Checkpoint blockade can reverse T-cell exhaustion and promote antitumor responses. Although blocking the PD-1 pathway has been successful in Hodgkin lymphoma, response rates have been modest in ...B-cell non-Hodgkin lymphoma (NHL). Coblockade of checkpoint receptors may therefore be necessary to optimize antitumor T-cell responses. Here, characterization of coinhibitory receptor expression in intratumoral T cells from different NHL types identified TIGIT and PD-1 as frequently expressed coinhibitory receptors. Tumors from NHL patients were enriched in CD8
and CD4
T effector memory cells that displayed high coexpression of TIGIT and PD-1, and coexpression of these checkpoint receptors identified T cells with reduced production of IFNγ, TNFα, and IL2. The suppressed cytokine production could be improved upon
culture in the absence of ligands. Whereas PD-L1 was expressed by macrophages, the TIGIT ligands CD155 and CD112 were expressed by lymphoma cells in 39% and 50% of DLBCL cases and in some mantle cell lymphoma cases, as well as by endothelium and follicular dendritic cells in all NHLs investigated. Collectively, our results show that TIGIT and PD-1 mark dysfunctional T cells and suggest that TIGIT and PD-1 coblockade should be further explored to elicit potent antitumor responses in patients with NHL.
Sentinel lymph nodes are the first nodes draining the lymph from a breast and could reveal early changes in the host immune system upon dissemination of breast cancer cells. To investigate this, we ...performed single‐cell immune profiling of lymph nodes with and without metastatic cells. Whereas no significant changes were observed for B‐cell and natural killer (NK)‐cell subsets, metastatic lymph nodes had a significantly increased frequency of CD8 T cells and a skewing toward an effector/memory phenotype of CD4 and CD8 T cells, suggesting an ongoing immune response. Additionally, metastatic lymph nodes had an increased frequency of TIGIT (T‐cell immunoreceptor with Ig and ITIM domains)‐positive T cells with suppressed TCR signaling compared with non‐metastatic nodes, indicating exhaustion of effector T cells, and an increased frequency of regulatory T cells (Tregs) with an activated phenotype. T‐cell alterations correlated with the percentage of metastatic tumor cells, reflecting the presence of metastatic tumor cells driving T effector cells toward exhaustion and promoting immunosuppression by recruitment or increased differentiation toward Tregs. These results show that immune suppression occurs already in early stages of tumor progression.
Sentinel lymph nodes (SLN) are the first nodes draining the lymph from a breast and could reveal early changes in the host immune system upon dissemination of breast cancer (BC) cells. We used single‐cell mass cytometry and functional analyses of metastatic and non‐metastatic LN and revealed a dysfunctional and suppressed immune response. Metastatic LN had an increased frequency of TIGIT‐positive T cells with suppressed TCR signaling compared with non‐metastatic nodes and an increased frequency of Tregs with an activated phenotype. The alterations were correlated with metastatic tumor burden in the LN of patients with BC.
Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of ...acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody—clone m971—targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.
Neither the number of HIV-1 proviruses within individual infected cells in HIV-1—infected patients nor their genetic relatedness within individual infected cells and between cells and plasma virus ...are well defined. To address these issues we developed a technique to quantify and genetically characterize HIV-1 DNA from single infected cells in vivo. Analysis of peripheral blood CD4+ T cells from nine patients revealed that the majority of infected cells contain only one copy of HIV-1 DNA, implying a limited potential for recombination in virus produced by these cells. The genetic similarity between HIV populations in CD4+ T cells and plasma implies ongoing exchange between these compartments both early and late after infection.
T cells modified to express chimeric antigen receptor (CAR) targeting CD19 (CD19CAR) have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic ...leukemia. CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19− tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-cell acute lymphoblastic leukemia and B-NHL. Here, we confirmed previous data by showing that, overall, B-NHL has high expression of CD37. A second-generation CD37CAR was designed, and its efficacy in T cells was compared with that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon coculture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the 2 CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19− subpopulation, CD37CAR T cells efficiently controlled tumor growth and prolonged survival, whereas CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells also can effectively eradicate B-cell lymphoma tumors when CD19 antigen expression is lost and support further clinical testing for patients with relapsed/refractory B-NHL.
•CD37 is highly expressed across multiple types of B-cell lymphoma, representing a potential target for CAR-based therapy.•T cells expressing CD37CAR are efficiently redirected and can control B-cell lymphoma tumor progression in xenograft models.
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Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell ...non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3- Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.
MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl
, that alters the two-helix 'brace' that ...connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl
homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, ...we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4 ⁺ T cells CD45RO ⁺/CD27(⁺/⁻). The HIV-1 infection frequency of CD4 ⁺ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.
Polycomb repressive complex 2 (PRC2) is a chromatin modifier that regulates stem cells in embryonic and adult tissues. Loss-of-function studies of PRC2 components have been complicated by early ...embryonic dependence on PRC2 activity and the partial functional redundancy of enhancer of zeste homolog 1 (Ezh1) and enhancer of zeste homolog 2 (Ezh2), which encode the enzymatic component of PRC2. Here, we investigated the role of PRC2 in hematopoiesis by conditional deletion of suppressor of zeste 12 protein homolog (Suz12), a core component of PRC2. Complete loss of Suz12 resulted in failure of hematopoiesis, both in the embryo and the adult, with a loss of maintenance of hematopoietic stem cells (HSCs). In contrast, partial loss of PRC2 enhanced HSC self-renewal. Although Suz12 was required for lymphoid development, deletion in individual blood cell lineages revealed that it was dispensable for the development of granulocytic, monocytic, and megakaryocytic cells. Collectively, these data reveal the multifaceted role of PRC2 in hematopoiesis, with divergent dose-dependent effects in HSC and distinct roles in maturing blood cells. Because PRC2 is a potential target for cancer therapy, the significant consequences of modest changes in PRC2 activity, as well as the cell and developmental stage-specific effects, will need to be carefully considered in any therapeutic context.
•Inactivation of Suz12 results in a rapid and marked exhaustion of the HSC pool.•Lymphoid development is completely dependent on PRC2, but numerous myeloid lineages develop in the absence of PRC2.
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