Cell-depleted Mice Research Articles

Potentiating the radiation-induced type I interferon antitumoral immune response by ATM inhibition in pancreatic cancer.

Radiotherapy induces a type I interferon-mediated (T1IFN-mediated) antitumoral immune response that we hypothesized could be potentiated by a first-in-class ataxia telangiectasia mutated (ATM) inhibitor, leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound, AZD0156, on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced antitumoral immune responses and sensitized tumors to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells, as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN, leading to both innate and subsequent adaptive antitumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon Treg depletion.

Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell-mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell-targeted immunotherapy in mice, we find that CD4+ Foxp3- conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3- Tconv cells within tumors adopt a Treg cell-like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10-dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell-targeted therapies.

Conditional NKT Cell Depletion in Mice Reveals a Negative Feedback Loop That Regulates CTL Cross-Priming.

NKT cells are unconventional T cells whose biological role is incompletely understood. Similar to TH cells, activated NKT cells can cause dendritic cell (DC) maturation, which is required for effective CTL responses. However, it is unclear whether and how NKT cells affect CTLs downstream of the DC maturation phase. This is partially due to the lack of techniques to conditionally deplete NKT cells invivo. To overcome this problem, we have developed two approaches for this purpose in mice: the first is based on mixed bone marrow chimeras where Jα18 knockout and depletable CD90 congenic bone marrow is combined, and the second used PLZFCre × iDTR bone marrow chimeras, which target innate-like T cells. Using these tools, we found that NKT cell depletion at 20 h, that is, after initial DC activation, did not render CTLs helpless, as CD40L signaling by non-NKT cells sufficed. Instead, NKT cell depletion even augmented CD8 T cell expansion and cytotoxicity by mechanisms distinct from reduced STAT6 signaling. These findings revealed a negative feedback loop by which NKT cells control CTL cross-priming downstream of DC maturation. The techniques described in this study expand the toolbox to study NKT cells and other unconventional T cell subsets invivo and uncovered a hidden immunoregulatory mechanism.

Inhibition of microfold cells ameliorates early pathological phenotypes by modulating microglial functions in Alzheimer’s disease mouse model

BackgroundThe gut microbiota has recently attracted attention as a pathogenic factor in Alzheimer’s disease (AD). Microfold (M) cells, which play a crucial role in the gut immune response against external antigens, are also exploited for the entry of pathogenic bacteria and proteins into the body. However, whether changes in M cells can affect the gut environments and consequently change brain pathologies in AD remains unknown.MethodsFive familial AD (5xFAD) and 5xFAD-derived fecal microbiota transplanted (5xFAD-FMT) naïve mice were used to investigate the changes of M cells in the AD environment. Next, to establish the effect of M cell depletion on AD environments, 5xFAD mice and Spib knockout mice were bred, and behavioral and histological analyses were performed when M cell-depleted 5xFAD mice were six or nine months of age.ResultsIn this study, we found that M cell numbers were increased in the colons of 5xFAD and 5xFAD-FMT mice compared to those of wild-type (WT) and WT-FMT mice. Moreover, the level of total bacteria infiltrating the colons increased in the AD-mimicked mice. The levels of M cell-related genes and that of infiltrating bacteria showed a significant correlation. The genetic inhibition of M cells (Spib knockout) in 5xFAD mice changed the composition of the gut microbiota, along with decreasing proinflammatory cytokine levels in the colons. M cell depletion ameliorated AD symptoms including amyloid-β accumulation, microglial dysfunction, neuroinflammation, and memory impairment. Similarly, 5xFAD-FMT did not induce AD-like pathologies, such as memory impairment and excessive neuroinflammation in Spib−/− mice.ConclusionTherefore, our findings provide evidence that the inhibiting M cells can prevent AD progression, with therapeutic implications.

Social stress induces autoimmune responses against the brain.

Clinical studies have revealed a high comorbidity between autoimmune diseases and psychiatric disorders, including major depressive disorder (MDD). However, the mechanisms connecting autoimmunity and depression remain unclear. Here, we aim to identify the processes by which stress impacts the adaptive immune system and the implications of such responses to depression. To examine this relationship, we analyzed antibody responses and autoimmunity in the chronic social defeat stress (CSDS) model in mice, and in clinical samples from patients with MDD. We show that socially stressed mice have elevated serum antibody concentrations. We also confirm that social stress leads to the expansion of specific T and B cell populations within the cervical lymph nodes, where brain-derived antigens are preferentially delivered. Sera from stress-susceptible (SUS) mice exhibited high reactivity against brain tissue, and brain-reactive immunoglobulin G (IgG) antibody levels positively correlated with social avoidance behavior. IgG antibody concentrations in the brain were significantly higher in SUS mice than in unstressed mice, and positively correlated with social avoidance. Similarly, in humans, increased peripheral levels of brain-reactive IgG antibodies were associated with increased anhedonia. In vivo assessment of IgG antibodies showed they largely accumulate around blood vessels in the brain only in SUS mice. B cell-depleted mice exhibited stress resilience following CSDS, confirming the contribution of antibody-producing cells to social avoidance behavior. This study provides mechanistic insights connecting stress-induced autoimmune reactions against the brain and stress susceptibility. Therapeutic strategies targeting autoimmune responses might aid in the treatment of patients with MDD featuring immune abnormalities.

Glycolysis-Driven IFN-γ Expression in T-Cell Via Histone Lactylation Promotes Acute Graft-Versus-Host Disease

Background Cell metabolism is a highly regulated process essential for sustaining life and driving evolution. Cellular metabolites and metabolic flux intricately govern post-translational modifications of proteins. In the context of acute graft-versus-host disease (aGVHD), T cells undergo enhanced glycolysis, resulting in increased lactate production. Consequently, lactate may act as a substrate for lysine lactylation (Kla), influencing gene expression by modulating histone biology. This study aims to assess T cell glucose metabolism during aGVHD, investigate the role of histone lactylation, and explore its impact on T cell functionality. Methods We conducted a comparative analysis of histone lactylation between aGVHD patients and individuals undergoing allo hematopoietic stem cell transplantation (control). Subsequently, we established a murine bone marrow transplant model and isolated T cells for analysis. Glucose metabolism status was assessed using extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) assays, along with the 2-NBDG glucose uptake assay. To comprehensively investigate histone lactylation in T cells, we employed the cleavage under targets and tagmentation (CUT&Tag) technique to explore its regulatory effects on gene expression. To investigate the impact of lactatylation on aGVHD pathology, we constructed an aGVHD model by applying T cells with a specific deletion of LDHA. Results By analyzing the serum lactate at the onset of aGVHD, we found that patients with grade III-IV aGVHD had significantly higher lactate levels compared to those with grade I-II aGVHD ( Figure 1A). In light of this finding, we further examined the histone lactylation levels in T cells from aGVHD patients. The results demonstrated an enhanced histone lactylation in these T cells compared to controls ( Figure 1B). In the aGVHD mice, the ECAR of T cells exhibited a significant increase ( Figure 1C). Intriguingly, the OCR were also significantly higher in the aGVHD mice compared to the bone marrow T cell-depleted (BM-TCD) mice ( Figure 1D). Additionally, the 2-NBDG revealed a significantly higher fluorescence intensity in aGVHD mice ( Figure 1E). Through analysis of T cell RNA-seq data, we discovered upregulation of most glycolytic enzymes in aGVHD mice similar to human data ( Figure 1F). Notably, LDHA, glucose transporter 1 ( Slc2a1), and monocarboxylate transporter 4 ( Slc16a3) were highly upregulated ( Figure 1G). The upregulation of LDHA in T cells from aGVHD mice prompted us to investigate the presence of histone lactylation. The results revealed that histone in T cells from aGVHD mice exhibited a higher level of lactylation ( Figure 1H). Because lactylation was particularly prominent at the H4K12 site, we conducted CUT&Tag experiments. We found promoters of key regulatory genes of the Th1 subset, such as Tbx21 and Ifng, along with several genes associated with IFN-γ production, including Stim2, Crtam, Il18rap, Fasl, and Phf11a, Cxcr3 were all lactylated ( Figure 1I). Specifically, the Call Peak analysis confirmed the elevated H4K12la levels on the promoters Tbx21 and Ifng ( Figure 1J). The expression of IFN-γ and CXCR3 in CD4 + and CD8 + T cells of aGVHD mice was significantly higher than in BM-TCD mice ( Figure 1K). To confirm the role of histone lactylation in IFN-γ generation, we intervened with T cells using the p300 lactylation writer inhibitor (A485). The results demonstrated a significant reduction in histone lactylation upon A485 treatment, which was effectively restored by the addition of neutral lactate sodium (Nala) ( Figure 1L). Notably, A485 treatment led to a decrease in IFN-γ + T cells, while Nala administration resulted in a substantial recovery of IFN-γ expression ( Figure 1M). Additionally, the infusion of Cd4creLdhafl/fl T cells into recipient mice improved the survival of aGVHD mice ( Figure 1N). Conclusion This study represents the first global investigation of T cell histone lactylation in aGVHD. Importantly, we have uncovered a novel link between histone lactylation and the regulation of IFN-γ-related gene expression in the context of aGVHD. Considering the heightened glycolysis and the pivotal role of IFN-γ in aGVHD pathology, it is plausible to postulate that T cell-produced lactate induces histone lactylation, thereby promoting the transcription of IFN-γ-related genes and contributing to the development of aGVHD.

End-Stage Idiopathic Pulmonary Fibrosis Lung Microenvironment Promotes Impaired NK Activity.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. In this study, we characterized the NK phenotype in IPF lungs via flow cytometry using 5-dodecanoylaminofluorescein di-β-d-galactopyranoside, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media (CM), and the bleomycin-induced pulmonary fibrosis mouse model was used to assess the invivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in CM from IPF lower lobe lung fibroblasts induced a senescent-like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF, and NKs presented decreased migration toward their CM. In addition, NK cell-depleted mice treated with bleomycin showed increased collagen deposition and accumulation of different populations of senescent cells compared with controls. The IPF lung microenvironment induces a dysfunctional NK phenotype limiting the clearance of lung senescent cells and the resolution of lung fibrosis. We propose that impaired NK activity could be one of the mechanisms responsible for perpetuating the accumulation of senescent cells in IPF lungs.

Proteolipid Protein-Induced Mouse Model of Multiple Sclerosis Requires B Cell-Mediated Antigen Presentation.

The pathogenic role B cells play in multiple sclerosis is underscored by the success of B cell depletion therapies. Yet, it remains unclear how B cells contribute to disease, although it is increasingly accepted that mechanisms beyond Ab production are involved. Better understanding of pathogenic interactions between B cells and autoreactive CD4 T cells will be critical for novel therapeutics. To focus the investigation on B cell:CD4 T cell interactions invivo and invitro, we previously developed a B cell-dependent, Ab-independent experimental autoimmune encephalomyelitis (EAE) mouse model driven by a peptide encompassing the extracellular domains of myelin proteolipid protein (PLPECD). In this study, we demonstrate that B cell depletion significantly inhibited PLPECD-induced EAE disease, blunted PLPECD-elicited delayed-type hypersensitivity reactions invivo, and reduced CD4 T cell activation, proliferation, and proinflammatory cytokine production. Further, PLPECD-reactive CD4 T cells sourced from B cell-depleted donor mice failed to transfer EAE to naive recipients. Importantly, we identified B cell-mediated Ag presentation as the critical mechanism explaining B cell dependence in PLPECD-induced EAE, where bone marrow chimeric mice harboring a B cell-restricted MHC class II deficiency failed to develop EAE. B cells were ultimately observed to restimulate significantly higher Ag-specific proliferation from PLP178-191-reactive CD4 T cells compared with dendritic cells when provided PLPECD peptide in head-to-head cultures. We therefore conclude that PLPECD-induced EAE features a required pathogenic B cell-mediated Ag presentation function, providing for investigable B cell:CD4 T cell interactions in the context of autoimmune demyelinating disease.

#4455 DISCOVERY AND VALIDATION OF NOVEL RENAL EPITHELIAL SENESCENCE ASSOCIATED BIOMARKERS

Abstract Background and Aims Cellular senescence is characterised by irreversible cell cycle arrest and marked changes in transcriptional and secretory activity. It can occur as a physiological component of healthy organismal development or in response to a range of cellular insults including DNA damage, oncogenic mutations and metabolic stress. Renal tubular senescence in response to ageing and injury is proposed as a driver of kidney fibrosis. Senescent cell depletion in mice improves outcomes in multiple organs including the kidney. There are currently no non-invasive biomarkers for quantifying renal senescence available. We are using a multi-omics approach and utilising human renal proximal tubular epithelial cells (hRPTECs) in culture, a murine model of renal senescence and human samples, to identify urinary biomarkers of renal tubular senescence and to determine if they can predict decline in kidney function. Method In vitro: We optimised a model of induced senescence in hRPTECs in vitro using 10Gy irradiation and treatment with MDM2-antagonist Nutlin 3A. Transcriptomic studies using bulk RNAseq were performed comparing senescent cells with proliferating controls (each group n = 5). Genes with >x2 fold-change between groups and adjusted p values <0.01 were regarded as differentially expressed. In vivo: Senescence was induced using 7Gy total body irradiation (TBI) in mice whilst the Bcl2/w/xL inhibitor ABT-263, was used to deplete senescent cells. We performed LC-MS proteomic studies on urine from mice comparing healthy controls with mice exposed to TBI with or without subsequent senolytic therapy. In human kidney biopsy samples from 55 patients with CKD, we performed immunofluorescence staining for senescence marker p21CIP1, proliferation marker Ki67 (absent in senescent cells) and tubular markers CD10 and CKPAN and quantified tubular senescence using machine learning algorithms in the software QuPath (see figure). Tubular cells were classified as senescent when p21CIP1 positive and Ki67 negative and expressed as a percentage of all tubular cells. Results Irradiated and Nutlin 3A treated cells had increased mRNA levels of CDKN1A and reduced LMNB1 and MKI67 in keeping with senescence induction. Other transcripts including CXCL8 and IL6 rose in irradiated cells compared to controls but fell in Nutlin 3A treated cells. 1272 genes where differentially expressed in irradiated cells compared to controls; 760 of these genes were differentially expressed in the same direction in Nutlin 3A treated samples. Over representation analysis highlighted pathways relating to the cell cycle, consistent with senescence induction. LC-MS studies on murine urine samples identified 15 proteins that fell in mice exposed to TBI compared to healthy controls but reverted towards baseline with senolytic treatment. By combining my datasets with publicly available data, we identified several candidate biomarkers of senescence. This includes urokinase plasminogen activator surface receptor, a protein linked with senescence and ageing as well as other novel senescence markers (not named due to pending patent applications). In the patients where senescence was quantified, the mean age at the time of biopsy was 54 years (range 19-81 years) and the mean eGFR was 56 ml/min/1.73 m2 [s.d. 34]). Renal tubular senescence correlated with age (rho = 0.64, p<0.001) and inversely with baseline eGFR (rho = -0.51, p<0.001). LC-MS and ELISA studies in matched urine samples are ongoing to determine which molecules most closely correlate with senescence histologically in the human kidney; these will be presented at the meeting. Conclusion Through a combination of transcriptomic studies and LC-MS proteomics, we have identified several candidate urinary biomarkers of senescence; ongoing studies will determine which molecules correlate with renal senescence histologically. Further studies in a cohort of >380 patients with >4 years follow-up will determine if the most promising biomarkers predict renal outcomes and will also be presented at the meeting.

Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer’s disease

BackgroundBrain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer’s disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis.MethodsAppNL−G−F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL−G−F mice, with evaluation after 3 days. Second, to assess the antibodies’ ability to halt the progression of Aβ pathology, 3 months old AppNL−G−F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL−G−F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining.ResultsNeither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies.ConclusionsBoth RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy.

CD301b +DCs orchestrate type 2 immune responses to helminth infection in the lung

Abstract Helminth parasites induce potent host type 2 immune responses (T2I) that promote parasite clearance and prevent reinfection. The parasite-induced T2I involve both innate and adaptive immune cell components, but how the activation of different cell types is coordinated during the initiation of T2I is incompletely understood. Dendritic cells (DCs) are best known for their role in bridging innate and adaptive immunity by sensing immunological insults and presenting antigens to T cells. We previously found that CD301b +DCs, a major subset of migratory type 2 conventional DCs in peripheral organs, are specifically required for the differentiation of T helper type 2 (Th2) cells in the skin- and lung-draining lymph nodes following infection with Nippostrongylus brasiliensis (Nb) in mice, but their overall role in host immune responses against Nb infection remains unclear. Here, we show that the depletion of CD301b +DCs results in reduced T2I in the lung and impaired worm clearance. The reduction in type 2 cytokine production and eosinophil influx was observed as early as 2 days post-infection before the onset of adaptive immunity, as well as in CD4T cell-depleted mice, suggesting that CD301b +DCs play a critical role in the induction of innate T2I independently of their role in Th2 cell priming. Moreover, CD301b +DC-depleted mice exhibited ameliorated lung hemorrhage with reduced neutrophil infiltration and IL-17 production early after infection. These data suggest the role of CD301b +DCs in co-regulating innate type 2 and type 3 inflammation mechanisms during the early phase of Nb infection in the lung. This work was supported by NIH grant R01AI132576.

Alteration of secretory cell lineages and immune cell migration induced by acute tuft cell ablation in mouse intestine

Background: Intestinal tuft cells have only been recently investigated thoroughly due to their roles in type 2 immunity and chemosensory function. This rare population of intestinal epithelium is implicated in extensively interacting with other cell types and involved in nutrient absorption. Doublecortin-like kinase 1 (DCLK1) is widely used for tuft cell identification in the mouse intestine. However, the effects of tuft cell deletion on mouse intestines have not been fully understood. Here, we investigated the effects of tuft cell deletion on the epithelial cell lineages and overall impact on mouse intestine. Methods: Tuft cell deletion was induced in Dclk1-IRES-GFP-CreERT2/+; Rosa-DTA (Dclk1-DTA) mice by a single dose of tamoxifen injection. Cre-negative DTA/+ mice and DTA-negative Dclk1-CreERT2 mice of littermates received tamoxifen injection at same time as controls. Mouse bodyweight was monitored daily, and intestinal tissues were sampled 2, 4, or 7 days after the tamoxifen injection. Immunostaining of intestinal tissue sections was performed for lysozyme (LYZ), trefoil factor 3 (TFF3), cluster of differentiation 3 (CD3), DCLK1, mast cell protease 1 (MCPT1), Ly6G, and F4/80. Image analysis was conducted using Qupath. Results: Dclk1-DTA mice showed a significant decrease in bodyweight to 11% of their original weight on day 4 after tamoxifen injection. They showed significantly shortened small intestinal length compared to control mice, however, colonic length showed no significant difference. Immunofluorescent staining of induced Dclk1-DTA mouse tissues revealed that DCLK1+ tuft cells were decreased on day 2, almost completely deleted on day 4, and recovered on day 7. Overall brush border structures defined by villin and SGLT1 staining were intact. Correlated with the tuft cell reduction, mislocalized Paneth-like cells, identified with LYZ+ granules, were observed in the middle or upper part of villi with increasing frequency on day 4 and decreasing on day 7. To confirm goblet/Paneth intermediate cells, multiplexed immunofluorescent (MxIF) staining was employed with LYZ and TFF3. Double-positive (LYZ+/TFF3+) cells were significantly increased in the small intestine of tuft cell-depleted mice on day 4. Intraepithelial lymphocytes, mucosal neutrophils, and F4/80+ macrophages/dendritic cells did not show significant changes after the tuft cell deletion. This indicates that there is no inflammatory reactions following tuft cell deletion. Conclusion: Acute ablation of intestinal tuft cell may cause nutrient malabsorption and changes in mucosal defense response in vivo due to the alteration of cell differentiation lineage and the decrease in absorptive area. Our findings support the importance of tuft cell function in nutrient sensing and mucosal defense mechanism. NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 6391: Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction

Abstract [Purpose] Tumors with low T cell infiltrates (poor immunogenicity) display lower probability of clinical response to immune checkpoint inhibitors. To achieve higher immunotherapy efficacy, it is imperative to establish a novel strategy for improving immunogenicity. Abacavir (ABC), an anti-HIV drug, binds to the specific allele of human leukocyte antigen (HLA-B*57:01) and activate CD8+ T cells by presenting altered abnormal peptides. In this study, we considered the immune interaction between ABC and HLA-B*57:01 improving immunogenicity in poorly immunogenic tumors leading to a potent anti-cancer effect. [Methods] Mice were inoculated subcutaneously with tumor cells into the right flanks and interperitoneally treated with either ABC 5 mg or vehicle (water) for consecutive 9 days (B16F10) or 13 days (3LL) after tumor inoculation. Each tumor size was measured using vernier calipers on every 2 days. Activation, proliferation, and IFN-γ secretion of tumor infiltrating CD8+ T cells were analyzed by FACS. [Results and Discussion] ABC treatment showed significant tumor growth inhibition associated with CD8+ T cell infiltration into tumor in mice inoculated with HLA-B*57:01 expressing B16F10 melanoma or Lewis lung carcinoma (3LL) cells, but neither in HLA-B*57:03 (negative allele)-expressing nor control tumors inoculated mice. Moreover, the tumor infiltrating CD8+ T cell were immune-activated and proliferated, with secreting IFN-γ. In contrast, ABC-induced anti-tumor effect was abrogated in CD8+ T cell-depleted mice, IFN-γ KO mice, and CXCR3-blocked mice. The combination therapy with anti-PD-1 antibody treatment induced better inhibition of tumor growth than ABC monotherapy in HLA-B*57:01 expressing B16F10 inoculated mice. These results suggested that ABC/HLA-B*57:01 interaction could improve tumor immunogenicity associated with CD8+ T cell-mediated anti-tumor effect in poorly immunogenic tumors. This finding lays a foundation to develop a novel drug-induced cancer immunotherapy targeting immune resistant tumors. Citation Format: Takeshi Susukida, So-ichiro Sasaki, Tomohiro Shirayanagi, Shigeki Aoki, Kousei Ito, Yoshihiro Hayakawa. Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction [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 6391.

Abstract 6333: Repurposing NSAIDs for chemo-immunotherapy: mechanisms beyond COX inhibition

Abstract It has been well-established that some nonsteroidal anti-inflammatory drugs (NSAIDs) have antitumor activities. The beneficial effects of these NSAIDs are mainly attributed to their ability to inhibit the production and activity of cyclooxygenases (COX1 and COX2) and prostaglandin (PGE2), which are known to promote tumor progression, angiogenesis, metastasis and immunosuppression. We recently reported that administration of indomethacin, a prototypical NSAID, can enhance the antitumor efficacy of adoptive T cell therapy in animal models. However, currently adoptive T cell therapy as a treatment option is only suitable for selected patients with certain types of cancer. Here, we set out to explore the utility of indomethacin in a chemo-immunotherapy setting that is readily applicable to a wide range of cancers. Cyclophosphamide (CTX) is a widely used antineoplastic chemotherapeutic agent with immunostimulatory activities when used at low-medium doses. We show that indomethacin had no impact on tumor growth but can significantly enhance the antitumor effect of CTX in multiple mouse tumor models. The beneficial effect of indomethacin was diminished in immunodeficient mice or CD8 T cell-depleted wild-type mice, indicating the requirement for the host immunity. Furthermore, the CTX and indomethacin regimen can sensitize tumors to anti-PD1 immunotherapy. scRNAseq analysis of tumor samples revealed the impact of indomethacin on the tumor microenvironment. Our data also indicate that the immunopotentiating effect of indomethacin can be independent of PGE2 inhibition, and involves the TRAIL-DR5 axis in promoting tumor regression. Other potential mechanisms, including modulation of chronic interferon signaling in tumor cells, have also been investigated. Altogether, our study sheds light on the multiple mechanisms underlying indomethacin’s beneficial effects, and highlights the potential of NSAID administration as a readily applicable and cost-effective approach to augment the efficacy of chemo-immunotherapy. Citation Format: Ogacheko D. Okoko, Md Y. Gazi, Xin Wang, Caitlin Brandle, Mercy Kehinde-Ige, Gary A. Piazza, Huidong Shi, Gang Zhou. Repurposing NSAIDs for chemo-immunotherapy: mechanisms beyond COX inhibition [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 6333.

B cells as modulators of HPV+ oropharyngeal cancer in a preclinical model.

Among the different immune cells present within tumors, B cells also infiltrate human papillomavirus-positive (HPV+) oropharyngeal tumors. However, the role of B cells during programmed death-1 (PD-1) blockade in HPV+ oropharyngeal cancer needs to be better defined. By using the preclinical mouse model for HPV+ oropharyngeal cancer (named mEER), we characterized B cells within tumors and determined their functional role in vivo during PD-1 blockade. We determined that treatment naïve tongue-implanted tumors, which we have previously demonstrated to be sensitive to PD-1 blockade, contained high infiltration of CD8+ T cells and low infiltration of B cells whereas flank-implanted tumors, which are resistant to PD-1 blockade, contain a higher frequency of B cells compared to T cells. Moreover, B cell-deficient mice (µMt) and B cell-depleted mice showed a slower tumor growth rate compared to wild-type (WT) mice, and B cell deficiency increased CD8+ T cell infiltration in tumors. When we compared tongue tumor-bearing mice treated with anti-PD-1, we observed that tumors that responded to the therapy contained more T cells and B cells than the ones that did not respond. However, µMt mice treated with PD-1 blockade showed similar tumor growth rates to WT mice. Our data suggest that in untreated mice, B cells have a more pro-tumorigenic phenotype potentially affecting T cell infiltration in the tumors. In contrast, B cells are dispensable for PD-1 blockade efficacy. Mechanistic studies are needed to identify novel targets to promote the anti-tumorigenic function and/or suppress the immunosuppressive function of B cells in HPV+ oropharyngeal cancer.

B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice.

The current study reveals that in chronic TB, the B cell-deficient μMT strain, relative to wild-type (WT) C57BL/6 mice, displays in the lungs lower levels of inflammation that are associated with decreased CD4+ T cell proliferation, diminished Th1 response, and enhanced levels of interleukin (IL)-10. The latter result raises the possibility that B cells may restrict lung expression of IL-10 in chronic TB. These observations are recapitulated in WT mice depleted for B cells using anti-CD20 antibodies. IL-10 receptor (IL-10R) blockade reverses the phenotypes of decreased inflammation and attenuated CD4+ T cell responses in B cell-depleted mice. Together, these results suggest that in chronic murine TB, B cells, by virtue of their capacity to restrict expression of the anti-inflammatory and immunosuppressive IL-10 in the lungs, promote the development of a robust protective Th1 response, thereby optimizing anti-TB immunity. This vigorous Th1 immunity and restricted IL-10 expression may, however, allow the development of inflammation to a level that can be detrimental to the host. Indeed, decreased lung inflammation observed in chronically infected B cell-deficient mice, which exhibit augmented lung IL-10 levels, is associated with a survival advantage relative to WT animals. Collectively, the results reveal that in chronic murine TB, B cells play a role in modulating the protective Th1 immunity and the anti-inflammatory IL-10 response, which results in augmentation of lung inflammation that can be host-detrimental. Intriguingly, in tuberculous human lungs, conspicuous B cell aggregates are present in close proximity to tissue-damaging lesions manifesting necrosis and cavitation, suggesting the possibility that in human TB, B cells may contribute to the development of exacerbated pathology that is known to promote transmission. Since transmission is a major hindrance to TB control, investigating into whether B cells can shape the development of severe pulmonic pathological responses in tuberculous individuals is warranted.

Contact lens-induced corneal parainflammation involving Ly6G+ cell infiltration requires IL-17A and γδ T cells

PurposePreviously, using a murine model, we reported that contact lens (CL) wear induced corneal parainflammation involving CD11c+ cells after 24 h and Ly6G+ cells (neutrophils) after 5–6 days. Here, we investigated the role of IL-17 and γδ T cells in the CL-induced neutrophil response. MethodsCL-wearing C57BL/6 wild-type (WT) mice were compared to lens-wearing IL-17A/F single or double gene knock-out mice, or mice treated with UC7-13D5 monoclonal antibody to functionally deplete γδ T cells. Contralateral eyes served as no lens wear controls. Corneal Ly6G+ and γδ T cell responses were quantified as was expression of genes encoding pro-inflammatory cytokines IL-17A/F, IL-β, IL-18 and expression of IL-17A/F protein. ResultsAfter 6 days lens wear, WT corneas showed Ly6G+ cell infiltration while remaining free of visible pathology. In contrast, lens-wearing corneas of IL-17AF (−/−), IL-17A (−/−) mice and γδ T cell-depleted mice showed little or no Ly6G+ cell infiltration. No Ly6G+ cell infiltration was detected in contralateral eye controls. Lens-wearing WT corneas also showed a significant increase in γδ T cells after 24 h that was maintained after 6 days of wear, and significantly increased cytokine gene expression after 6 days versus contralateral controls: IL-18 & IL-17A (∼3.9 fold) and IL-23 (∼6.5-fold). Increased IL-17A protein (∼4-fold) was detected after 6 days lens wear. γδ T cell-depletion abrogated these lens-induced changes in cytokine gene and protein expression. ConclusionTogether, these data show that IL-17A and γδ T cells are required for Ly6G+ cell (neutrophil) infiltration of the cornea during contact lens-induced parainflammation.

Abstract TP204: Elimination Of Senescent Cells Prevents The Development Of Intracranial Aneurysm Rupture In Mice

Introduction: Aging is one of the risk factors for intracranial aneurysm rupture. Aging causes various changes in cellular homeostasis, including cellular senescence. Cellular senescence is defined as a state of irreversible cellular growth arrest. Senescence cells secrete pro-inflammatory senescence-associated secretory phenotypes (SASPs). SASPs affect surrounding cells and contribute to a vicious cycle of senescence and induce inflammation. Recent studies strongly suggest that inflammation promotes aneurysm rupture. Therefore, the accumulation of senescence cells may induce aneurysm rupture due to pro-inflammatory effects of SASPs. We hypothesized that the elimination of senescence cells reduces the aneurysm rupture. To test our hypothesis we used p16-3MR mice. P16-3MR mouse is a transgenic mouse model, in which senescence cells can be eliminated by treatment of ganciclovir (GCV). In previous reports, the elimination of senescence cells by GCV in p16-3MR mice improved senescence-related diseases. In this study, we tested whether the elimination of senescence cells by GCV in p16-3MR mice reduces the intracranial aneurysm rupture rate. Methods: We induced intracranial aneurysms by combining systemic hypertension and a single injection of elastase into the cerebrospinal fluid in mice. We compared the vehicle to GCV group in p16-3MR aged male mice (10-15 months, vehicle vs GCV: 9 vs 6). We started GCV treatment the same day after aneurysm induction. Aneurysm rupture rate was used as the primary endpoint. Results: There was no statistical difference in the formation rate of aneurysms between the vehicle and the GCV group. However, the aneurysmal rupture rate was significantly lower in the senescence cell-depleted mice (P < 0.05). Additionally, the symptom-free curve (Kaplan-Meier analysis curve) showed a significant reduction of aneurysm rupture in the GCV-treated group (P < 0.05). Conclusions: The elimination of senescence cells reduced the aneurysm rupture rate in a mouse model of the aneurysm. Our results suggest that the accumulation of senescence cells induces aneurysm rupture. Therefore, the pharmacological prevention of the accumulation of senescence cells may be a new therapeutic target in aneurysm rupture.

TNFR2+ regulatory Tcells protect against bacteremic pneumococcal pneumonia by suppressing IL-17A-producing γδ Tcells in the lung.

Streptococcus pneumoniae is a pathogen of global morbidity and mortality. Pneumococcal pneumonia can lead to systemic infections associated with high rates of mortality. We find that, upon pneumococcal infection, pulmonary Treg cells are activated and have upregulated TNFR2 expression. TNFR2-deficient mice have compromised Treg cell responses and highly activated IL-17A-producing γδ Tcell (γδT17) responses, resulting in significantly enhanced neutrophil infiltration, tissue damage, and rapid development of bacteremia, mirroring responses in Treg cell-depleted mice. Deletion of total Treg cells predominantly activate IFNγ-T cell responses, whereas adoptive transfer of TNFR2+ Treg cells specifically suppress the γδT17 response, suggesting a targeted control of γδT17 activation by TNFR2+ Treg cells. Blocking IL-17A at early stage of infection significantly reduces bacterial blood dissemination and improves survival in TNFR2-deficient mice. Our results demonstrate that TNFR2 is critical for Treg cell-mediated regulation of pulmonary γδT17-neutrophil axis, with impaired TNFR2+ Treg cell responses increasing susceptibility to disease.

Reduced Number and Immune Dysfunction of CD4+ T Cells in Obesity Accelerate Colorectal Cancer Progression

Obesity, a known risk factor for various types of cancer, reduces the number and function of cytotoxic immune cells in the tumor immune microenvironment (TIME). However, the impact of obesity on CD4+ T cells remains unclear. Therefore, this study aimed to clarify the impact of obesity on CD4+ T cells in the TIME. A tumor-bearing obese mouse model was established by feeding with 45% high-fat diet (HFD), followed by inoculation with a colon cancer cell line MC38. Tumor growth was significantly accelerated compared to that in mice fed a control diet. Tumor CD4+ T cells showed a significant reduction in number and an increased expression of programmed death-1 (PD-1), and decreased CD107a expression and cytokine such as IFN-γ and TNF-α production, indicating dysfunction. We further established CD4+ T cell-depleted HFD-fed model mice, which showed reduced tumor infiltration, increased PD-1 expression in CD8+ T cells, and obesity-induced acceleration of tumor growth in a CD4+ T cell-dependent manner. These findings suggest that the reduced number and dysfunction of CD4+ T cells due to obesity led to a decreased anti-tumor response of both CD4+ and CD8+ T cells to ultimately accelerate the progression of colorectal cancer. Our findings may elucidate the pathogenesis for poor outcomes of colorectal cancer associated with obesity.