicting a patient’s survival and response to ICI. Several studies showed that NSCLC patients with smoking history had a better response than never smokers due to increased TMB caused by carcinogens in cigarette smoke. However, the roles of tumor microenvironment especially immune microenvironment between smokers and never smokers in response to ICI are poorly understood. We hypothesize that the functions of T cells are influenced by the exposure of cigarette smoke prior to the ICI treatment which affect patients’ response to ICI. Method: A cohort of 216 patients diagnosed with NSCLC who had received at least one dosage of Immune Checkpoint Inhibitor (ICI) at Atrium Wake Forest Baptist Hospital were split into three groups: current smokers, former smokers, and never-smokers. Both PFS and OS analyses were performed to examine whether smoking history is correlated with the ICI response. To establish an in vivo model that recapitulates the patients with different smoking history, we treated mice with cigarette extract (CSE) followed by accessing the peripheral T cell functions by FACS analyses using antibodies targeting GZMB, TNF and IFNG. LL/2 and CMT167 cells will be inoculated to those CSE treated mice by flank injection once we observe a stable activation of T cells compared to control group. Tumor growth will be measured and the amount of tumor infiltrated CD4 and CD8 T cells will be examined by immunocytochemistry at the end point. Results: We found it took around 9 weeks of CSE treatment to observe a significant increase of CD8 and CD4 populations in CSE treated mice. We also treated mouse CD8 T cells isolated from the spleen with CSE and observed a strong activation of GZMB, TNF and IFNG by FACS analyses. Future directions: We will test the efficacy of anti-tumor effect of anti-PD-1 ab in tumor bearing mice exposed with or without CSE prior to the immune therapy. We will also establish a mice model that mimics the former smokers by halting the CSE treatment after 9 weeks of CSE exposure followed by examine the amount of memory T cells. We believe our study will provide additional mechanisms of how cigarette smoke affects the response of ICI by affecting the properties of immune cells. Citation Format: Margaret Rose Smith, Yuezhu Wang, Jimmy Ruiz, Jason Grayson, Yin Liu, Fei Xing. Novel lung cancer mouse model to study the effects of cigarette smoke on immune therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1347.

Abstract 1718: Characterization of infiltrating immune cells after irradiation using 10x genomics single cell sequencing

Abstract Background: Radiation therapy is delivered to approximately 50% of all cancer patients. It has long been known that radiation causes not only DNA strand breaks, apoptosis, and necrosis, but also immunogenic modulation. Lately, there has been renewed interest in the potential of irradiation in colorectal cancer, not only as an adjuvant and palliative treatment, but also as an immune stimulant. Materials and Methods: CT26 murine colorectal carcinoma cells were subcutaneously implanted on the flank of BALB/c mice. Tumors were exposed to sham, single dose or fractionated radiotherapy regimens via a 300kV, 10mA X-ray with additional filtration to give a radiation quality of 2.3mm Cu half-value layer (HVL). Mice were anaesthetized, restrained and delivered a single uniform dose of radiation at a rate of 0.696Gy/min with the whole mouse being lead shielded except for a 1cm hole in the lead above the tumor site. Tumors were harvested at different timepoints post irradiation and dissociated into single cell suspensions. The tumor microenvironment and immune response within the tumors were analyzed with flow cytometry comparing the different treatment regimens. The individual CD45+ cell profile was investigated using 10x gene expression. 10x sequencing was demultiplexed using CellRanger and data analyzed using Seurat and ACTINN. Results: Following the initial cytotoxicity, flow cytometry showed a clear immune stimulatory effect of radiotherapy with immune cells infiltrating the irradiated tumors. The level of infiltration was dependent on the treatment scheduling. For single cell assessments, we observed approximately 8000 reads/cell and T-SNE cluster analysis using Seurat identified 16 clusters in control and treated samples. Identification of clusters by ACTINN demonstrated that CD8+ T-cells were increased 8-fold in irradiated samples whereas monocytes numbers decreased 3-fold. In irradiated tumors, the main population of CD8+ T-cells with increased numbers also expressed NKG7. Conclusion: These data provide evidence that radiotherapy can enhance immune cell trafficking to locally treated CT26 tumors. Furthermore, 10x genomics single cell sequencing revealed increased levels of cytotoxic T-cells expressing NKG7, a protein implicated in enhanced killing of extracellular targets. This response could prime the tumors such that they may be exploited by immune modulating drugs. Citation Format: Frida Ponthan, Valentina Ubertini, Gary Beale. Characterization of infiltrating immune cells after irradiation using 10x genomics single cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1718.

Abstract 4218: 6-gingerol upregulated CD8+ T cells function in mice model of colorectal cancer via modulation of programmed cell death protein 1/programmed death-ligand1 and cytotoxic T-lymphocyte-associated protein 4 receptor signaling

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States, with about 149,500 new cases of CRC diagnosed in 2021. Although various treatments are available for CRC, survival rates of CRC patients remain very poor. In the last few decades, Inhibition of immune checkpoint proteins have been shown to be a promising strategy for CRC treatments, thus allowing T cells to destroy CRC cells. 6-gingerol, an active compound present in fresh ginger have been shown to possess anti-cancer and immunomodulatory properties. We have previously described the anti-metastatic effect 6-gingerol in CRC. However, there is paucity of information on the effect 6-gingerol on CD8+ T cell function and immune checkpoints proteins in CRC. Therefore, this study investigated the effect of 6-gingerol on CD8+ T cells function and immune check point proteins in using mice model of CRC and system biology approach. Male BALB/c mice were divided into four groups of 10 mice each. Group 1 mice serve as control, group 2 mice received 6-gingerol 10mg/kg/day by oral gavage for 10 weeks. Group 3 (CRC model) mice received a single dose of azoxymethane 10mg/kg (Intraperitoneal) and on the seventh day they received three cycles of dextran sulfate sodium (DSS) 2% (W/V) in drinking water (each cycle is defined as one week of 2% DSS followed by two weeks of plain drinking water) Group 4 mice received 6-gingerol 10mg/kg/day by oral gavage in combination with single dose of azoxymethane 10mg/kg (iP) and three cycles of 2% DSS (W/V) in drinking water. Administration of 6-gingerol to AOM/DSS treated mice increase the percentage (%) gated cells population of CD8+Tcells and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) with concomitant decrease in % gated population of Programmed cell death protein 1 (PD-1) and B-7 proteins as compared to AOM/DSS only treated mice. Furthermore, there was decrease expression of PD-L1 on colon tissues of 6-gingerol treated mice when compared with AOM/DSS treated mice colon. Also, using system biology analysis, we observed a high binding energy of 6-gingerol with PD-1/PD-L1 Complex, PD-L1, Dimeric PD-L1 and CTLA-4/B7-2 complex proteins. The result obtained from this study showed that 6-gingerol inhibited immune check point proteins and upregulated CD8+ T cells function in colorectal cancer via modulation of PD-1/PD-L1 and CTLA4 receptor signaling, and as such 6-gingerol could be a promising natural product for CRC immunotherapy. Citation Format: Babajide O. Ajayi, Anuoluwapo Adeshina. 6-gingerol upregulated CD8+ T cells function in mice model of colorectal cancer via modulation of programmed cell death protein 1/programmed death-ligand1 and cytotoxic T-lymphocyte-associated protein 4 receptor signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4218.

Abstract 560: Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs

Abstract Background: We have previously established the ability of CD3-directed lentiviral vectors encoding for CD19 or CD22 CARs to mediate robust anti-tumor immunity in humanized lymphoreplete mouse models. We now present additional mechanistic data for this novel subcutaneous (SC) CAR-T approach. Methods: Human PBMCs loaded with a self-inactivating lentiviral vector (LV) encoding CD19 CAR with a synthetic driver element were injected SC into autologous PBMC humanized NSG MHC I/II double knock out (DKO) mice. The LV was packaged with a modified envelope with the ability to target and activate CD3+ T cells. To first track the site of CAR formation in vivo following SC injection, PBMCs were loaded with an LV encoding a CD19 CAR and luciferase, and bioluminescence imaging (BLI) was performed. Additionally, histopathology of the site of injection and distal organs were examined. Evaluation for CAR+ cells was performed through immunohistochemistry and PCR detection. To examine the tropism of the CD3-directed CD19 CAR LV and characterize CAR+ cells, the phenotype of in vivo expanded CAR+ cells was evaluated. Further characterization of CAR+ cells was performed with in vitro studies. Results: Following SC injection of LV-loaded PBMCs, the first evidence of transgene expression utilizing BLI for luciferase was detected four to five days following SC injection. At the same time point, histologic examination of the SC site of injection revealed the formation of tertiary lymphoid structures (TLS) consisting of human CD8+ and CD4+ T cells, CD68+ macrophages, CD68+ dendritic cells, and a few CD20+ B cells. On day 13 post-SC injection, BLI detected the presence of CAR+ cells systemically beyond the site of injection and within subcutaneous Raji tumors implanted on the contralateral side. On day 14 post-SC injection, histologic examination showed sustained TLS within the SC tissue without signs of dermal acute inflammation or ulceration and evidence of CAR+ cells appearing in the spleen. CAR+ cells exhibited robust anti-tumor immunity with expansion into peripheral blood. CAR+ cells consisted of a distinct population of CD8+ T cells with NK-like features (TaNKs) and a CD3+ CD8+ CD56+ NKG2D+ cell phenotype. In vitro transduction of CD56 NK cell-depleted PBMC with the CD3-directed LV also led to CAR-TaNK formation. Conclusion: The subcutaneous injection of CD3-directed LV-loaded PBMCs leads to the formation of tertiary lymphoid structures at the site of injection and the development of distinct CD3+ CD8+ CD56+ NKG2D+ CAR-TaNK cells. These cells possess enhanced systemic proliferative capacity compared to traditional ex vivo manufactured 41BB CAR-T cells in a lymphoreplete mouse model and the ability to eliminate target cells in vivo with low numbers of starting cells (10,000 cells). Citation Format: Ani Kundu, Dongming Zhang, Frederic Vigant, Junyi Zhang, Greg Schreiber, Ewa Jaruga-Killeen, Alissa Kerner, Michelle Andraza, Wei Zhang, John Henkelman, Renata Soares, Gregory I. Frost, Sid P. Kerkar. Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 560.

Abstract 3527: Novel bacteria strains, CJRS-10671 and CJRS-10672, enhance anti-tumor efficacy in LLC1 syngeneic model and humanized PDX mice model

Abstract Background: Immune checkpoint inhibitor has emerged as remarkable therapeutic that improves the anti-cancer effect of patients. However, response rate is low in a large proportion of patients. The overall efficacy of immune checkpoint inhibitor therapy remains unsatisfactory. Recently, live biotherapeutic products (LBPs) have emerged as potential therapeutics to overcome the limitation of immune checkpoint inhibitor. Here, we demonstrated the efficacy of CJRS-10671 and CJRS-10672 in tumor bearing mice models. Methods: To evaluate anti-tumor effects, 109 CFU/mouse CJRS-10671 was administered via oral gavage twice daily (BID) alone or in combination with anti-PD-1 (10 mg/kg, QOD, i.p.) for 10 days in a Lewis Lung Carcinoma (LLC1) syngeneic tumor model. Tumor growth was measured by calipers 3 times per week. We analyzed immune cell profiles by flow cytometry. In lung squamous cell carcinoma patient derived xenograft (PDX) model,109 CFU/mouse CJRS-10671 and CJRS-10672 were orally administered twice daily (BID) alone or in combination with pembrolizumab (10 mg/kg, Q5D, i.p.) for 35 days. Tumor growth was measured by calipers 3 times per week. We analyzed immune cell profiles and single cell RNA sequencing. Results: In LLC1 syngeneic tumor model, CJRS-10671 alone suppressed tumor growth comparable to that of immune checkpoint inhibitor (anti-PD-1 antibody) only treatment group. Combined administration of CJRS-10671 and immune checkpoint inhibitor significantly suppressed tumor growth (p<0.05). We found that combination of CJRS-10671 with anti-PD-1 antibody increased the population of CD8+IFN-γ+ cells in splenocytes and tumor-infiltrating lymphocytes (TILs).In humanized PDX model, CJRS-10671 alone significantly suppressed tumor growth compared with that of vehicle control and pembrolizumab only treatment (p<0.001). CJRS-10672 alone suppressed tumor growth compared with that of vehicle control (p<0.001) which is comparable with the efficacy of pembrolizumab alone. Combined administration of CJRS-10672 and pembrolizumab further significantly suppressed tumor growth than pembrolizumab alone (p<0.01). None of the mice in pembrolizumab showed more than 75% tumor growth inhibition cut-off (TGI-75%). Fifty percent of the mice in CJRS-10671 single treated group showed over TGI-75%. The groups of pembrolizumab combination with CJRS-10672, pembrolizumab combination with CJRS-10671, CJRS-10672 alone were observed 25%, 20%, and 11.1%, respectively (>TGI-75%).We confirmed that administration of CJRS-10671 increased population of CD8+ central memory T cells in tumor. Also, TGI (%) directly related features were mostly antigen presentation and monocyte origin cells, especially macrophage in immune-profiling analysis. Furthermore, we observed that CJRS-10671 and CJRS-10672 were associated with signaling of damage-associated molecular pattern (DAMP) and especially CJRS-10671 was related to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling by single cell RNA sequencing analysis. Conclusion: CJRS-10671 and CJRS-10672, single administration or combination with anti-PD-1 antibody, have potential anti-tumor effect. Citation Format: Arim Min, Bo-eun Kwon, Hyunjeong Kim, Hyunkyung Park, Jiyoung Lee, Kyoung-Ho Pyo, Byoung Chul Cho. Novel bacteria strains, CJRS-10671 and CJRS-10672, enhance anti-tumor efficacy in LLC1 syngeneic model and humanized PDX mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3527.

Abstract 288: An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics

Abstract Background: Molecular changes underlying immune cell signaling in response to new therapy development are crucial to validate the clinical efficacy of immunotherapeutics. There are several preclinical tools including patient derived cell lines, organoid and xenograft (PDX) models to assess the efficacy of drugs. However, it is important to recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. Here we report, an ex vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal and immune cell components to assess treatment-mediated changes in molecular and transcriptional profiles of tumor resident immune cells using a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) platform. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids measuring 150 µm in size with intact tumor immune microenvironment were prepared from fresh tumor samples of colon and lung tissues using proprietary technology developed at Nilogen Oncosystems. No enzymatic digestion, propagation or reassembly was used during the preparation of the tumoroids. Hundreds of tumoroids originated from different parts of each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with or without immunostimulatory agents for 48h. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells. Results: Following sequencing of FACS sorted viable CD45 populations, the FAST-Q output files were uploaded into Cell Ranger for analysis with Loupe Browser. Multimodal analysis of transcriptomes or proteomics at the single-cell level revealed significant changes in the top 100 gene expression levels specific to CD4 and CD8+ T cells, NK cells and B cells signaling and activation upon stimulation with immunostimulatory agents. In addition, several pro and anti-inflammatory chemokines and cytokines were upregulated in response to ex vivo treatment in both colon and lung tumoroids, which coincided with marked changes in the activation status of tumor resident immune cells, as detected by multiparameter flow cytometry analysis. Conclusions: These results demonstrate Nilogen’s 3D fresh tumoroid model reflects the significant impact on activation and proliferation of tumor resident immune cells within the intact tumor microenvironment at the transcriptional level. We believe this model system can serve as a clinically relevant tool for accelerating immuno-oncology drug development. Citation Format: Jonathan P. Waterman-Smith, Jared Ehrhart, Brittany Bunch, Krithika Kodumudi, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 288.

DIPG-22. Modifying the tumor microenvironment with a TIM-3 monoclonal antibody as a therapeutic strategy for DIPGs

Abstract Diffuse Midline Glioma, encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. TIM-3 (HAVCR2) is a member of the T-cell immunoglobulin and mucin domain protein family. It is expressed on multiple immune cell types, including T cells, NK cells, myeloid populations, and microglia, regulating adaptive and innate immunity. In silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Single-cell sequencing analyses of DIPG biopsies uncover TIM-3 expression, especially in microglia. In vivo efficacy studies showed that treatment with AbTIM-3 significantly increased overall survival in two DIPG immunocompetent orthotopic models, led to long-term survivors (50%), and showed immune memory. TIM-3 treatment led to a significant increase in the tumor microenvironment of microglia, granulocytes, NK, and CD8+ cells and higher levels of IFNγ, GrzB and TNFα corresponding with an NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population which causes an increase in the pro-inflammatory CD8/Treg ratio. CD4, CD8 or NK cell depletion leads to a significant but not a total loss of treatment efficacy. CD4+ and CD8+ cells were aumented in treated draining lymph nodes and expressed higher amounts of pro-inflamattory cytokines than control-mice. Population analysis and depletion experiments demonstrated the relevance of NK, CD4, CD8 and myeloid populations in the response to anti-TIM-3 therapy. Interestingly, the depletion of the different immune populations combined or using immunodeficient Rag2 mice, did not completely abrogate the treatment efficacy. These results suggest the concurrence of an additional mechanism of action that together with the immune response leads to a robust anti-DIPG effect. In conclusion, these data demonstrate that TIM-3 is a potential target for the treatment of DIPG.

Dexamethasone and brain metastasis-infiltrating CD8+ T cells.

e15075 Background: Nearly all patients who develop large and/or symptomatic brain metastases receive corticosteroids such as dexamethasone (Dex) to treat or prevent symptoms like nausea, headaches, or focal neurologic deficits. Dex has immunosuppressive effects such as induction of T cell apoptosis, so it’s possible that this treatment may impair the anti-tumor immune response. To address this question, we performed a secondary analysis of our in-press data describing the phenotype of brain metastasis-infiltrating CD8+ T cells to determine whether the duration of Dex therapy prior to surgical resection correlated with phenotypes we described our cohort. Methods: Fresh brain metastases specimens were weighed, and immune cells were isolated. CD45+ and CD8+ lymphocytes were counted via flow cytometry from 35 and 34 samples, respectively. A subset of samples were analyzed by high-parameter spectral flow cytometry to gain phenotypic information. Dex treatment was later extracted from the electronic medical record, and Spearman’s correlation coefficients (CC) were calculated to evaluate the association between duration of Dex therapy and brain metastasis-infiltrating lymphocyte density or phenotype. Results: Patients were on Dex for a median of 3 days (4mg q6 hours) prior to surgery. On univariate analysis, the CC between CD45+ lymphocytes or CD8+ T cells per gram of tumor and duration of Dex treatment was -0.185 (P=0.288) and -0.122 (P=0.49), respectively. The CC between frequency of PD-1+ CD8+ T cells and duration of Dex was -0.064 (p=0.784, 21 patients). In our previous work, we used the FLOWSOM algorithm to identify distinct populations of CD8+ T cells that were enriched in brain metastases compared to blood. We described two such populations, one with a tissue residence phenotype, and the other with terminally-differentiated phenotype. The CC between frequency of these populations and duration of Dex was 0.043 (p=0.889) and 0.238 (p=0.433), respectively (13 patients). We also tested for correlation between frequency of individual PD-1+ CD8+ T cell populations and duration of Dex treatment (13-18 patients). The CC for Tim3+, CD39+, CTLA4+, CD127+, Tcf-1+, CD28+, and GZMB+ populations of PD-1+ CD8+ T cells were 0.037 (p=0.891), 0.096 (p=0.705), -0.094 (p=0.738), 0.011 (p=0.971), -0.026 (p=0.918), 0.384 (p=0.128), and 0.236 (p=0.358), respectively. Conclusions: Within this cohort of patients who were on Dex for a median of 3 days prior to surgery, duration of pre-surgery Dex did not significantly affect density of brain metastasis-infiltrating CD45+ lymphocytes or CD8+ T cells or frequency of PD-1+ CD8+ T cells.

Immune cell differences between patients in different stages of monoclonal plasma cell disorders.

8065 Background: Multiple myeloma (MM) is an incurable plasma cell malignancy. Precursor conditions include monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma (SMM). Immune surveillance is paramount for keeping the malignancy in check, whereas a dysfunctional immune system is suspected to promote disease progression. Methods: We performed flow cytometric immune panel analyses including T-cell, B-cell, natural killer (NK) cell and dendritic cell (DC) subsets in MGUS, SMM, and MM patients between 2018-2021. Immune panels were analyzed on fresh peripheral blood samples drawn at diagnosis. The patient population were MGUS (n=28), SMM (n=19) and MM (n=94). Samples from MGUS and SMM patients were combined into a single group (asymptomatic) and were compared to MM patients (symptomatic). Lymphocytes were identified by CD45+ expression resolving against side-scattered light on a bivariate plot. B cell and NK cell antigen expression profiles were assessed from this lymphocyte population. T cell subsets were further determined by CD3+. The percent gated for each marker was summarized by cohort using the appropriate descriptive statistics and compared using the Mann-Whitney U exact test. Analyses were performed in SAS v9.4 (Cary, NC) using a two-sided α=0.05. Results: Compared to MGUS/SMM patients, MM patients had a significant lower proportion of naive B cells, NK cells, and cytolytic NK cells, and a significant higher proportion of cytolytic T cells at diagnosis (Table). Consistent with prior studies, we demonstrated an increase in exhaustion markers. There was a trend toward a higher population of CD8+ exhausted T cells in MM patients compared to MGUS/SMM patients. Since the follow up for this study was short, survival data is not mature. Conclusions: This study demonstrates the immune imbalance that occurs between myeloma precursor conditions and MM. There is a shift in certain cell subsets in the immune microenvironment that alters tumor immunosurveillance and tumor cell killing favoring disease progression. In data not shown, we are currently analyzing the bone marrow microenvironment and its relation to disease progression. Follow up studies could assess if therapeutic intervention could reverse the imbalance, restore homeostatic equilibrium with the goal of controlling disease long-term. [Table: see text]

Dihydroartemisinin inhibits the growth of pancreatic cells by inducing ferroptosis and activating antitumor immunity

Dihydroartemisinin (DHA) exhibits a direct antitumor effect in various tumor models. However, the mechanism of DHA inducing ferroptosis and activating antitumor immunity remains obscure. Therefore, our study was dedicated to investigate the effect of DHA on ferroptosis and tumor microenvironment and elucidate the underlying molecular mechanism. PDAC orthotopic tumor model was used to investigate tumor proliferation and the population of immune cell in vivo, including M2-type macrophages (M2), myeloid-derived suppressor cells (MDSCs), CD4+T cells, CD8+T cells, NK cells and NKT cells. Levels of GPX4, SLC7A11, P53 and ALOX12 were determined by Real-time PCR and Western blot. CCK8 assay was performed to detect cell viability, and the ferroptosis was distinguished by flow cytometry. Our results showed that DHA inhibited pancreatic cancer cell proliferation. In addition, DHA induced cell ferroptosis by up-regulating the expression of P53 and ALOX12, which was blocked by baicalein (a selective ALOX12 inhibitor). However, DHA also up-regulated the expression of GPX4 and SLC7A11. On the other hand, DHA significantly decreased the suppressive expansion of M2 and MDSCs. Moreover, DHA increased the immune cell population of CD8+T cells, NK cells and NKT cells in the tumor tissues of the tumor-bearing mice. Whereas, the DHA treatment did not affect the frequencies of M2, MDSCs, CD4+T, CD8+T, NK and NKT cells in the spleen. Our research provided experimental evidences on the activity and mechanism of ferroptosis induced by DHA and revealed that DHA regulated tumor local immunosuppressive microenvironment.

Type 1 Diabetes: A Promising Dialogue between Promiscuous T Cell Receptor and H‐2Db Peptide Complex

Obliteration of the beta cells mediated by CD8 T cells in the islets of Langerhans leads to the development of autoimmune disease, type 1 diabetes (T1D). During the kickoff of T1D, CD8 T cells play a pivotal role in the destruction of these pancreatic beta cells. The CD8 T cell clone AI4, previously isolated from the islets of a young diabetes‐prone NOD mouse, is found to be the most pathogenic among the population of CD8 T cell clones. Multiple natural and synthetic ligands for AI4 have been identified. These ligands include an unusually short seven‐residue peptide derived from the insulin A chain (InsA14‐20) that is recognized by AI4 in the context of H‐2Db but lacks a C‐terminal anchor residue. High diabetogenic activity of AI4 is accredited to the promiscuity, or cross reactivity, of its T cell receptor (TCR). We adopted the structural biology studies for understanding the mechanism of cross‐reactivity of the AI4 TCR with its multiple ligands. Unraveling the three‐dimensional structure of H‐2Db with AI4 ligands will help us to unveil the distinct mechanisms involved in the recognition and the cross‐reactivity of the AI4 TCR. In previous studies, our group has already reported two structures of the H‐2Db peptide complexes recognized by AI4. In our present work we have solved two additional structures of H‐2Db with AI4 ligands and several others are in progress. In addition to these studies, we have cloned, expressed and purified the AI4 TCR in a soluble form, which should allow us to obtain its structure alone and in complex with its multiple ligands. Our approach to study the interaction of promiscuous TCRs with their multiple ligands will allow us to uncover the likely multiple mechanisms that underlie TCR cross‐reactivity in autoimmunity.

ХАРАКТЕРИСТИКА ИММУНОЛОГИЧЕСКИХ НАРУШЕНИЙ У БОЛЬНЫХ ХРОНИЧЕСКИМ РЕЦИДИВИРУЮЩИМ АФТОЗНЫМ СТОМАТИТОМ

This work is devoted to the study of general and local immunity in chronic recurrent aphthous stomatitis. The relevance of the study of immunological mechanisms and their role in the development of aphthous stomatitis is associated with an increase in the number of relapses, the duration of the course and the complexity of the treatment. The study found an increase in the concentration of all classes of immunoglobulins, a decrease in the concentration of gamma-interferon, an increase in the production of TNF-α in the oral fluid, an increase in antiviral protection factors (CD16, CD4, CD25), a population of CD20, B-lymphocytes in blood serum.

Single-cell TCR and mRNA sequencing of antigen-specific T cells reveal spatial trajectories from single time points with HLA and VDJ bias

Abstract Yellow fever (YF) vaccination induces massive CD8 expansion, comprising up to 10% of all circulating CD8 T cells. We hypothesize that HLA type and VDJ gene usage implicate the highly biased response towards certain immunodominant epitopes. PBMCs were collected from healthy human donors 3 weeks after vaccination with the yellow fever vaccine, Stamaril, and enriched for YF-CD8 T cells using HLA class I tetramers. We focused on the immunodominant epitopes NS4B and NS2A, and the less dominant epitope NS3. Cells were analyzed by single-cell T cell receptor (TCR) and mRNA sequencing, yielding detailed gene phenotypes for around 2000–3000 quality cells from each donor. Data was analyzed using SeqGeq, VDJ explorer, Seurat and Monocle2. From three donors, we performed in depth analyses on both TCR gene use bias across epitope specificity, and established pseudotime cell trajectories from a single sampling time point. We found that within the CD8 population from the same donor, VDJ usage was highly biased for the target antigen. Based on mRNA expression of a panel of 259 T-cell relevant genes, we performed unsupervised clustering, that shows cells with different epitope targets cluster interchangeably, indicating similar functional patterns. However, cells specific for the immunodominant epitopes had lower TCR clonal divergence between naïve-like and activated clusters, indicating a higher degree of clonal expansion compared to less immunodominant clones. Taken together, we show antigen-driven T cell responses are biased based on donor HLA-types, which could implicate development and success of T-cell driven immunotherapies. Supported by grants from Independent Research Fund Denmark (1029-00004B)

Human lung tissue-resident memory CD8+ T cells are transcriptionally, epigenetically, and phenotypically diverse

Abstract Tissue-resident memory CD8+ T cells (TRM) are strategically located in peripheral tissues, especially at mucosal surfaces, where they can provide protection against invading pathogenic microbes. In the lungs, TRM play a critical role in limiting disease and transmission of respiratory viruses; however, the heterogeneity present in TRM populations in the human lung remains largely unexplored. Here we show that human lungs harbor transcriptionally, epigenetically, and phenotypically distinct populations of memory CD8+ T cells expressing the tissue residency-associated markers CD69 and CD103. High-dimensional flow cytometry and single-cell RNA sequencing of memory CD8+ T cells isolated from human lungs shows that heterogeneity exists both between and within CD69+ CD103− and CD69+ CD103+ subsets, with transcriptional diversity among the CD69+ CD103− subset being most prominent. Single-cell ATACseq demonstrates that similar levels of epigenetic diversity exist between and within these different TRM subsets. However, flow cytometry and single-cell RNAseq of influenza- and SARS-CoV-2-specific lung TRM demonstrates that heterogeneity between CD69+ CD103− and CD69+ CD103+ subsets is largely eliminated when the cells are specific for a common antigen. Together, these data illuminate underappreciated and unexplored aspects of heterogeneity in TRM populations in humans. Supported by grants from NIH/NIAID (75N93019R00028) and NIH/NHLBI (R35 HL150803)

Molecular regulation of CD8 T cell activity and heterogeneity in pancreatic cancer

Abstract T cell dysfunction or ‘exhaustion’ is a hallmark of solid malignancies. Despite the critical importance of CD8 T lymphocytes in controlling tumor growth and immunotherapy efficacy, the mechanisms governing T cell exhaustion remain unclear. Here, we focused on resolving the heterogeneity and molecular programming of exhausted CD8 T cell states in the context of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a discouraging 5-year survival rate of approximately 8%. PDAC tumors commonly present as immunologically ‘cold’, devoid of protective T cell populations, and are typically resistant to promising immunotherapies such as immune checkpoint blockade or adoptive cell therapy. Therefore, understanding the unique microenvironmental and CD8 T cell-intrinsic signals governing T cell exhaustion and accumulation in PDAC tumors may reveal crucial insight for engineering CD8 T cells for optimal immunotherapy efficacy. Here, we developed preclinical PDAC models for profiling tumor-specific murine CD8 T cell responses. Through application of spectral flow cytometry and multimodal single-cell sequencing approaches, we have uncovered impressively diverse antigen-specific CD8 T cell states in PDAC tumors. Select CD8 T cell populations identified within the PDAC tumor microenvironment included canonical populations of terminally-exhausted and progenitor-exhausted CD8 T cells as well as unexpected and uncharacterized CD8 T cell populations. Accordingly, we also have identified critical transcription factors controlling key features of CD8 T cell heterogeneity and persistence in PDAC tumors. These findings may ultimately provide insight for tailoring CD8 T cell fate and function in PDAC. Supported by 2022 AAI Intersect Fellowship for Computational Scientists and Immunologists

The biological basis of disease recurrence in psoriasis: a historical perspective and current models.

SummaryA key challenge in psoriasis therapy is the tendency for lesions to recur in previously affected anatomical locations after treatment discontinuation following lesion resolution. Available evidence supports the concept of a localized immunological ‘memory’ that persists in resolved skin after complete disappearance of visible inflammation, as well as the role of a specific subpopulation of T cells characterized by the dermotropic CCR4+ phenotype and forming a local memory. Increasing knowledge of the interleukin (IL)‐23/T helper 17 (Th17) cell pathway in psoriasis immunopathology is pointing away from the historical classification of psoriasis as primarily a Th1‐type disease. Research undertaken from the 1990s to the mid‐2000s provided evidence for the existence of a large population of CD8+ and CD4+ tissue‐resident memory T cells in resolved skin, which can initiate and perpetuate immune responses of psoriasis in the absence of T‐cell recruitment from the blood. Dendritic cells (DCs) are antigen‐presenting cells that contribute to psoriasis pathology via the secretion of IL‐23, the upstream regulator of Th17 cells, while plasmacytoid DCs are involved via IL‐36 signalling and type I interferon activation. Overall, the evidence discussed in this review indicates that IL‐23‐driven/IL‐17‐producing T cells play a critical role in psoriasis pathology and recurrence, making these cytokines logical therapeutic targets. The review also explains the clinical efficacy of IL‐17 and IL‐23 receptor blockers in the treatment of psoriasis.