Genetic Id2 loss uncouples Tfh differentiation from effector activity, positioning Id2 inhibition as a novel therapeutic strategy to selectively disrupt pathogenic Tfh-GCB crosstalk in cGVHD.
Abstract
Chronic graft-versus-host disease (cGVHD) is a severe complication of hematopoietic stem cell transplantation (HSCT) complication driven by immune dysregulation. A key feature is aberrant T follicular helper (Tfh) and germinal center B (GCB) cells, which promote pro-inflammatory cytokine release, pathogenic antibody production, and tissue fibrosis. Leveraging cGVHD patient T cell single-cell RNA sequencing data from the Kean Lab, we selected Id2, a transcription factor that inhibits Tfh differentiation while supporting antibody production, as a target for the treatment of cGvHD.
We used CRISPR/Cas9 to generate Id2-knockout (KO) naïve murine T cells and adoptively transferred into pre-conditioned B10.BR recipients of C57BL/6 bone marrow (BM) ± T cells to generate murine cGVHD model with bronchiolitis obliterans (BO) lung disease.
Id2-KO T cells maintained ≥90% KO efficiency until study end (d49). Mice receiving Id2-KO vs control T cells had significantly improved pulmonary function (p < 0.0001), normalizing resistance, compliance, and elastance to BM-only (no disease) levels. Consistent with cGVHD amelioration, a 2-fold reduction in lung collagen deposition (p < 0.0001) and 4-fold decrease in serum allo-reactive antibody (p = 0.0286) were noted. On D49, despite similar GCB and Tfh ratios to control, Id2-KO T cells had enhanced Tfh differentiation: Bcl6+T cells increased from 4.9% to 10.8% (p = 0.0038) with 1.8-fold higher Bcl6 MFI(p = 0.0005). Id2-KO Tfh cells were functionally impaired with a 50% reduction in IL-4 production (p = 0.0078) and a 1.9-fold decrease in SLAMF1+ Tfh cells (from 63.3% to 33%, p < 0.0001), indicating disrupted Tfh-GC B cell interactions.
Id2 restrains Tfh differentiation while supporting their functional capacity to help GCB cells produce pathogenic antibody. Genetic Id2 loss uncouples Tfh differentiation from effector activity, positioning Id2 inhibition as a novel therapeutic strategy to selectively disrupt pathogenic Tfh-GCB crosstalk in cGVHD
NIH
Transplantation Immunology (TRAN)
Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic transplantation. cGVHD pathophysiology involves cooperation between T follicular helper cells (TFHs) and germinal center B cells (GCBs), allo- and autoantibody depositions in cGVHD tissues, and fibrosis. We evaluated human CD19–directed chimeric antigen receptor (CAR19) T cell therapy in a clinically relevant murine cGVHD model with bronchiolitis obliterans syndrome (BOS). Although CD8+ CAR19 T cells effectively reduced peripheral B cell and GCB frequencies, pulmonary function was unimproved. In contrast, a single infusion of CAR19 CD4+ regulatory T cells (Tregs) mitigated ongoing pulmonary disease and modulated germinal centers (GCs) associated with reduced TFH frequencies compared with control Tregs but without measurable B cell depletion. Compared with EGFR Treg infusion, mice receiving CAR19 Tregs exhibited enhanced suppression of B cell activation and preserved splenic architecture, and CAR19 Treg infusion provided greater opportunities for interaction with CD19+ B cells at the B cell follicle boundary zones. Taken together with the absence of detectable B cell cytolysis, these findings were most consistent with GC suppression rather than B cell depletion as the dominant mechanism. Overall, our findings suggest that CAR19 Tregs represent a promising and safe cGVHD/BOS therapeutic strategy, offering immunosuppressive benefits and improved disease outcomes that may be more limited with CD8+ CAR19 T cell treatment.
Sujeong Jin, Michael C. Zaiken, Cameron McDonald-Hyman et al.· JCI Insight· 0 citations
Ocular graft-versus-host disease severity correlates with inflammation and fibrotic damage in the lacrimal gland (LG). Long-living stem-like progenitor exhausted TCF1+ PD-1+ T cells (Tpex) have been reported to maintain and replenish the reservoir of exhausted-like effector T cells (Texef). In chronic settings, the cumulative cytotoxicity of CD8+ Texef mediates tissue damage. A comprehensive characterization of the phenotypic and transcriptomic profiles of these pathogenic exhausted populations has not yet been reported.
To this end, we used an established minor MHC-mismatched murine model of sclerodermatous cGVHD (B10.D2 (H-2d) into BALB/c (H-2d), allogeneic (allo) cGVHD) using syngeneic (syn) controls (Balb/c into Balb/c) assessed at 14-, 21-, and 42-days post-transplant. Here, we longitudinally investigated transcriptional changes in exocrine tissue immune population.
Microscopy identified increased lymphocyte infiltration in the lacrimal gland and conjunctiva. Single cell sequencing was performed on lacrimal gland. Datasets were processed using a robust integrative approach based on scVI deep learning architecture. We identified four distinct exhausted T cell subpopulations expanded in allo LG. Exhaustion, cytotoxic and residency scores were highest for these four subsets. Proportional distribution analysis revealed a substantial early increase in Tcf7+, Sell+, Il7r+, Slamf6+, Pdcd1+ CD8+ Tpex cells in the allo group. Importantly, allo lacrimal glands were marked by the expansion of Tcf7− CD8+ Texef cells that were not present in the syn group. Cell-cell communication analyses inferred an interaction between Tfrchi acinar cells and exhausted T cells through Ceacam1-Havcr2, signaling shown to drive terminal exhaustion of Texef.
Overall, scRNAseq results evince a major shift in T-cell fate towards a pathogenic exhausted-like phenotype possibly stemming from an early Tcf7+ expressing precursor exhausted population in cGVHD-affected LG.
Intramural program of the NIDCR, NIH
Transplantation Immunology (TRAN)
C. Basset, Marit Aure, Rubina Sharma et al.· Journal of Immunology· 0 citations
Hematopoietic cell transplantation (HCT) is a curative therapy for blood cancers but is complicated by graft-versus-host disease (GVHD), where donor CD4+ T helper (Th) cells drive gut pathology. Defining factors that drive pathogenic Th cell differentiation in the gut promises to inform therapies that mitigate GVHD without impacting the beneficial anti-cancer activity of HCT therapy. In our work, we identified GATA3 as a key transcription factor that may facilitate the development of gut pathogenic Th cells after HCT. We hypothesized that Th cell deletion of GATA3 will impair pathogenic Th cell differentiation and improve clinical outcomes in a murine GVHD model.
Using a murine allo-HCT model, we transplanted T cell-depleted bone marrow from WT C57BL/6 donors and splenocytes from C57BL/6-Gata3fl/fl Rosa26ERT2-Cre donors into irradiated BALB/c recipients. Donor cell GATA3 deletion was induced via tamoxifen treatment of recipient mice. Disease severity, intestinal pathology, donor cell trafficking, cytokine/granzyme expression, and innate immune infiltration were assessed by clinical scoring, histology, flow cytometry, and ex vivo Th cell stimulation.
GATA3 deletion reduced intestinal GVHD pathology and impaired early donor T cell trafficking and persistence in the gut. While IFN-γ and IL-17A production were largely unaffected in early timepoints, GATA3 was required for heightened granzyme A expression, sustained GM-CSF production and chronic GM-CSF-driven intestinal eosinophilia at later timepoints. Analysis of human GVHD samples exhibited enrichment of eosinophil chemotactic pathways, mirroring murine findings.
GATA3 promotes intestinal GVHD by supporting donor T cell gut infiltration, granzyme A expression, and sustained GM-CSF-dependent eosinophilia. These findings identify GATA3 as a key regulator of pathogenic Th responses and a potential therapeutic target for mitigating intestinal GVHD.
Purdue Institute for Cancer Research
Transplantation Immunology (TRAN)
Franklin J Yeo, Mengbo Wang, Sungtae Park et al.· Journal of Immunology· 0 citations
Chronic graft-versus-host disease (cGVHD) is an alloimmune complication of allogeneic hematopoietic stem cell transplantation driven by donor—recipient antigen mismatch. The oral mucosa is a frequent target; however, the cellular circuitry and spatial architecture that sustain oral cGVHD remain incompletely defined.
Using single-cell RNA sequencing, we observed a marked enrichment of macrophage subsets in cGVHD oral mucosa. Spatial transcriptomics mapped CD68+ macrophages preferentially along the epithelial basement membrane. Ultrahigh-plex spatial phenotyping further delineated a significant expansion of proinflammatory (M1-like) macrophages, defined by HLA-DR, CD86 expression, accompanied by elevated IL-17 within lesional regions.
These data suggest an IL-17—driven axis in M1 macrophage recruitment and activation that may amplify mucosal inflammation. Spatial proximity analyses demonstrated juxtaposition of M1 macrophages with CD4+ regulatory T cells (Tregs) in cGVHD tissue, yet TIGIT+ Tregs were comparatively reduced, consistent with impaired regulatory—myeloid crosstalk. Ligand—receptor inference identified upregulation of JAK-STAT signaling, including increased CXCL10—CXCR3 interactions, suggesting chemokine-guided positioning of effector cells. Clinically, strategies that expand Tregs or antagonize CSF1R to limit macrophage activity have shown benefit in cGVHD, underscoring the therapeutic relevance of these pathways. Co-culture assays with macrophage-Treg cells are needed to probe reciprocal effects on cytokine production, Treg suppressive capacity, and macrophage polarization with verification in cGVHD patients.
Collectively, these studies may define macrophage—Treg crosstalk as an important determinant of immune disequilibrium and tissue injury in oral cGVHD.
Intramural program of the NIDCR, NIH
Transplantation Immunology (TRAN)
Rubina Sharma, Ana Costa da Silva, Noemi Kedei et al.· Journal of Immunology· 0 citations
Abstract Background Head and neck squamous cell carcinoma (HNSCC) exhibits a profoundly immunosuppressive tumor microenvironment (TME) enriched for myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and dysfunctional CD8⁺ T cells, limiting therapeutic benefit from immune checkpoint blockade. Macrophage migration inhibitory factor (MIF) is elevated in HNSCC and linked to poor outcomes, yet the cellular source and functional role of tumor-promoting MIF in shaping antitumor immunity remain unclear. Methods We used a myeloid-specific MIF knockout mouse (mMIF KO) in an orthotopic MOC2 HNSCC model and evaluated immune function using ex vivo co-cultures of tumor-derived MDSCs with naïve T cells under Treg-skewing, basal, or Th1-polarizing conditions. Results Myeloid-restricted MIF deletion significantly reduced tumor growth and increased CD8⁺ T cell infiltration, accompanied by reduced CTLA4, TIGIT, and TIM3 expression, with elevated PD1 consistent with antigen-engaged effector activation. High-dimensional immune profiling revealed expansion of cytotoxic CD8⁺ T cell states (GranzymeBhi/Perforinhi) and contraction of IL10–producing CD4⁺ regulatory (Tr1-like) populations, findings corroborated by decreased intratumoral CD4⁺FoxP3⁺Tregs. Within the myeloid compartment, MIF deletion selectively depleted polymorphonuclear MDSCs (PMN-MDSCs), including CSF1Rhi and PD-L1hi subsets. Functionally, tumor-derived MDSCs lacking MIF showed impaired capacity to induce Tregs and to promote CD8⁺ T cell exhaustion in ex vivo co-cultures, effects partially overcome by TGFβ/Th1-polarizing signals. Conclusions These findings identify myeloid-derived MIF as a central regulator of the MDSC–Treg–CD8⁺ axis in HNSCC and support myeloid-targeted MIF inhibition as a strategy to reprogram the TME and enhance responses to immune checkpoint blockade.
Suvekshya Shrestha, Felipe F. Lamenza, Natalie Kazmierowicz et al.· Oncoimmunology· 0 citations
Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation leading to increased microvascular permeability, pulmonary edema, and organ failure. ARDS commonly complicates septic patients, and accounts for 10% of intensive care unit admissions with a 35-45% mortality and no effective pathological-pharmacological treatment available. Bone marrow-derived macrophages (BMDMs) contribute to pulmonary damage in ARDS by adopting a pro-inflammatory phenotype and producing inflammatory mediators in the lung tissue. V-domain Ig Suppressor of T-cell Activation (VISTA), a negative checkpoint regulator expressed across the hematopoietic compartment, has been shown to promote an anti-inflammatory phenotype in macrophages and maintain a protective effect on survival in response to both septic and ARDS challenge.
Using an indirect ‘double hit’ ARDS mouse model of fixed pressure hemorrhagic shock (Hem) followed by a polymicrobial sepsis insult through the cecal ligation and puncture (CLP) procedure, we examined VISTA function in BMDMs from wild-type (WT) and VISTA deficient (VISTAKO) mice. Bone marrow samples were harvested from Hem/CLP challenged mice and BMDMs were cultured and polarized in-vitro for characterization through flow cytometry analysis and functional assays.
Early data shows that VISTA KO BMDMs exhibit higher pro-inflammatory markers and MHCII expression in both Hem/CLP and sham control mice compared to WT macrophages. Lastly, VISTA KO Hem/CLP BMDMs exhibit higher production of inflammatory cytokines such as IL-6 and TNF-α even when polarized with IL-4 compared to Hem/CLP WT macrophages.
Our results highlight the role of VISTA function in BMDMs in the development of pulmonary damage seen during ARDS. Future studies will analyze how anti-inflammatory cytokine production is affected by VISTA and if VISTA agonistic treatment can alleviate increased proinflammatory function exhibited by BMDMs during ARDS.
R35 GM118097
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
J. Renedo, Fernando Gutierrez Garcia, Yaping Chen et al.· Journal of Immunology· 0 citations
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