Extending the residence period of allogeneic iPSC-derived invariant natural killer T cells in humanized mice by editing HLA class I expression

Modeling immune responses to autologous and allogeneic human stem cell-derived islet grafts in vivo 2332710

Stem cell-derived β cells offer a promising strategy for Type 1 diabetes (T1D) treatment, yet the mechanisms driving immune infiltration and graft rejection remain unclear. Using human immune system (HIS) mice, we sought to compare immune responses to autologous versus allogeneic iPSC-derived islet grafts and to test whether increasing the frequency of autoreactive T cells is sufficient to trigger insulitis in autologous grafts. HIS mice were generated by engrafting human fetal liver CD34+ cells and thymic tissue with HLA-A2, -DR4 and -DQ8 alleles. Autologous or fully mismatched allogeneic iPSC-derived islets were transplanted under the kidney capsule. In subsets of mice, human T cells transduced with multiple islet antigen-reactive (IAR) TCRs were adoptively transferred. Fourteen-eighteen weeks after grafting, iPSC-derived islets were analyzed by imaging mass cytometry (IMC) and flow cytometry. Allogeneic grafts exhibited dense CD45+ immune infiltration, CD8+ T-cell enrichment, macrophage accumulation, and marked fibrosis and cellular proliferation. In contrast, autologous grafts showed minimal infiltration, limited fibrosis, and restricted proliferation. Despite robust engraftment and splenic persistence of IAR-TCR+ T cells, no detectable infiltration of these cells into autologous grafts was observed. Circulating human C-peptide remained stable in both settings, correlating with β-cell differentiation efficiency. While allogeneic grafts undergo strong immune infiltration and tissue remodeling, autologous stem cell-derived islet grafts remain largely protected, even in the presence of autoreactive T cells, indicating that additional stimuli may be required to trigger autoimmune β-cell targeting in vivo. ADA Postdoctoral Fellowship #1-25-PDF-01, JDRF 2-SRA-2022-1220-S-B Immune Mechanisms of Human Disease (HUM)

Memory T Cells in MHC-Deficient Humanized Mice

Immune cell development from human pluripotent stem cells.

This review summarizes research with a focus on clinical translation of iPSC-derived immune cells, as well as highlights continued challenges and prospects of this field.

T-cell receptor repertoires against HLA class I-restricted minor histocompatibility antigens are highly diverse with a small subset of public clonotypes

Background Allogeneic hematopoietic stem cell transplantation (alloHSCT) can be a curative treatment for hematological diseases. After HLA-matched alloHSCT, donor T cells may recognize minor histocompatibility antigens (MiHAs), which are polymorphic HLA-binding peptides on patient cells that are absent from donor cells due to genetic differences. Donor T cells can induce beneficial anti-tumor effects if MiHAs are targeted on malignant hematopoietic cells in the patient, while graft-versus-host disease (GvHD) may develop if MiHAs are targeted on patients’ healthy non-hematopoietic tissues. Methods We previously isolated T-cell clones from patients responding to donor lymphocyte infusions (DLIs) after HLA-matched alloHSCT, and identified HLA class I-restricted MiHAs. To investigate MiHA-specific T-cell responses in patients, we here sequenced the T-cell receptors (TCRs) of MiHA-specific T-cell clones and identified 394 distinct TCRs against 122 MiHAs. We used the collection of identified TCRs to measure frequencies of matched MiHA-specific TCRs in 39 patients responding to DLI with antitumor responses accompanied with no (n=9), limited (n=8) or severe (n=22) GvHD. Results The data showed higher MiHA-specific TCR frequencies in patients with severe GvHD, which were mainly driven by clonal expansion. Moreover, within the diverse MiHA-specific TCR repertoires in these patients, we identified five public TCRs against four MiHAs with identical CDR3 regions and several TCRs targeting MiHAs with similar, but not identical, CDR3 regions. Conclusion Patients with severe GvHD have high MiHA-TCR frequencies mainly driven by clonal expansion, and that MiHA-specific TCR repertoires in patients responding to DLI after alloHSCT are highly diverse with a few public clonotypes.

A synthetic NKG2A engager enables long-term persistence of HLA-deficient allogeneic engineered Tregs.

Allogeneic cell therapies offer a scalable and off-the-shelf alternative to the autologous approach, but immune rejection, particularly by natural killer (NK) cells following human leukocyte antigen (HLA) ablation, remains a major barrier to their persistence. Here, we report an improved synthetic NKG2A engager to selectively inhibit NKG2A⁺ NK cells while avoiding activation of NKG2C⁺ subsets, thereby overcoming a key limitation of the natural ligand HLA-E. Engineered regulatory T cells (EngTregs) lacking HLA and expressing the engager were protected from in vitro NK cell-mediated cytotoxicity more effectively than previously reported NK inhibitory strategies. In humanized mouse models, EngTregs persisted for up to 12 weeks, whereas unprotected cells were rapidly rejected. Incorporation of the engager into a clinically compatible dual-AAV EngTregs preserved Treg identity and function while conferring resistance to immune rejection. Together, these findings establish the improved NKG2A engager as an effective synthetic immune-evasion strategy and provide a clinically translatable approach to enable durable persistence of off-the-shelf EngTreg therapies.

Scalable expansion of human iNKT cells: single-cell profiling and in vivo control of GvHD with preserved GvL activity

Invariant natural killer T (iNKT) cells can limit graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), but their scarcity in peripheral blood limits therapeutic development. Current clinical-grade human iNKT expansion protocols mainly rely on IL-2, require prior iNKT-cell sorting, last 6–8 weeks, and predominantly expand CD4+ iNKT cells, whereas human CD4− iNKT cells are more strongly associated with GVHD control in patients and uniquely regulate antigen-presenting cells and T-cell activation. We developed a scalable culture system to preferentially expand human CD4− iNKT cells directly from total peripheral blood mononuclear cells (PBMCs) using alpha-galactosylceramide (α-GalCer) and optimized cytokine conditions. IL-15 was the most effective cytokine. The optimized 14-day protocol generated a mean of 3.8×107 iNKT cells from 2×107 PBMCs, including 74% CD4− iNKT cells. Single-cell transcriptomic profiling identified eight major iNKT subsets, differentiation trajectories during expansion, and distinct IL-2- versus IL-15-associated transcriptional programs. IL-15-expanded iNKT cells induced apoptosis of monocyte-derived dendritic and leukemic cells in vitro, controlled xeno-GVHD, and preserved graft-versus-leukemia (GVL) activity in preclinical mouse models. This platform enables reproducible production of human CD4− iNKT cells at clinically relevant scale and position IL-15–expanded iNKT cells as a compelling immunotherapy candidate for allo-HSCT.