Adoptive Immunotherapy Research Articles

Adjuvant cytokine-induced killer cell immunotherapy in hepatocellular carcinoma: Extended follow-up of a randomized controlled trial and post-treatment immune cell profiling.

518 Background: Notwithstanding the pressing need for adjuvant therapy for hepatocellular carcinoma (HCC), most adjuvant therapies, including atezolizumab/bevacizumab, have failed. Meanwhile, adjuvant immunotherapy utilizing autologous cytokine-induced killer (CIK) cells for HCC improved recurrence-free survival (RFS) in a previously reported randomized controlled trial (RCT) and real-world data. This study aimed to assess the longer-term outcomes of the RCT and elucidate the underlying mechanisms of sustained effects of CIK cell treatment. Methods: This study comprised two parts: a long-term follow-up of the preceding RCT and an analysis of immune cells in patients who received adjuvant CIK cell treatment. In the original RCT, 226 patients who underwent curative treatment for stage I or II HCC were randomly allocated to either the CIK (n=114, 16 injections of 6.4×10 9 CIK cells over an 11-month period) or control group (n=112). The follow-up period was extended to 9 years after the enrollment of the last patient. The primary endpoint was recurrence-free survival (RFS). The secondary endpoints included cancer-specific survival (CSS) and overall survival (OS). Parallelly, a prospective study was conducted to investigate post-treatment changes of immune cells in peripheral blood of 7 patients, who received repeated transfer of CIK cells after curative treatment for HCC, using flow cytometry. Results: In the extended follow-up of the RCT (median follow-up=115.7 months, interquartile range=74.2–130.5 months), the CIK group sustained a significantly prolonged RFS (median=43.5 vs 27.4 months; hazard ratio [HR]=0.74, 95% confidence interval [CI]=0.55–0.99, P =0.045) and CSS (median=unreached; HR=0.49, 95% CI=0.25–0.95, P=0.04) compared to the control group. Adjuvant CIK cell therapy reduced the risk of overall death by 30%, although it did not achieve statistical significance (median=unreached; HR=0.70, 95% CI=0.44–1.11, P=0.1). A preliminary analysis of peripheral blood immune cells revealed that the CIK cell treatment tended to increase the frequencies of CD8 + and CD4 + classical memory cells. Conclusions: Adjuvant CIK cell treatment demonstrated significantly enhanced RFS and CSS in the prolonged follow-up of the RCT extending to 9 years in patients who received curative treatment for HCC. The CIK group also demonstrated a consistent trend of improved OS. The increase in memory T cell populations might be linked to the sustained off-treatment anti-tumor efficacy of CIK cell treatment.

Construction and characterization of a novel secreted MsC-CAR-T cell in solid tumors

The CD47-SIRPα signaling has been acknowledged as a significant immune checkpoint and CD47 blocking has been proved as a potential therapeutic strategy for the treatment of solid tumor. However, the potential application of CAR-T cells secreted antibody fragment simultaneously in solid tumor is rarely explored. In this study, we searched bioinformatic databases and investigated the characteristics of CD47 in solid tumors. Then we consulted bioinformatic databases to design, optimize and construct a novel MsC-CAR which could target MAGE-A1 and self-secrete CD47-scFv. The engineering T cells containing MsC-CAR were transfected, verified and characterized. The tumor-inhibitory role of MsC-CART cells was further determined in vitro and in vivo. The results showed that MsC-CARs were successfully constructed and MsC1-CARs demonstrated the preferable features of recognizing MAGE-A1 and secreting CD47-scFv. Engineering T cells transfecting with MsC1-CAR (MsC1-CART cells) exerted the prominent tumor-inhibitory effectiveness, both in different cancer cell lines and LUAD xenograft tumors. The present data highlighted that MsC1-CART cells elaborately combined the adoptive cellular immunotherapy and immune checkpoint inhibitor therapy, may represent a new direction for the treatment of MAGE-A1 positive solid tumors.

Advances in cellular therapies for children and young adults with solid tumors.

Adoptive immunotherapy brings hope to children and young adults diagnosed with high-risk solid tumors. Cellular (cell) therapies such as chimeric antigen receptor (CAR) T cell, CAR natural killer (NK) cell, and T cell receptor (TCR) T cell therapy are potential avenues of targeted therapy with limited long-term toxicities. However, development of cell therapies for solid tumors is in its nascent stages. Here, we will review the current clinical experience, barriers to efficacy, and strategies to improve clinical response and patient access. Cell therapies are shown to be generally safe and well tolerated. Strategies to optimize antitumor activity have now moved into early-phase trials. The immunosuppressive tumor microenvironment remains a major barrier to efficacy, and efforts are underway to gain better understanding. This will inform future treatment strategies to enhance the antitumor activity of cell therapies. Clinical experiences to date provide important insights on how to leverage cell therapies against solid tumors. Key factors in advancing the field include a better understanding of immune cell biology, tumor cell behavior, and the tumor microenvironment. Lastly, improving access to novel cell therapies remains an important consideration in the conduct of clinical trials and for future implementation into standard practice.

Advances and prospects of cell-penetrating peptides in tumor immunotherapy

Cell-penetrating peptides (CPPs) have been shown to have superior material transport ability because poor infiltration of activated lymphocytes into tumors is one of the crucial factors limiting the therapeutic effect of tumor immunotherapy. Numerous studies have investigated the potential application of CPPs in tumor immunotherapy. This review delves into the crucial role that CPPs play in enhancing tumor immunotherapy, emphasizing their impact on various immunotherapy strategies, such as cytokine therapy, adoptive cell therapy, cancer vaccines, and immune checkpoint inhibitors. We also discuss the practical application challenges associated with enhancing the efficiency of CPPs in terms of their stability and targeting ability. In conclusion, the combination of CPPs with tumor immunotherapy is a promising strategy that has potential for precision administration and requires further research for optimal implementation.

Wild-type bone marrow cells repopulate tissue resident macrophages and reverse the impacts of homozygous CSF1R mutation.

Adaptation to existence outside the womb is a key event in the life of a mammal. The absence of macrophages in rats with a homozygous mutation in the colony-stimulating factor 1 receptor (Csf1r) gene (Csf1rko) severely compromises pre-weaning somatic growth and maturation of organ function. Transfer of wild-type bone marrow cells (BMT) at weaning rescues tissue macrophage populations permitting normal development and long-term survival. To dissect the phenotype and function of macrophages in postnatal development, we generated transcriptomic profiles of all major organs of wild-type and Csf1rko rats at weaning and selected organs following rescue by BMT. The transcriptomic profiles revealed subtle effects of macrophage deficiency on development of all major organs. Network analysis revealed a common signature of CSF1R-dependent resident tissue macrophages that includes the components of complement C1Q (C1qa/b/c genes). Circulating C1Q was almost undetectable in Csf1rko rats and rapidly restored to normal levels following BMT. Tissue-specific macrophage signatures were also identified, notably including sinus macrophage populations in the lymph nodes. Their loss in Csf1rko rats was confirmed by immunohistochemical localisation of CD209B (SIGNR1). By 6-12 weeks, Csf1rko rats succumb to emphysema-like pathology associated with the selective loss of interstitial macrophages and granulocytosis. This pathology was reversed by BMT. Along with physiological rescue, BMT precisely regenerated the abundance and expression profiles of resident macrophages. The exception was the brain, where BM-derived microglia-like cells had a distinct expression profile compared to resident microglia. In addition, the transferred BM failed to restore blood monocyte or CSF1R-positive bone marrow progenitors. These studies provide a model for the pathology and treatment of CSF1R mutations in humans and the innate immune deficiency associated with prematurity.

Current Landscape of Adoptive Cell Therapy and Challenge to Develop "Off-The-Shelf" Therapy for Hepatocellular Carcinoma.

Adoptive cell therapy (ACT) is a type of immunotherapy in which autologous or allogeneic immune cells, such as tumor-infiltrating lymphocytes or engineered lymphocytes, are infused into patients with cancer to eliminate malignant cells. Recently, autologous T cells modified to express a chimeric antigen receptor (CAR) targeting CD19 showed a positive response in clinical studies for hematologic malignancies and have begun to be used in clinical practice. This article discusses the current status and promise of ACT research in hepatocellular carcinoma (HCC), focusing on challenges in off-the-shelf ACT using primary cells or induced pluripotent stem cells (iPSCs) with or without genetic engineering. Early clinical trials of autologous GPC-3-, MUC1-, or CEA-targeted CAR-T cell therapies are underway for HCC. There is a growing demand for the development of off-the-shelf therapies due to the high cost and manufacturing issues associated with autologous CAR-T. The development of ACT from various cell sources, such as NK cells, NKT cells, macrophages, and γδ T cells without MHC restriction other than T cells has been proposed. Advances in genome editing, including HLA gene knockout to avoid GvHD, and strategies to enhance efficacy in overcoming the suppressive tumor microenvironment, are used to create universal 'off-the-shelf' CAR-T cells which can be used immediately as therapeutic products from healthy donors or iPSC-derived immune cells. Despite several limitations, cell-based immunotherapy is expected to become a key cancer treatment modality for both hematologic malignancies and solid tumors including HCC, thanks to technological advancements overcoming these challenges.

Modeling the response to interleukin-21 to inform natural killer cell immunotherapy.

Natural killer (NK) cells are emerging agents for cancer therapy. Several different cytokines are used to generate NK cells for adoptive immunotherapy including interleukin (IL)-2, IL-12, IL-15 and IL-18 in solution, and membrane-bound IL-21. These cytokines drive NK cell activation through the integration of signal transducers and activators of transcription (STAT) and nuclear factor-kappa B (NF-κB) pathways, which overlap and synergize, making it challenging to predict optimal cytokine combinations for both proliferation and cytotoxicity. We integrated functional assays for NK cells cultured in a variety of cytokine combinations with mathematical modeling using feature selection and mechanistic regression models. Our regression model successfully predicts NK cell proliferation for different cytokine combinations and indicates synergy of activated STATs and NF-κB transcription factors between priming and post-priming phases. The use of IL-21 in solution in the priming of NK cell culture resulted in an improved NK cell proliferation, without compromising cytotoxicity potential or interferon gamma secretion against hepatocellular carcinoma cell lines. Our work provides an integrative framework for interrogating NK cell proliferation and activation for cancer immunotherapy.

Implications of packed red bloods cells production and transfer on post transfusion hemoglobin increase.

Blood loss resulting in severe anemia is the most common indication for postoperative allogenic red blood cell (RBC) transfusions. In high-income countries, the majority of transfusions is received by elderly patients. Preservatives extend the storage of RBCs, though concerns exist about potential harm from transfusing older RBCs. This study tested the hypothesis that RBC storage duration effects hemoglobin increase in patients older than 70years who underwent non-cardiac surgery. Observations on surgical cohorts from two study sites of the LIBERAL-Trial were collected. Transfusion events and hemoglobin between 2018 and 2022 assessments in addition to manufacturing and product specific quality review information were evaluated. A total of 1626 transfusion events in 505 patients were analyzed. A linear mixed effects model was used to estimate the effect size of different predictors on hemoglobin increment upon red blood cell transfusion. No statistically significant effect of the RBC unit storage duration was found. Confounding variables resulting in higher hemoglobin increase included lower hemoglobin values prior to transfusion, the length of Hb measurement intervals before and after transfusion, as well as the method of RBC cell separation in line with different manufacturer hemoglobin values. The aspired increase in hemoglobin can be achieved with red blood cell concentrates of any storage duration. In general, elderly patients exhibit a sufficient hemoglobin rise following transfusion. However, if this is associated with improved outcomes cannot be answered.

Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

Background: Chimeric antigen receptor (CAR) T cell therapy has shown significant promise in treating hematological malignancies, yet its application in solid tumors, particularly those expressing the epidermal growth factor receptor (EGFR), remains limited. This study investigates the potential of CAR-engineered peripheral blood mononuclear cells (PBMCs) as a novel adoptive cell therapy against EGFR-positive cancers. Methods: Lentiviral transduction at an MOI of 50 was performed to generate specific anti-EGFR second generation CAR-effector cells. The transduced PBMCs were stimulated with cytokines and CD3/CD28 beads to enhance their proliferation and activation. Flow cytometric and real-time cell analysis were performed at various effector-to-target ratios to explore the cytotoxic potential of CAR-PBMCs. Results: CAR-PBMCs exhibited improved targeting and cytotoxicity against EGFR-positive cancer cell lines MDA-MB-468 and SK-BR-3, compared to untransduced controls, with unsignificant effects on allogeneic PBMCs. Conclusion: CAR-PBMCs hold considerable potential as a therapeutic strategy for EGFR-positive solid tumors, warranting further clinical investigation.

P0090 Synergistic reduction of inflammatory cytokines with obefazimod and etrasimod in combination treatment vs. either monotherapy in a mouse model of inflammatory bowel disease

Abstract Background Obefazimod (Obe) is an investigational, oral, once-daily small molecule that enhances expression of microRNA-124 and has shown both efficacy and safety in Phase 2 studies in patients with moderately to severely active ulcerative colitis (UC) [1, 2]. Due to a therapeutic ceiling of 10-20% placebo adjusted remission rates among available monotherapy strategies, combination therapies are gaining interest as a treatment strategy [3]. We present early preclinical data on the combination of Obe and the sphingosine-1-phosphate receptor modulator etrasimod (Etra) in a mouse model of inflammatory bowel disease. Methods The efficacy of Obe and Etra, as monotherapies and in combination, in preventing colitis was assessed using the mouse T-cell adoptive transfer (TCT) model. CD4+CD45high T-cells from immunocompetent C57BI/6J mice were transferred into RAG2 knockout mice. Treatments were administered daily (10 ml/kg, p.o.) from Day 0 (1h before cell transfer) until Day 55: vehicle (0.5% methylcellulose), Obe (40 or 100 mg/kg, Obe40, Obe100), Etra (3 mg/kg), Obe40 + Etra, Obe100 + Etra. Body weight (BW), disease activity index (DAI, based on BW loss, fecal consistency, and fecal blood), and levels of inflammatory cytokines in the colon and blood were measured to assess disease severity and inflammation. Results Obe40 alone did not protect against BW loss and colitis (as measured by DAI), while Etra alone prevented BW loss but did not fully prevent colitis. Instead, the combination Obe40 + Etra improved BW and the response on DAI. Obe100 alone protected against BW loss and colitis. The combination Obe100 + Etra did not improve BW and DAI compared to Obe100 alone. At day 55, in the colon tissue, Obe100 induced a statistically significant decrease in TNFα, IL-17 and IFNγ, while Etra did not. The combination of Obe100 + Etra increased the reduction of these cytokines in the colon. At day 55, in the blood, Obe100 induced a statistically significant reduction in IL-17 levels, while Etra induced a significant decrease in TNFα levels. The combination therapy induced a synergistic reduction of TNFα, IL-17, IL-6 and IFNγ. Conclusion: In the mouse TCT model, the combination of Obe and Etra improved BW and DAI and reduced CD4+ cell numbers and inflammatory cytokines in both colon and blood, showing a synergistic reduction of cytokine levels in the blood. These promising findings suggest that this combination could provide enhanced therapeutic potential for treating inflammatory conditions like UC.

P0057 METTL3 potentiates aged CD4+T cell response via programming effector differentiation in elderly ulcerative colitis

Abstract Background The burden of IBD in the elderly is increasing globally. However, the mechanisms underlying the age-related IBD susceptibility remain unclear. Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to inflammatory and autoimmune disease. As a fundamental mRNA modification, N6-methyladenosine (m6A) participates in various pathological processes. However, the role of METTL3-mediated m6A RNA modification in elderly IBD remains unclear. Herein, we sought to determine the role of immunosenescence in age-dependent colitis and to explore the underlying mechanisms. Methods Young (2-3 months), middle-aged (10-12 months), and aged (20-24 months) mice were established models of colitis by 2% dextran sulfate sodium salt (DSS) treatment. We detected the senescence-associated-β-galactosidase (SA-β-Gal) activity in lymphocytes isolated from colon of mice. Single-cell and bulk RNA-seq of colonic immune cells in mice with different ages were performed to investigate the precise molecular mechanisms. CD4+CD25-CD45RBhi T cell adoptive transfer model was used to further analyze the role of senescent CD4+T cells in colitis. Mettl3fl/lfCd4Cre mice were obtained to explicit role of METTL3 in regulating CD4+T cell senescence. Results Ageing increased the severity of DSS-induced colitis. CD4+T cell senescence was implicated in the progression of aging-related experimental colitis, exhibiting an increase of SA-β-Gal and accumulation of CD4+ effector memory T (TEM) cells in colon. Aged colonic CD4+ T cells generate higher levels of IFN-γ and IL-17 comparing to young colonic CD4+ T cells. Single-cell analysis revealed that aging increased colonic CD4+ TEM cells, which were associated with T cell-mediated cytotoxicity and cytokine-mediated signaling pathway. Adoptive transfer of aged CD4+CD25-CD45RBhi T cells to Rag-/- mice induced more severe colitis compared with young CD4+CD25-CD45RBhi T cells transfer. Aged CD4+T potentiates Th1/Th17/Tfh differentiation and exacerbates the progression of colitis. METTL3 was increased in aged colonic CD4+T cells. Inhibiting METTL3 in CD4+T cell protects from cellular senescence, effector differentiation and colitis. Conclusion These results provide a significant insight into the contribution of senescent CD4+ T cells to age-dependent colitis and provide an attractive possibility that targeting T cell specifically might be a potential strategy to treat elderly IBD patients.

P1339 CJRB-201, a promising candidate for microbiome-based therapy with potent anti-inflammatory effects in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal condition characterized by immune dysregulation driven by microbiome dysbiosis, with a reduction of beneficial bacteria such as Faecalibacterium, a dominant commensal bacterium comprising 5-10% of the human gut microbiome. Its depletion is a hallmark of Crohn's disease and other autoimmune-related conditions, making it an emerging candidate as a next-generation probiotic. Methods CJRB-201 was selected using our BI platform (Ez-Mx), based on more than 500 metagenomic datasets from IBD patients and healthy controls. The immune regulatory efficacy of CJRB-201 was evaluated through in vitro and ex vivo assays, focusing on key cytokine ratios (IL-10/IL-1β, IL-10/TNF, IL-10/IFN-γ) in macrophages and T cells. The induction of T regulatory (T reg) cell populations was evaluated ex vivo and in germ-free (GF) mice. Three pathogenetically different IBD animal models (DSS-induced, IL-10 KO GF, T cell transfer) were examined to assess its efficacy. Lastly, the SHIME system (Simulator of the Human Intestinal Microbial Ecosystem) was applied to test various fibers that promote the growth of CJRB-201. Results Among 34 tested anti-inflammatory strains, including those from CJ Bioscience, 4D Pharma, and our Ez-Mx platform, CJRB-201 emerged as one of the most effective. Notably, CJRB-201 and its conforming species showed the highest induction of T reg populations ex vivo among the sixty Faecalibacterium strains, primarily driven by its cell pellet form rather than the supernatant. Administration of CJRB-201 resulted in successful colonization in the colon of GF mice, leading to an enhanced T reg cell population. In the three IBD models with both preventative and therapeutic approaches, CJRB-201 demonstrated significant improvements across five key disease features (body weight, disease activity index, colon length, histopathology, and inflammatory markers such as cytokines and fecal lipocalin-2). Interestingly, CJRB-201 outperformed other potential single and consortia strains and demonstrated efficacy comparable to the α-IL-12p40 antibody, particularly in the T cell transfer model (Figure 1). Lastly, we identified the fiber that stabilizes the engraftment of CJRB-201 in the SHIME system. Conclusion CJRB-201 exhibits superior anti-inflammatory efficacy in multiple immune assays and IBD animal models, significantly improving preclinical and inflammatory markers. Given its ability to enhance T reg populations and the promising potential of the identified fiber in supporting its colonization, CJRB-201 holds strong promise as a microbiome-based therapeutic for IBD patients.

P0081 Novel in vitro protocol to isolate and expand murine polyclonal Treg cells for efficacy testing in murine models of IBD

Abstract Background Regulatory T cells (Treg) are a subset of CD4+ T cells that play a crucial role in maintaining immune tolerance and preventing autoimmunity. In the context of Inflammatory Bowel Disease (IBD), dysregulation of Treg function has been implicated in the pathogenesis of the disease. Studies have shown that polyclonal Treg have the potential to suppress inflammatory responses and ameliorate colitis in preclinical models. This has led to a growing interest in harnessing the therapeutic potential of polyclonal Treg cells as a potential treatment for IBD. To support this new research and effectively produce Treg, we prompted the in-house development of a new platform to isolate, in vitro expand, and evaluate murine regulatory T cells in mouse models of IBD. Methods We used a two-step purification method using a pre-enrichment of CD4/CD25/CD62L-postive cells followed by FACS sorting and alternative cell culture methods to establish a protocol for the scalable isolation and expansion of naïve murine polyclonal Treg (muTreg). We then assessed the efficacy of the muTreg in the CD45RBhigh naïve CD4+ T cell transfer model of colitis. Results Quality check of the isolated muTreg showed a high purity that was sustained over a 7-day in vitro expansion period (over 96% FOXP3+) with an over 10-fold expansion. muTreg also conserved their functional activity and showed a 70% suppression of naïve T cells in vitro. Upon administration into the T cell transfer model, muTreg led to a dose-dependent improvement of colitis, as demonstrated by a decrease of inflammation markers such as colon weight, pro-inflammatory cytokine/chemokines, and colon histopathological score. muTreg also showed a strong, dose dependent suppression of effector CD4+ T cells in vivo despite a significant loss of FOXP3 expression under inflammatory conditions (over 60% loss of FOXP3 expression). Conclusion In summary, we successfully developed a novel platform for isolating and expanding muTreg and demonstrated their ability to alleviate T cell-induced colitis in a dose-dependent manner. Thus, our preclinical data lay the foundations for the investigation of the effect of Treg therapies in pre-clinical models and pave the way to study antigen specific CAR Treg cells or CRISPR-edited Treg in murine in vivo IBD models.

P0060 GPR15L counteracts chronic colitis by shaping the intestinal microbiota

Abstract Background The G protein-coupled receptor 15 (GPR15) is expressed on gut-homing T cells and the interaction of the GPR15 ligand GPR15L with GPR15 has recently been shown to crucially control integrin-dependent adhesion to the endothelium. However, the impact of GPR15L, which is predominantly expressed by colonic epithelial cells, on experimental colitis and inflammatory bowel disease (IBD) is currently not clear. Methods We studied GPR15L in experimental colitis with Gpr15l-deficient and -overexpressing mice as well as with rectal application of recombinant GPR15L. We performed phenotypical and molecular disease characterization using endoscopy, IVIS in vivo imaging, histology, immunofluorescence, FISH, qPCR, and multiplex ELISA. We also performed bulk RNA and metagenomic sequencing. We analyzed the expression of GPR15L in samples from IBD patients and non-IBD controls using qPCR and immunofluorescence and correlated it with clinical information. We explored the function of GPR15L in vitro using radial diffusion assays and antimicrobial assays. Results Acute DSS colitis was aggravated in Gpr15l-deficient mice compared to Gpr15l-proficient mice, while it was attenuated in Gpr15l-transgenic mice. This was T cell-independent, since the phenotype was conserved in Rag-/-Gpr15l-/- mice. Moreover, it was independent of Gpr15 in T cell transfer colitis with Gpr15-deficient and -proficient donor cells. RNA sequencing of colon tissue from Gpr15l-/- and Gpr15l+/+ mice with DSS colitis showed differential regulation in genes related to mucosal barrier function and host-microbiota crosstalk. Accordingly, 16S rRNA sequencing revealed a substantial difference in the intestinal microbial diversity of Gpr15l-/- and Gpr15l+/+ mice. Shotgun metagenomics and radial diffusion assays revealed that GPR15L acts as a selective anti-microbial peptide on community and single strain level, respectively, that predominantly restrains the growth of pathogenic bacteria. Rectal supplementation of recombinant GPR15L in Gpr15l-/- mice rescued increased disease severity in acute DSS colitis and reduced bacterial abundance and translocation. Patients with IBD showed reduced expression of GPR15L compared to controls. Furthermore, the expression of GPR15L was lower in patients with high disease severity compared to those with low severity. Finally, flare-free survival in patients with high GPR15L expression was substantially longer than in patients with low GPR15L expression. Conclusion GPR15L counteracts experimental colitis and seems beneficial in human IBD. Mechanically, it acts as an anti-microbial peptide to promote the homeostasis of the intestinal microbiota. Thus, GPR15L emerges as a potential future therapeutic approach for IBD.

DOP074 JAK inhibition to target fibroblasts and IBD-related fibrosis

Abstract Background Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD). Uncontrolled accumulation of extracellular matrix deposited by fibroblasts is thought to be the underlying cause of fibrogenesis. Janus kinases (JAK) inhibitors are a relatively novel therapeutic strategy in IBD which show a robust anti-inflammatory effect. We hypothesize that JAK-inhibitors might not only play a role in IBD by reducing inflammation, but also offer a therapeutic potential to treat intestinal fibrosis. In this study we explored the effects of JAK inhibition on intestinal fibroblasts in vitro and in vivo. Methods In vitro studies were performed with human CCD-18Co and primary colonic fibroblasts. Levels of activated JAKs and STATs were detected by western blot after cytokine stimulation and were inhibited using tofacitinib (10uM) or upadacitinib (10uM). For the T cell transfer colitis model, Rag-/- mice (n=5-12 mice/group) were used. After engraftment of donor T cells the severity of colitis was evaluated with the mouse endoscopy score (MEICS) and mice were treated with upadacitinib (20mg/kg, 4x/week intraperitoneal injection). Intestinal fibroblast subsets were analyzed using anti-CD90, anti-αSMA, anti-podoplanin, anti-FAP, anti-PDGFR-α and -β, by flow cytometry. Sirius red staining was performed to measure collagen deposition. Levels of activated JAKs and downstream STATs were detected by immunofluorescent staining. Results Re-analysis of our previously published single-cell RNA sequencing data from fibrostenotic tissue of Crohns disease patients identified that a pathogenic subset of activated pro-inflammatory FAP+ fibroblasts was driven by the activation of STAT3 [1]. Inflammatory cytokines activated the JAK-STAT pathway in human fibroblasts and both upadacitinib and tofacitinib strongly inhibited pJAK1, pJAK2, pSTAT1 and pSTAT3 in a concentration dependent manner. Mice treated with upadacitinib showed a significant lower MEICS compared to controls (2.4 vs 5.3, Figure 1). Interestingly, specific intestinal fibroblast subsets, e.g. CD90+PDGFR-β+ fibroblasts were less abundant upon JAK-inhibition. pSTAT3 expression in the colon seemed to change after JAK inhibition. Furthermore, upadacitinib was able to restore the amount and distribution of collagen in the colon. Conclusion Our study is the first to describe that the JAK-STAT pathway can be activated in intestinal fibroblasts and JAK-inhibitors including upadacitinib/ tofacitinib suppress this pathway in vitro. Furthermore, human data show activation of the JAK-STAT pathway in FAP+ fibroblasts and in mice, JAK inhibition has an effect on fibroblasts subsets and collagen deposition. This shows the potential of JAK-inhibitors to target more than immune cells.

P0088 3D tissue-level analysis of intestinal T cell dynamics reveals dual effects of etrasimod on recruitment and tissue exit in experimental colitis

Abstract Background Sphingosin-1-phosphate (S1P) receptor (S1PR) agonists are a novel class of anti-trafficking agents approved for the treatment of ulcerative colitis. These compounds induce T lymphocyte sequestration in secondary lymphoid organs, reducing their availability in peripheral blood and presumably in inflamed intestinal tissue. However, direct evidence for their effects on T cell dynamics within intestinal tissue is lacking. Moreover, while S1PR-dependent mechanisms regulate T cell egress from the gut, it remains unclear whether S1PR agonists modulate this process. This limited understanding of tissue-specific effects presents a crucial barrier to comprehending their full therapeutic mechanism. Methods We developed a comprehensive volumetric imaging platform to investigate tissue-level mechanisms of colitis within the complex three-dimensional architecture of the intestine. Using the T cell transfer colitis model, mice were treated with etrasimod or vehicle control. Full-thickness colon samples underwent tissue clearing and whole-mount immunofluorescence for T cell markers. Advanced light-sheet and volumetric confocal microscopy, combined with machine learning-based image analysis, enabled three-dimensional reconstruction of T cell distributions and lymphatic networks, allowing for quantitative histocytometry and spatial mapping of immune cell dynamics. Results T cell transfer colitis induced substantial lymphocyte infiltration in the colon, with T cells forming both distinct clusters and diffuse distributions throughout the tissue. The presence of numerous T cells within lymphatic vessels indicated active tissue egress, suggesting that T cell accumulation reflects a dynamic balance between recruitment and exit (Figure 1). Etrasimod treatment not only reduced overall T cell infiltration but also markedly altered their spatial distribution pattern. Notably, we observed a decreased proportion of T cells within lymphatic vessels, coupled with increased accumulation of T cells in proximity to lymphatic structures, indicating a previously unrecognized effect on tissue exit dynamics. Conclusion Our advanced volumetric imaging approach revealed novel insights into a potential dual mechanism of etrasimod in inflammatory bowel disease. Beyond reducing lymphocyte recruitment, etrasimod appears to create a tissue exit block, effectively trapping T cells within the intestinal compartment. These findings raise important questions about how enforced tissue residency influences local inflammatory circuits and may guide optimization of therapeutic strategies.

DOP068 Vedolizumab counteracts Parkinson’s disease-related brain pathology driven by gut-primed IBD T cells

Abstract Background The gut-brain axis has been increasingly recognized as a critical pathway linking Inflammatory Bowel Diseases (IBD) with neurodegenerative diseases. Chronic inflammation resulting from IBD can extend beyond the gut, and epidemiological data have also linked to the increased prevalence of neurodegenerative conditions like Parkinson’s disease (PD). However, the exact mechanisms underlying this interconnection are largely unclear. We therefore investigated how gut-primed T cells from IBD patients may drive neurodegenerative processes seen in PD. Methods Peripheral blood T cells from IBD patients were analyzed for co-expression of gut-homing integrin α4β7 and brain-homing integrin β1. Using gut-specific photoconversion in a Kaede T cell Transfer Colitis mouse model, we assessed the infiltration of gut-primed T cells into the brain tissue. To model and elucidate the interplay of T cells with the brain tissue ex vivo, we developed an innovative three-dimensional co-culture system involving human stem cell-derived Brain Organoids (hBrO) and peripheral blood T cells from IBD patients and controls. Vedolizumab, an anti-α4β7 integrin antibody, was used to assess its potential in blocking T cell accumulation and subsequent neurotoxicity in hBrO cultures. Results Gut-primed T cells from IBD patients co-expressed brain-homing integrins, suggesting that they are able to enter brain tissue. Clear evidence of gut-to-brain T cell trafficking was obtained in the Kaede T cell Transfer Colitis mouse model, confirming the capacity of gut-primed T cells to migrate across the blood-brain barrier under conditions of chronic intestinal inflammation. In the hBrO co-culture model, neurons expressed MAdCAM-1, and MAdCAM-1 expression in neurons was also found in single cell RNA sequencing of postmortem brain tissue. Consistently, α4β7+ IBD T cells accumulated specifically in the vicinity of neurons, leading to neuronal cell death and aSyn aggregation (a hallmark of PD) in hBrO. In line, Vedolizumab treatment reduced IBD T cell accumulation in hBrO and preserved neural cell integrity. Conclusion Our data, for the first time, show a T cell trafficking-driven axis of gut-to-brain communication with pathogenic potential for the central nervous system in the context of IBD. Moreover, our findings suggest that neurotoxic T cell-neuron interaction is mediated by MADCAM1 and α4β7 integrin. Thus, our study reveals a novel pathway, by which IBD T cells may contribute to neurodegeneration, linking chronic gut inflammation to brain tissue pathology and synucleinopathy. Blocking α4β7-mediated T cell-induced neurotoxicity and aSyn pathology presents a potential therapeutic approach for IBD patients at risk of neurodegenerative diseases such as PD.

P0199 Development of a high-throughput multiplex immunofluorescence and an AI-assisted image analysis tool to study T cells dynamics in pre-clinical rodent models of IBD

Abstract Background T cell therapies are emerging as a new approach to treat and cure patients suffering from immune diseases where T cell homeostasis is no longer maintained. For the evaluation of treatments in pre-clinical rodent models, tools are required that enable fast, observer-independent and high-resolution analysis of different T cells in tissue sections. Multiplex staining of tissue samples provides a valuable opportunity for comprehensive analysis of cellular dynamics with enhanced spatial resolution and tissue context compared to traditional staining and flow cytometry methods. We have developed a high-throughput multiplex immunofluorescence panel for detecting different T cell types and activation status in mouse models of inflammatory bowel disease (IBD). Moreover, we used an AI-assisted method to enhance image analysis, enabling automated, accurate, and quantification of complex cellular interactions. Methods For the development and optimization of the various stainings we used formalin-fixed paraffin embedded (FFPE) mouse colon sections from an efficacy study with murine polyclonal regulatory T cells (Tregs) in the T cell transfer mouse model of IBD, enabling access to sections with healthy versus diseased pathology, as well as samples where Tregs were transferred to reduce colon pathology. Multiplex staining was performed with antibodies (such as CD3, FOXP3, CD25, CD69, CTLA-4 and KI67) using a Leica BOND RX autostainer, by applying sequential primary antibody staining, followed by secondary HRP-conjugated antibodies and Opal-TSA chemistry. Whole slide images (WSI) were generated by a Phenoimager HT (Akoya) and analyzed using Visiopharm software, with DAPI+ nuclei detection via AI-based application. Results We successfully developed a multiplex panel that enables high throughput staining and spatial resolution analysis of over six markers. We were able to determine the activation status of different subtypes of T cells and their specific localization in the colon. We could quantify infiltration of activated effector T cells (Teffs) in both the mucosa and submucosa in colon samples with high pathological scores, as well as FOXP3+ cells in samples from mice treated with Tregs. Conclusion We developed an Opal-based multiplex immunofluorescence method to detect T cells in mouse colon samples, focusing on activation and proliferation markers. Additionally, an AI-assisted image analysis tool that provided fast, reproducible, and quantitative results with spatial resolution. This innovative multiplex method holds promise for adaptation to different mouse models of colitis and other rodent models related to immune diseases, furthering its potential impact in the area of T cell therapy research.

DOP031 Anti-IL-7 receptor plus anti-IL12/23 combination induces complete histological normalization in chronic colitis

Abstract Background The signaling networks perpetuating chronic inflammatory bowel disease remain unclear. Interleukin-7 (IL-7) has been previously reported to trigger the expansion of effector Th17 cells and anti-IL-7 receptor (IL-7R) antagonism to render IL23-differentiated Th17 cells susceptible to apoptosis1. We previously reported that IL-7R pathway is locally dysregulated and over-expressed in the colon mucosa of severe IBD patients and reproducibly associated with unresponsiveness to anti-TNF or anti-α4β7 integrin therapies2. We developed a humanized anti-IL7R lusvertikimab with a good PK/PD and tolerance profile in healthy volunteers3 and recently announced positive phase 2 efficacy results in ulcerative colitis after a 10 week-induction treatment period (randomized placebo-controlled CoTikiS study: NCT04882007). Methods Here, we investigated at preclinical level the additive effect of blocking IL-7R in combination with anti-IL12/23 antagonist antibody in an anti-TNF refractory adoptive T cell transfer chronic colitis mouse model as well as in-vitro on human Th17 cell differentiation in combination with anti-IL23 antibody. Results T cell-recipient mice developed a colitis characterised by the development of diarrhoea and body weight loss with post-mortem examination confirming epithelial hyperplasia and inflammatory cell infiltration into the mucosa. Anti-IL7R monotherapy at this dose was suboptimal in this model. Anti-IL-12/23 blockade in monotherapy was efficient but not enough alone to achieve complete remission at macroscopic and histological level. In contrast, administration of anti-IL-7R in combination with anti-IL12/23 mAb was associated with a strong and significant reduction in all measured colitis symptoms, macroscopic large bowel changes and all histopathological endpoints (hyperplasia, colitis severity, inflammation). When mice were treated in combination, the group was statistically different from the anti-IL12/23 monotherapy group and no statistical difference have been measured with the non-colitis group, illustrating this combination achieved complete remission including at histological mucosal healing. In vitro, we confirmed that IL-7 drives human Th17 differentiation despite IL23 blockade and that combining IL7R and IL23 blockade was efficient to prevent Th17 generation in the presence of both IL7 and IL23. Conclusion Our findings show that anti-IL-7R is well tolerated in combination with anti-IL12/23 mAb in mice and act in synergy to achieve a profound control of chronic colitis and complete histological normalization compared to monotherapy.

Syngeneic natural killer cell therapy activates dendritic and T cells in metastatic lungs and effectively treats low-burden metastases.

Natural killer (NK) cells can control metastasis through cytotoxicity and IFN-γ production independently of T cells in experimental metastasis mouse models. The inverse correlation between NK activity and metastasis incidence supports a critical role for NK cells in human metastatic surveillance. However, autologous NK cell therapy has shown limited benefit in treating patients with metastatic solid tumors. Using a spontaneous metastasis mouse model of MHC-I+ breast cancer, we found that transfer of IL-15/IL-12-conditioned syngeneic NK cells after primary tumor resection promoted long-term survival of mice with low metastatic burden and induced a tumor-specific protective T cell response that is essential for the therapeutic effect. Furthermore, NK cell transfer augments activation of conventional dendritic cells (cDCs), Foxp3-CD4+ T cells and stem cell-like CD8+ T cells in metastatic lungs, to which IFN-γ of the transferred NK cells contributes significantly. These results imply direct interactions between transferred NK cells and endogenous cDCs to enhance T cell activation. We conducted an investigator-initiated clinical trial of autologous NK cell therapy in six patients with advanced cancer and observed that the NK cell therapy was safe and showed signs of effectiveness. These findings indicate that autologous NK cell therapy is effective in treating established low burden metastases of MHC-I+ tumor cells by activating the cDC-T cell axis at metastatic sites.