Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD.
© 2023 The Author(s).

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.Copyright © 2022 Elsevier Inc. All rights reserved.

Citrobacter rodentium is a gastrointestinal infection that requires early IL-22 from group 3 innate lymphoid cells (ILC3) for resistance. The role of vitamin D in the clearance of C. rodentium infection was tested in vitamin D sufficient (D+) and vitamin D deficient (D-) wildtype (WT) and Cyp27B1 (Cyp) KO mice (unable to produce the high affinity vitamin D ligand 1,25(OH)2D, 1,25D). Feeding Cyp KO mice D- diets reduced vitamin D levels and prevented synthesis of 1,25D. D- (WT and Cyp KO) mice had fewer ILC3 cells and less IL-22 than D+ mice. D- Cyp KO mice developed a severe infection that resulted in the lethality of the mice by d14 post-infection. T and B cell deficient D- Rag KO mice also developed a severe and lethal infection with C. rodentium compared to D+ Rag KO mice. D- WT mice survived the infection but took significantly longer to clear the C. rodentium infection than D+ WT or D+ Cyp KO mice. Treating infected D- Cyp KO mice with IL-22 protected the mice from lethality. Treating the D- WT mice with 1,25D reconstituted the ILC3 cells in the colon and protected the mice from C. rodentium. IL-22 treatment of D- WT mice eliminated the need for vitamin D to clear the C. rodentium infection. Vitamin D is required for early IL-22 production from ILC3 cells and protection from enteric infection with C. rodentium.

Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-γ-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-γ expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-γ production, but also the protective function of iNKT cells in arthritis.

Donor lymphocyte infusion (DLI) followed by hematopoietic stem cell transplantation has served as an effective prevention/treatment modality against the relapse of some hematologic tumors, such as chronic myeloid leukemia (CML). However, the therapeutic efficacies of DLI for other types of leukemia, including acute lymphocytic leukemia (ALL), have been limited thus far. Therefore, we examined whether increasing the reactivity of donor T cells by gene modification could enhance the therapeutic efficacy of DLI in a murine model of ALL. When a CTLA4-CD28 chimera gene (CTC28) in which the intracellular signaling domain of CTLA4 was replaced with the CD28 signaling domain was introduced into CD4 and CD8 T cells in DLI, the graft-versus-tumor (GVT) effect was significantly increased. This effect was correlated with an increased expansion of donor CD8 T cells in vivo, and the depletion of CD8 T cells abolished this effect. The CD8 T cell expansion and the enhanced GVT effect were dependent on the transduction of both CD4 and CD8 T cells with CTC28, which emphasizes the role of dual modification in this therapeutic effect. The CTC28-transduced T cells that expanded in vivo also exhibited enhanced functionality. Although the potentiation of the GVT effect mediated by the CTC28 gene modification of T cells was accompanied by an increase of graft-versus-host disease (GVHD), the GVHD was not lethal and was mitigated by treatment with IL-10 gene-modified third-party mesenchymal stem cells. Thus, the combined genetic modification of CD4 and CD8 donor T cells with CTC28 could be a promising strategy for enhancing the therapeutic efficacy of DLI.