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AbstractBackgroundPeritoneal metastasis (PM) is one of the most challenging clinical problems in gastric cancer (GC), largely due to its high recurrence rate and poor response to current therapies. Increasing evidence indicates that remodeling of the extracellular matrix (ECM) plays an important role in therapeutic failure. However, how specific stromal–immune interactions contribute to PM heterogeneity and immunotherapy resistance remains unclear. In this study, we investigated how ECM composition—particularly the accumulation of hyaluronic acid (HA)—influences the immune microenvironment and therapeutic responses in GC-associated PM.MethodsWe combined histopathological assessment, analyses of patient-derived specimens, single-cell transcriptomic profiling, and murine models of PM to delineate ECM remodeling patterns and immune cell dynamics in therapy-sensitive and therapy-resistant lesions. In addition, functional assays and pharmacological approaches were used to examine HA–CD44 signaling and its impact on CD4+ T cell differentiation and responsiveness to immune checkpoint blockade.ResultsTherapy-sensitive PM lesions were characterized by enrichment of elastic fibers, whereas therapy-resistant lesions showed collagen accumulation. Notably, HA deposition emerged as a key feature distinguishing these ECM states and was closely associated with differential therapeutic outcomes. Elevated HA levels activated CD44-dependent signaling in CD4+ T cells, driving regulatory T cell (Treg) differentiation through a CD44–IQGAP1–RAC1–SMAD3 signaling pathway and thereby establishing an immunosuppressive microenvironment. Importantly, pharmacological inhibition of CD44 reduced Treg expansion and markedly enhanced the antitumor efficacy of anti-PD-1 therapy in murine PM models.ConclusionsOur findings identify HA–CD44 signaling as a critical link between ECM remodeling and immune evasion in GC PM. Targeting ECM-driven immunosuppressive mechanisms may represent a promising strategy to overcome therapeutic resistance and improve the efficacy of immunotherapy in this aggressive disease.

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases, yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage, limited by toxicity from likely excessive signaling. By contrast, mouse Tregs-IL2pa exhibit sustained expansion, long-term survival in immunocompetent mice for over a year, and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype, Treg-specific demethylation, and a diverse TCR repertoire. In vivo, prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly, compared with control Treg, human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability, durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery, Regulatory T cells, Autoimmune diseases, Interleukins

FOXP3+ regulatory T cells (Tregs) play a central role in the regulation of the immune system. Human Tregs preferentially express a FOXP3 isoform known as delta 2, which lacks exon 2. In addition to FOXP3, Tregs also express isoforms of other Treg-related molecules, such as CTLA-4 and IKZF-2. It is hypothesized that Tregs possess a unique isoform repertoire based on their unique gene and isoform expression profiles, which include FOXP3. Here, we identified a Treg-specific unique isoform repertoire confirmed by long-read high-throughput isoform sequencing called Iso-seq, which is uniquely capable of providing data on genome-wide isoform usage. Notably, while conventional T cells (Tconvs) do not exhibit this pattern, Tregs preferentially express the full-length FOXP3 isoform. Interestingly, the preferential expression of ICOS and PD-L1 upon T-cell receptor (TCR) stimulation was noted in activated Tregs but not in Tconvs or non-activated Tregs. Moreover, using a PD-L1 antibody blockade on Tregs did not diminish FOXP3 expression; however, it significantly reduced the suppressive function. Therefore, Tregs may have a unique isoform repertoire, which becomes pronounced upon polyclonal TCR stimulation.