Dendritic cells are a rare lineage, consisting of cDC1, cDC2, and the scarce (< 0.3% of blood) DC3. Uniquely, DC3s share monocyte and cDC2 features, marked by co-expression of CD1c and CD14. They promote Th1 and Th17 cells, associate with autoimmunity, and induce CD8+CD103+ tissue-resident memory T-cells that may exacerbate disease. Contrarily, DC3s can be immunosuppressive in a tumor microenvironment. This duality could provide an immunotherapy platform if sufficient-numbers can be obtained.
HSPCs were generated from iPSCs via embryoid bodies; HSPCs were cultured for 2 weeks on murine MS5 stromal cells with SCF, FLT3L, and ascorbic acid. To induce regulatory iDC3s (iDC3regs), CD1c+ isolated cells were treated for 24hrs with CHIR99021 (Wnt agonist), L-kyneurine, retinoic acid and IFNγ, IL-10, and/or TGFβ (γ10β), the latter tested alone and in combination. Suppressive function was measured via AlloMLRs.
iDC3s emerged with a fold-increase of 1096±123 iDC3s per iPSC in 4 maturation states: CD45RA+CD14- (immature), CD45RA-CD14-, CD45RA-CD14+ CD163-, and CD14+CD163 + (mature). Isolated CD1c+ iDC3s cultured with GM-CSF, known to support DC3s, not FLT3L, gradually promoted maturation to a CD14+CD163+ over 3 days. γ10β-iDC3regs were most suppressive, mediated through PDL1 and IDO1. Interestingly, γ10β rapidly induced two states: CD14+CD163- and CD14+CD163+ that resisted inflammatory exposure (TNF+/IL1β/PGE2, LPS, CpG, R848, or GMCSF/IL4). Bulk RNA-seq of these populations is underway to define the regulatory profile and to compare public DC3 datasets. Studies in humanized mouse graft-versus-host disease models will test iDC3regs in vivo regulatory capacity; Studies examining tumor antigen-loaded and activated iDC3s will test their anti-tumor potential.
We demonstrate efficient generation of iDC3s, define their developmental trajectory, and regulatory potential. These findings reveal how DC3s operate across disease contexts.
Kidz1stFund, P01CA065493-28
Transplantation Immunology (TRAN)
Jordan Fink, Ke Yao, Lie Ma et al.· Journal of Immunology· 0 citations
The clinical successes of chimeric antigen receptor (CAR) T cells represent a major shift in immunotherapy. However, there is also increasing emphasis on potential long-term effects of CAR T cell therapy, especially using preclinical xenogeneic models. It has been previously demonstrated that only naïve, and not memory, peripheral blood human T cells can mediate a rapid and acute xenogeneic graft-versus-host disease (xenoGVHD). Here, we demonstrate that simply by altering the donor T cells in the process of generating CAR T cells, in which they are all memory phenotype, that the xenoGVHD outcome was markedly altered. Following tumor clearance with CAR T cell administration, we observed a significantly delayed (up to 200 days post-transfer with some donors) occurrence of lethal xenoGVHD, marked by profound scleroderma and multi-organ pathology consistent with chronic, not acute, GVHD. Notably, this novel chronic xenoGVHD occurred in the absence of B cells, which are classically associated with mediating chronic GVHD pathology. TCR-repertoire constriction during disease and the lack of disease using MHCI/II double-knockout NSG recipient mice confirmed the observed pathology was xenoGVHD and mediated by human-TCR:murine-MHC interactions. Interestingly, despite the consistent expansion of CAR-positive T cells during early tumor-clearance, a later emergence of CAR-negative populations during xenoGVHD also resulted. Our findings highlight xenoGVHD as a problem that makes long-term assessment of CAR T efficacy or toxicity highly problematic in xenograft models due to the artefact of xenoreactivity which is not representative of autologous clinical usage, and the profound effect that T cell alterations have in GVHD pathophysiology.
Wahed A. Firoz, Michael K. Sheng, Pedro Ruivo et al.· Blood Advances· 0 citations
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)
Yujie Zhao, Eun Ko, Cameron McDonald-Hyman et al.· Journal of Immunology· 0 citations
