MHC-II Expression Research Articles

TLR9 promotes monocytic myeloid-derived suppressor cell induction during JEV infection

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9−/− mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9−/− mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.

Irradiated microparticles suppress prostate cancer by tumor microenvironment reprogramming and ferroptosis

Immunogenic cell death (ICD) plays a crucial role in triggering the antitumor immune response in the tumor microenvironment (TME). Recently, considerable attention has been dedicated to ferroptosis, a type of ICD that is induced by intracellular iron and has been demonstrated to change the immune desert status of the TME. However, among cancers that are characterized by an immune desert, such as prostate cancer, strategies for inducing high levels of ferroptosis remain limited. Radiated tumor cell-derived microparticles (RMPs) are radiotherapy mimetics that have been shown to activate the cGAS-STING pathway, induce tumor cell ferroptosis, and inhibit M2 macrophage polarization. RMPs can also act as carriers of agents with biocompatibility. In the present study, we designed a therapeutic system wherein the ferroptosis inducer RSL-3 was loaded into RMPs, which were tested in in vitro and in vivo prostate carcinoma models established using RM-1 cells. The apoptosis inducer CT20 peptide (CT20p) was also added to the RMPs to aggravate ferroptosis. Our results showed that RSL-3- and CT20p-loaded RMPs (RC@RMPs) led to ferroptosis and apoptosis of RM-1 cells. Moreover, CT20p had a synergistic effect on ferroptosis by promoting reactive oxygen species (ROS) production, lipid hydroperoxide production, and mitochondrial instability. RC@RMPs elevated dendritic cell (DC) expression of MHCII, CD80, and CD86 and facilitated M1 macrophage polarization. In a subcutaneously transplanted RM-1 tumor model in mice, RC@RMPs inhibited tumor growth and prolonged survival time via DC activation, macrophage reprogramming, enhancement of CD8+ T cell infiltration, and proinflammatory cytokine production in the tumor. Moreover, combination treatment with anti-PD-1 improved RM-1 tumor inhibition. This study provides a strategy for the synergistic enhancement of ferroptosis for prostate cancer immunotherapies.Graphical

Abstract PO5-13-09: Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting

Abstract Background: A CLIA-accredited assay that measures the expression and/or activation levels of 32 clinically actionable protein and phosphoprotein analytes in clinical tissue biopsy samples was launched in January 2021. The current intended use population for the assay is for patients with late/advanced stage breast cancer wherein molecular profiling is being utilized for clinical decision making. Assessment of physician ordering practices, clinical use dynamics as well as protein/phosphoprotein actionability frequencies were determined for clinically actionable biomarkers. Methods: A total of 219 cases have been processed to date, with mean turn-around time of 16.4 days. The assay utilizes laser capture microdissection to enrich tumor epithelium from FFPE core needle biopsy and resection material coupled to reverse phase protein array. Results for each of the 32 analytes are generated by comparing each patient to a reference population and reported both as a relative quartile and as a continuous percentile compared to the reference population (0-100%). For the purposes of this analysis, significant expression/activation cutpoints were set at the 40th percentile threshold. Results: To date, clinicians have requested the test most frequently to identify potential therapeutic targets in patients with HER2-negative disease (84% of all tested patients, 36% were triple-negative). Despite the majority of cases being HER2-negative, we found that phosphorylated (p) HER2 (Y1248) was found to be activated in 17% of all HER2-negative cases and 19% of triple negative breast cancer cases (TNBC). pHER2 was concurrently activated with pHER3 (Y1289) in 15% (12/80) of TNBC cases and 13% (24/187) of all HER2-negative cases. pHER2 presented concurrently with pEGFR (Y1068 or T654) in 14% (11/80) of TNBC cases and 14% (26/187) of all HER2- cases. Expression of TROP2 was observed in 16% of HER2-negative patients including 11% of ER+/HER2- and 23% of TNBC cancers. MHCII expression, a recently described candidate predictive biomarker of anti-PDL1/PD1 response in TNBC, was found to be expressed in 29% of TNBC patients. Activation frequencies of other clinically relevant drug targets in HER2-negative disease such AKT and mTOR pathway activation was also assessed. We found that pAKT (S473 and/or T308) was activated in 7% of ER+/HER2- cases and 14% of TNBC. Concurrent with AKT activation, at least one additional member of the AKT-mTOR pathway (mTORC1 (S2448), 4E-BP1 (S65), p70S6K (T389), or S6RP (S235/S236) was co-activated in 50% and 45% of ER+/HER2- and TNBC cancers respectively. Conclusion: This initial assessment of a CLIA LDT protein/phosphoprotein assay utilized to date found that physicians most often order the test for advanced stage HER2-negative breast cancer. Activation/expression of clinically important protein drug targets with potential actionability in HER2-negative disease was observed in a significant number of cases and demonstrates the power of the assay to elucidate actionable targets for potential therapeutic intervention. Citation Format: Claudius Mueller, Brian Corgiat, Emanuel Petricoin, Kayla Sparks, Joyce O'Shaughnessy, Chelsea Gawryletz, Matthew Schwartz, Justin Davis. Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-09.

Emergence of novel CD3+ Macrophages in Brown Adipose Tissue Following Adrenergic Stimulation

Abstract Macrophages play a vital role in maintaining tissue homeostasis and defending against infections and cancer. Brown adipose tissue (BAT) is essential for expending surplus energy during cold stress, while white adipose tissue functions as the primary energy reservoir. The sympathetic nervous system responds to cold stress by releasing norepinephrine (NE), activating BAT through β3-adrenergic receptor. BAT-infiltrated macrophages exert opposing effects on thermogenesis, regulating the interplay between BAT cells and sympathetic nerves to either inhibit thermogenesis by clearing NE or enhance it by supporting sympathetic innervation of BAT. In mouse interscapular BAT, we identified a novel subset of CD3+ macrophages (CD45+ CD3+ CD4+ CD8+ CD11b+ F4/80+), whose frequency increased with chronic adrenergic stimulation. Despite the potential for T cell-monocyte doublets in flow cytometry, CD3+ macrophages demonstrated a unique surface expression pattern, lacking T cell markers CD62L and TCRβ. Furthermore, they showed elevated levels of Ly6C, MHCII, F4/80, and CD11b compared to T cells and CD3- macrophages. CD3+ macrophages exhibit heterogeneity in MHCII expression, with subsets expressing low and high levels. Since MHCII-expressing macrophages play a crucial role in regulating sympathetic nerve-BAT interaction, further characterization of CD3+ macrophages is pivotal for understanding the intricate mechanisms that govern adaptive thermogenesis regulation.

Application of Deep Learning Models on Single-Cell RNA Sequencing Analysis Uncovers Novel Markers of Double Negative T Cells

Abstract Double negative T (DNT) cells, a unique subset of CD3+TCRαβ+ T lymphocytes lacking CD4, CD8, or NK1.1 expression, constitute 3-5% of the total T cell population in C57BL/6 mice. Traditional machine learning models such as principal component analysis in single-cell RNA sequencing (scRNA-seq) analysis have been utilized to characterize this subset, but only deep learning models such as Single Cell Variational Inference (SCVI) can capture nonlinear gene expression in the sequencing data. In this study, using the deep learning approach, we have uncovered novel markers of splenic DNT cells in C57BL/6 mice. We classified DNT cells into two subgroups, naïve DNT (nDNT) cells and activated DNT (aDNT) cells, which could be differentiated by the expression of Ly6C and MHC-II, respectively. In addition, our data identified CD153 and CD153 as unique markers for aDNT cells. SCVI analysis suggested, and flow cytometry analysis confirmed, that Ly49G2 and CCR2 are respective markers for two distinct subsets within nDNT and aDNT populations. In addition to cell surface markers, we further discovered that nDNT cells expressed higher Rgs2 that is required for T cell activation and that aDNT cells expressed higher Rbpj involved in Notch signaling. Together, our comprehensive analysis has uncovered and validated novel markers for different subpopulations of DNT cells that can be used in the phenotypic and/or functional characterization of these relatively rare cells in health and disease.

Regulation of MHC II by OPTN, a novel regulator of MARCH1 via mTOR/AKT2/STAT3 pathway ameliorates severe outcomes during herpesvirus infection.

Abstract Protective immune responses elicited by primary activation of CD4+ T cells via dendritic cells is an essential requirement to evade herpesvirus infections. Optineurin (OPTN), a selective autophagy adaptor protein, is a crucial host restriction factor for herpes simplex virus (HSV) infection. We observed a significant reduction in the basal level of MHCII expression in OPTN–/– DCs accompanied by a concomitant decrease in CD4+ T cell counts in OPTN–/– mouse model. We hypothesize that abrogation of antigen presentation via MHCII in DCs weakens adaptive immune responses, leading to severe HSV1 infection. In this study, we elucidate the mechanism of OPTN-mediated down-regulation of MHCII via AKT/mTOR/STAT3 pathway. Mechanistically, OPTN induces activation of mTORC2 sequentially upregulating AKT2 which adversely affects the activation of mTORC1 and STAT3 protein. This could be attributed to the dynamic interplay between AKT1 and AKT2. Our findings provide novel insights into how the absence of OPTN downregulates AKT2 and triggers STAT3 activation. Furthermore, aberrant activation of STAT3 influences transcriptional activity of MARCH1(an E3 ubiquitin ligase) via IL10. MARCH1 mediates ubiquitination of MHCII resulting in the reduction of MHCII expression in OPTN–/– and AKT2–/– DCs. This study represents the first comprehensive delineation of the signaling pathway involved in the regulation of MHCII, shedding light on the intricate interplay between OPTN, AKT, mTOR, STAT3, and MARCH1.

Elevated type I IFN signalling directly affects CD8+ T-cell distribution and autoantigen recognition of the skeletal muscles in active JDM patients

The link between type I IFN and adaptive immunity, especially T-cell immunity, in JDM still remained largely unclear. This study aimed to understand the effect of elevated type I IFN signaling on CD8+ T cell-associated muscle damage in juvenile dermatomyositis (JDM). This study used flow cytometry (FC) and RT‒PCR were used to examine the circulating cell ratio and type I IFN response. And scRNA-seq was used to examine peripheral immunity in 6 active JDM patients, 3 stable JDM patients, 3 juvenile IMNM patients and 3 age-matched healthy children. In vivo validation experiments were conducted using a mouse model induced by STING agonists and an experimental autoimmune myositis model (EAM). In vitro experiments were conducted using isolated CD8+ T-cells from JDM patients and mice. We found that active JDM patients showed an extensive type I IFN response and a decreased CD8+ T-cell ratio in the periphery (P < 0.05), which was correlated with muscle involvement (P < 0.05). Both new active JDM patients and all active JDM patients showed decreased CD8+ TCM cell ratios compared with age and gender matched stable JDM patients (P < 0.05). Compared with new pediatirc systemic lupus erythematosus (SLE) patients, new active JDM patients displayed decreased CD8+ T-cell and CD8+ TCM cell ratios (P < 0.05). Active JDM patient skeletal muscle biopsies displayed an elevated type I IFN response, upregulated MHC-I expression and CD8+ T-cell infiltration, which was validated in EAM mice. sc-RNAseq demonstrated that type I IFN signalling is the kinetic factor of abnormal differentiation and enhances the cytotoxicity of peripheral CD8+ T cells in active JDM patients, which was confirmed by in vivo and in vitro validation experiments. In summary, the elevated type I IFN signalling affected the differentiation and function of CD8+ T cells in active JDM patients. Skeletal muscle-infiltrating CD8+ T cells might migrate from the periphery under the drive of type I IFN and increased MHC I signals. Therapies targeting autoantigen-specific CD8+ T cells may represent a potential new treatment direction.

A protective effect of lower MHC-II expression in MOGAD

Myelin oligodendrocyte glycoprotein-antibody-associated disease (MOGAD) is a demyelinating central nervous system disorder. We aimed to uncover immune pathways altered in MOGAD to predict disease progression. Using nanostring nCounter technology, we analyzed immune gene expression in PBMCs from MOGAD patients and compare it with healthy controls (HCs). We found 35 genes that distinguished MOGAD patients and HCs. We then validated those results in a larger cohort including MS and NMOSD patients. Expressions of HLA-DRA was significantly lower in MOGAD patients. This reduction in HLA-DRA, correlated with a monophasic disease course and greater brain volume, enhancing our ability to predict MOGAD progression.

Irradiated tumour cell-derived microparticles upregulate MHC-I expression in cancer cells via DNA double-strand break repair pathway

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs.

Activation of MAL2 by RAD21 inhibits the expression of MHC-I in immune evasion of endometrial cancer.

CD8+ T cells are the primary mediators of anticancer immunity, and modulation of the CD8+ T cell response has been a central focus of immunotherapy to treat cancer. When CD8+ T cells specifically recognize antigenic peptides presented by the MHC-I on tumor cells, they become activated and kill the tumor cells. However, one pivotal mechanism through which tumor cells evade immune surveillance is to reduce their antigen presentation. To identify novel immunotherapeutic targets, we specifically focused on the role of MAL2 in immune evasion in endometrial cancer (EC) and the underlying mechanism. MAL2 was overexpressed in EC tissues and cells and its transcription was enhanced by RAD21. Knockdown of MAL2 or RAD21 inhibited malignant behavior and immune evasion of EC cells by repressing MHC-I expression and the cytotoxic effects of CD8+ cells. Conversely, MAL2 promoted immune evasion of EC cells and tumor growth in mice in the presence of RAD21 knockdown. These results indicate that RAD21 activation of MAL2 inhibits antigen processing and presentation of MHC-I, thereby inducing immune evasion of EC cells. We further suggest that RAD21 and MAL2 may serve as novel targets for EC immunotherapy.

Beneficial Effects of Dietary Fiber in Young Barley Leaf on Gut Microbiota and Immunity in Mice.

The health benefits of young barley leaves, rich in dietary fiber, have been studied for several decades; however, their beneficial effects on the intestinal microenvironment remain to be elucidated. To investigate the effects of young barley leaf-derived dietary fiber (YB) on the gut microbiota and immunity, mice were fed an AIN-93G diet containing cellulose or YB and subjected to subsequent analysis. The population of MHC-II-positive conventional dendritic cells (cDCs) and CD86 expression in the cDCs of Peyer's patches were elevated in the YB-fed mice. MHC-II and CD86 expression was also elevated in the bone marrow-derived DCs treated with YB. 16S-based metagenomic analysis revealed that the gut microbiota composition was markedly altered by YB feeding. Among the gut microbiota, Lachnospiraceae, mainly comprising butyrate-producing NK4A136 spp., were overrepresented in the YB-fed mice. In fact, fecal butyrate concentration was also augmented in the YB-fed mice, which coincided with increased retinaldehyde dehydrogenase (RALDH) activity in the CD103+ cDCs of the mesenteric lymph nodes. Consistent with elevated RALDH activity, the population of colonic IgA+ plasma cells was higher in the YB-fed mice than in the parental control mice. In conclusion, YB has beneficial effects on the gut microbiota and intestinal immune system.

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB403.

Abstract LB340: Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice

Abstract FDA approval for chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers has unleashed the potential of utilizing live T cells as therapeutics. However, CAR-mediated targeting of solid tumors is currently limited by a paucity of suitable tumor antigens that are both specific and highly expressed on the tumor cell surface. In this report we explore the possibility of harnessing tumor-specific expression of endogenous retroviruses (ERVs) as a universal target for CAR T cell therapy. We found that gp70, the envelope protein from a murine ERV, is highly expressed on the surface of tumor cell lines derived from a diverse array of murine tumor types. We utilized the B16 melanoma model as a representative poorly inflamed tumor that does not respond to existing immunotherapies to examine efficacy of gp70 CAR T cells. T cells engineered to express a gp70 CAR suppress tumor growth in vivo and recognize B16 tumor cells with improved sensitivity compared to T cells expressing two previously described dominant tumor antigens (gp100 or P15e) due to low expression of MHC-I on B16 tumors. Co-incubation of B16 tumor cells with gp70 CAR T cells results in upregulation of MHC-I on the tumor cell surface suggesting that in addition to killing tumor cells directly, gp70 CAR T cells could facilitate recognition of the tumor by endogenous T cells through local release T cell derived IFNγ. We postulate that utilization of CAR T cells specific for cell surface ERV envelope proteins offers a promising strategy that could be harnessed to achieve local release of T cell derived payloads specifically within the tumor microenvironment. Citation Format: Sophie M. Lehar, Srirupa Roy, Lisa Liao, Haiyin Chen, Yongmei Chen, Mayra Cruz Tleugabulova, Eric Janezic, Azadeh Madjidi, Tamaki Jones, Vincent Javinal, James Ziai, Ira Mellman, Jill Schartner, Andrey Shaw. Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB340.

IRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

Polyethylene glycol has immunoprotective effects on sciatic allografts, but behavioral recovery and graft tolerance require neurorrhaphy and axonal fusion.

JOURNAL/nrgr/04.03/01300535-202504000-00033/figure1/v/2024-07-06T104127Z/r/image-tiff Behavioral recovery using (viable) peripheral nerve allografts to repair ablation-type (segmental-loss) peripheral nerve injuries is delayed or poor due to slow and inaccurate axonal regeneration. Furthermore, such peripheral nerve allografts undergo immunological rejection by the host immune system. In contrast, peripheral nerve injuries repaired by polyethylene glycol fusion of peripheral nerve allografts exhibit excellent behavioral recovery within weeks, reduced immune responses, and many axons do not undergo Wallerian degeneration. The relative contribution of neurorrhaphy and polyethylene glycol-fusion of axons versus the effects of polyethylene glycol per se was unknown prior to this study. We hypothesized that polyethylene glycol might have some immune-protective effects, but polyethylene glycol-fusion was necessary to prevent Wallerian degeneration and functional/behavioral recovery. We examined how polyethylene glycol solutions per se affect functional and behavioral recovery and peripheral nerve allograft morphological and immunological responses in the absence of polyethylene glycol-induced axonal fusion. Ablation-type sciatic nerve injuries in outbred Sprague-Dawley rats were repaired according to a modified protocol using the same solutions as polyethylene glycol-fused peripheral nerve allografts, but peripheral nerve allografts were loose-sutured (loose-sutured polyethylene glycol) with an intentional gap of 1-2 mm to prevent fusion by polyethylene glycol of peripheral nerve allograft axons with host axons. Similar to negative control peripheral nerve allografts not treated by polyethylene glycol and in contrast to polyethylene glycol-fused peripheral nerve allografts, animals with loose-sutured polyethylene glycol peripheral nerve allografts exhibited Wallerian degeneration for all axons and myelin degeneration by 7 days postoperatively and did not recover sciatic-mediated behavioral functions by 42 days postoperatively. Other morphological signs of rejection, such as collapsed Schwann cell basal lamina tubes, were absent in polyethylene glycol-fused peripheral nerve allografts but commonly observed in negative control and loose-sutured polyethylene glycol peripheral nerve allografts at 21 days postoperatively. Loose-sutured polyethylene glycol peripheral nerve allografts had more pro-inflammatory and less anti-inflammatory macrophages than negative control peripheral nerve allografts. While T cell counts were similarly high in loose-sutured-polyethylene glycol and negative control peripheral nerve allografts, loose-sutured polyethylene glycol peripheral nerve allografts expressed some cytokines/chemokines important for T cell activation at much lower levels at 14 days postoperatively. MHCI expression was elevated in loose-sutured polyethylene glycol peripheral nerve allografts, but MHCII expression was modestly lower compared to negative control at 21 days postoperatively. We conclude that, while polyethylene glycol per se reduces some immune responses of peripheral nerve allografts, successful polyethylene glycol-fusion repair of some axons is necessary to prevent Wallerian degeneration of those axons and immune rejection of peripheral nerve allografts, and produce recovery of sensory/motor functions and voluntary behaviors. Translation of polyethylene glycol-fusion technologies would produce a paradigm shift from the current clinical practice of waiting days to months to repair ablation peripheral nerve injuries.

233 Evolutionary Analysis Identifies Early Responsive Macrophage Representing as Anti-tumor Myeloid Cell in Glioblastoma

INTRODUCTION: Glioblastoma (GBM) has no effective treatments. Although immunotherapy has shown promise in certain cancer types, it has not been effective against GBM, largely due to its highly immunosuppressive tumor microenvironment (TMEs), which is rich in tumor-associated macrophages/microglia (TAMs). TAMs in late-stage GBM contribute to T-cell exhaustion and worsen prognosis, but the role of TAMs in earlier stages of tumor development is unclear. METHODS: By employing genetically engineered mouse models and human samples, we used spatiotemporal single-cell transcriptomics to investigate TAM evolution during GBM progression. RESULTS: We identified a previously unknown macrophage state, termed early responsive macrophage (ERM), that exhibited antitumoral activity. ERMs were found to be abundant in the early stages of GBM progression and spatially enriched in the invading edges of GBMs. ERMs are characterized by upregulated immune response signatures, particularly pathways related to antigen processing and presentation, as well as genes associated with phagocytosis and chemotaxis. Mechanistically, we identified QKI, a transcriptional co-activator, as a key player that activates antigen processing and presentation and MHCII expression in ERMs. Loss of QKI in TAMs is associated with the transformation of ERMs into exhausted tumor-infiltrating macrophages (TIMs), which promote an immune-suppressive TME. Therapeutically, enhancement of QKI's transcriptional co-factor activity greatly improved the efficacy of immune checkpoint blockade. CONCLUSIONS: Our study sheds light on the evolutionary trajectory of TAMs in GBM and presents a novel therapeutic approach to enhance their antitumoral activity, thereby constraining tumor growth.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

Abstract 1369: PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection

Abstract Obesity has been established as a leading risk factor for many cancers and can drive tumor progression and metastasis. Paradoxically, obesity has in some cases been associated with better survival and improved response to immune checkpoint blockade therapies. The role of the immune system in the obesity-cancer connection and how obesity affects immunotherapy responses, however, have been unclear. Here we show that obesity enhanced PD-1 expression on macrophages to reduce phagocytosis and antigen presentation to T cells that correlated with reduced T cell expansion and function. This obesity associated immune dysfunction, however, primed for enhanced anti-tumor response to PD-1 blockade. Single cell RNAseq showed obesity remodels myeloid and T cell populations but does not impact non-immune cell populations such as fibroblasts and epithelial cells. Specifically, obesity increased the overall number of tumor-associated macrophages (TAM) while effector T cells were decreased in abundance. Interestingly, the frequency of macrophages expressing PD-1 increased, while the remaining T cells maintained similar or reduced PD-1 expression and appeared less activated. Further, T cell depletion from the tumor microenvironment, enhanced macrophage PD-1 expression and worsened PD1-associated TAM dysfunction in an obesity enhanced manor. Obesity associated cytokines and adipokines including IFN-ã, leptin and insulin induced PD-1 expression on macrophages. This expression is associated with mTOR and c-myc activation signaling pathways; as such inhibiting these signaling pathways blocked PD1 expression. Bulk RNA sequencing analysis revealed PD-1+ TAMs had an altered gene profile compared to PD-1− TAMs. PD1 expressing macrophages exhibited increased mitochondrial respiration and expression of oxidative phosphorylation, increased lipid uptake and increased cell cycling related genes. Conversely PD1- TAMs exhibited increased phagocytosis and antigen presentation. PD-1/PDL1 interaction directly regulated TAMs, as recombinant PDL-1 reduced glycolysis and phagocytosis in purified macrophages, and these effects could be reversed with blocking PD-1 antibody. Conversely, PD-1−/− TAMs had reduced lipid uptake but high rates of glycolysis, phagocytosis, and expression of MHC-II. Myeloid-specific PD-1 deficiency correlated with slower tumor growth and decreased LAG3 and increased CD69 on CD8 T cells. In addition, myeloid specific PD1 deficiency enhanced TAM antigen presentation through OVA specific CD8 T cell activation. These findings identify PD-1 as a metabolic regulator in TAM dysfunction and reveal a unique PD-1 mediated and macrophage-specific mechanism for immune tumor surveillance and checkpoint blockade. This may contribute to improved immunotherapy response in TAM-enriched tumors and obesity. Citation Format: Jackie Bader, Jeffrey Rathmell. PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1369.

Abstract 6678: Microbial regulation of anti-tumor immunity in colorectal cancer

Abstract Colorectal cancer (CRC) is the 3rd leading site of cancers and cause of cancer cell death. Although Immunotherapies revolutionized cancer therapy, the majority of CRC patients, particularly with metastatic disease, do not respond to the treatments. This necessitates a better mechanistic understanding of the tumor-immune cell interactions in the context of CRC. Microbiome influences multiple biological aspects of the host including immune responses against cancers. Here, we show that the gut microbiome influences cancer MHC-I and -II expression levels and restricts tumor progression and metastasis, resulting in longer survival in 3 different CRC models and two different mouse strains. We found that the anti-tumor effects are dependent on T cells and B cells. Interestingly, depletion of B cell negated anti-metastatic effects of gut microbiome. Mechanistically, MHC-II expression in cancer cells mediates the anti-tumor effects of the microbiome. MHC-II upregulation on human CRC organoids enhances cancer-immune cell interactions and associated apoptosis, demonstrating an anti-tumor role and significance of MHC-II in human CRC. Thus, gut microbes that enhance MHC-II antigen presentation promotes anti-tumor immunity and may be utilized to enhance efficacy of immunotherapy in metastatic CRC. Citation Format: Charlie Chung, Elif Ozcelik, Jill Habel, Jialin Zhang, Yihan Qin, Libia Garcia, Joseph Merrill, Taemoon Chung, Scott Lyons, Zeli Shen, James G. Fox, Peter Westcott, Semir Beyaz. Microbial regulation of anti-tumor immunity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6678.

Abstract 6347: To develop a tumor selective INF-γ-Yervoy fusion antibody to increase the therapeutic effect and safety of current Yervoy therapy in cancer

Abstract Introduction: Immune checkpoint inhibitor therapy is a highly promising strategy for clinical treatment of cancer. Among these inhibitors, yervoy stands out for its ability to induce cytotoxic T cell proliferation and activation by binding to CTLA-4. However, FDA has reported that yervoy would induce the over-activation of immune cells and cause the serious side effects. In additional, the therapeutic efficacy and application of Yervoy were still limited by the MHC expression of patient's tumor. Strategy: Therefore, we have developed a protease specific pro INF-γ-Yervoy to increase the therapeutic effect and safety of current immune checkpoint blockade therapy in cancer. We used the MMP-2/9 protease substrate as a linker between INF-γ and Yervoy. Using the concept of spatial shielding, IFNγ and Yervoy can block their functions from each other. Once the INF-γ-Yervoy arrives to the tumoral region, the INF-γ-Yervoy will be removed from Hinge Yervoy by the over-expressed MMPs protease locally so that the Yervoy will recruit its binding ability in order to increase the drug selectivity and improve the side effects. The INF-γ also can increase the MHC I expression in tumor to overcome the MHC-limitation of Yervoy. Result: On activation by MMP, IFNγ-yervoy not only restored yervoy’s binding ability but also released biologically active IFNγ. This finding confirms the reliability of IFNγ-yervoy’s functionality. In vivo studies demonstrated that IFNγ-yervoy enhances the therapeutic effect of yervoy against colorectal cancer by increasing CD8+ and CD4+ lymphocyte infiltration into the tumor area and inducing MHC-I expression in tumor cells. Mice treated with IFNγ-yervoy showed higher survival rates and body weight, as well as lower CD4+ and CD8+ lymphocyte activation rates in the blood and reduced organ damage. Conclusion: These results provide strong evidence that tumor-selective IFNγ-yervoy can improve the effectiveness of yervoy while reducing its side effects. It is likely that future immunotherapies would rely on such antibodies to activate local cancer cells or immune cells, thereby increasing the therapeutic effectiveness of cancer treatments and ensuring their safety. Keyword: Immune checkpoint inhibitor therapy, IFNγ, yervoy, rectal cancer, CTLA4 Citation Format: Chih-Hung Chuang. To develop a tumor selective INF-γ-Yervoy fusion antibody to increase the therapeutic effect and safety of current Yervoy therapy in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6347.