Alma-Martina Cepika

Type 1 regulatory T (Tr1) cells are CD4 ⁺ T cells with suppressive function that are induced from conventional T cells exposed to persistent or strong antigens. Human Tr1 cells are understudied; the regulators of their antigen-driven differentiation are unknown, and identifying them in tissues, where antigen interactions occur, is challenging. Here, we conducted a multiomic profiling of human antigen-induced Tr1 cells. Using CRISPR-based functional genomics, we uncovered essential roles of transcription factors IRF4, BATF, and MAF in human Tr1 differentiation, phenotype, and function. We also derived a Tr1 transcriptional signature that detects cells with a Tr1 phenotype in single-cell datasets from patients treated with Tr1 therapy and those with solid tumors. Cross-species analysis confirmed this signature identifies bona fide Tr1 cells induced in vivo in a murine solid tumor model. These findings provide a framework for development of Tr1-based and Tr1-targeting therapies and studies of Tr1 cell biology.

Few studies have tracked human CD4 ⁺ T cell clones through repeated infections. We used longitudinal single-cell RNA and T cell receptor (TCR) tracking to study the functional stability and memory potential of CD4 ⁺ T cell clonotypes during repeated Plasmodium falciparum ( Pf ) infections in Ugandan children and adults. Nearly all clonotypes displayed a strong preference for one of seven CD4 ⁺ subsets. This phenomenon of “clonal fidelity” was influenced by clonal expansion, linking T cell polarization and proliferation in vivo. Using clone tracking, we characterized subset-specific activation trajectories and identified antigen-specific clones. Type 1 regulatory T (T _R 1) cells accounted for nearly 90% of Pf -specific CD4 ⁺ T cells in blood. Tracking these clones longitudinally for hundreds of days, we observed malaria-induced expansion of T _R 1 effectors, long-term persistence of T _R 1 memory cells, and high-fidelity recall responses after reinfection. This work establishes clonal fidelity as a natural phenomenon and demonstrates the stable, long-term memory potential of T _R 1 cells.

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human-engineered cytotoxic CD4⁺ T cells. Single-cell RNA-seq of primary AML samples and CD4⁺ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4⁺ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing by primary ex vivo-isolated CD8⁺ T cells in vitro, and engineered CD4⁺ T cells in vitro and in vivo. While AML heterogeneity implies that multiple factors may determine their sensitivity to T cell killing, these data show that ICAM1 acts as an immune trigger, allowing T cell killing, and could play a role in AML patient survival in vivo.

Human adaptive immunity is orchestrated by effector and regulatory T (Treg) cells. Natural Tregs arise in the thymus where they are shaped to recognize self-antigens, while type 1 Tregs or Tr1 cells are induced from conventional peripheral CD4 ⁺ T cells in response to peripheral antigens, such as alloantigens and allergens. Tr1 cells have been developed as a potential therapy for inducing antigen-specific tolerance, because they can be rapidly differentiated in vitro in response to a target antigen. However, the epigenetic landscape and the identity of transcription factors (TFs) that regulate differentiation, phenotype, and functions of human antigen-specific Tr1 cells is largely unknown, hindering Tr1 research and broader clinical development. Here, we reveal the unique epigenetic signature of antigen-specific Tr1 cells, and TFs that regulate their differentiation, phenotype and function. We showed that in vitro induced antigen-specific Tr1 cells are distinct both clonally and transcriptionally from natural Tregs and other conventional CD4 ⁺ T cells on a single-cell level. An integrative analysis of Tr1 cell epigenome and transcriptome identified a TF signature unique to antigen-specific Tr1 cells, and predicted that IRF4, BATF, and MAF act as their transcriptional regulators. Using functional genomics, we showed that each of these TFs play a non-redundant role in regulating Tr1 cell differentiation, suppressive function, and expression of co-inhibitory and cytotoxic proteins. By using the Tr1-specific TF signature as a molecular fingerprint, we tracked Tr1 cells in peripheral blood of recipients of allogeneic hematopoietic stem cell transplantation treated with adoptive Tr1 cell therapy. Furthermore, the same signature identified Tr1 cells in resident CD4 ⁺ T cells in solid tumors. Altogether, these results reveal the epigenetic signature and the key transcriptional regulators of human Tr1 cells. These data will guide mechanistic studies of human Tr1 cell biology and the development and optimization of adoptive Tr1 cell therapies.

Biallelic mutations in the ACP5 gene cause spondyloenchondrodysplasia with immune dysregulation (SPENCDI). SPENCDI is characterized by the phenotypic triad of skeletal dysplasia, innate and adaptive immune dysfunction, and variable neurologic findings ranging from asymptomatic brain calcifications to severe developmental delay with spasticity. Immune dysregulation in SPENCDI is often refractory to standard immunosuppressive treatments. Here, we present the cases of two patients with SPENCDI and recalcitrant autoimmune cytopenias who demonstrated a favorable clinical response to targeted JAK inhibition over a period of more than 3 years. One of the patients exhibited steadily rising IgG levels and a bone marrow biopsy revealed smoldering multiple myeloma. A review of the literature uncovered that approximately half of the SPENCDI patients reported to date exhibited increased IgG levels. Screening for multiple myeloma in SPENCDI patients with rising IgG levels should therefore be considered.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation, and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations in chromatin profiles at loci encoding post-translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations of chromatin profiles at loci encoding post translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human engineered cytotoxic CD4 ⁺ T cells. Single-cell RNA-seq of primary AML samples and CD4 ⁺ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4 ⁺ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro . Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1 , a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing to primary ex vivo -isolated CD8 ⁺ T cells in vitro , and engineered CD4 ⁺ T cells in vitro and in vivo . Thus, ICAM1 on AML acts as an immune trigger, allowing T cell killing, and could affect AML patient survival in vivo . SIGNIFICANCE: AML is a common leukemia with sub-optimal outcomes. We show that AML transcriptional programs correlate with susceptibility to T cell killing. Killing resistance-associated AML programs are enriched in patients with poor survival. Killing-sensitive, but not resistant AML activate T cells and upregulate ICAM1 that binds to LFA-1 on T cells, allowing immune synapse formation which is critical for AML elimination. GRAPHICAL

Type 1 regulatory T cells suppress alloreactive immune cells after adoptive transfer in a CTLA-4 and PD-1–mediated manner.