The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors ...(IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (T
) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1
Eomes
T-bet
) BM-T
predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease.
Immunotherapy has revolutionized the management of numerous cancers; however, a substantial proportion that initially respond subsequently acquire means of immune escape and relapse. Analysis of ...recent clinical trials permits us to preliminarily understand how immunotherapies exert evolutionary pressures: selecting cancer subclones deficient in antigenicity and/or immunogenicity, thereby facilitating immune escape.
Single-cell transcriptomics has become the definitive method for classifying cell types and states, and can be augmented with genotype information to improve cell lineage identification. Due to ...constraints of short-read sequencing, current methods to detect natural genetic barcodes often require cumbersome primer panels and early commitment to targets. Here we devise a flexible long-read sequencing workflow and analysis pipeline, termed nanoranger, that starts from intermediate single-cell cDNA libraries to detect cell lineage-defining features, including single-nucleotide variants, fusion genes, isoforms, sequences of chimeric antigen and TCRs. Through systematic analysis of these classes of natural 'barcodes', we define the optimal targets for nanoranger, namely those loci close to the 5' end of highly expressed genes with transcript lengths shorter than 4 kB. As proof-of-concept, we apply nanoranger to longitudinal tracking of subclones of acute myeloid leukemia (AML) and describe the heterogeneous isoform landscape of thousands of marrow-infiltrating immune cells. We propose that enhanced cellular genotyping using nanoranger can improve the tracking of single-cell tumor and immune cell co-evolution.
After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important ...risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations.
Recent advances in cancer immunotherapy - ranging from immune-checkpoint blockade therapy to adoptive cellular therapy and vaccines - have revolutionized cancer treatment paradigms, yet the ...variability in clinical responses to these agents has motivated intense interest in understanding how the T cell landscape evolves with respect to response to immune intervention. Over the past decade, the advent of multidimensional single-cell technologies has provided the unprecedented ability to dissect the constellation of cell states of lymphocytes within a tumour microenvironment. In particular, the rapidly expanding capacity to definitively link intratumoural phenotypes with the antigen specificity of T cells provided by T cell receptors (TCRs) has now made it possible to focus on investigating the properties of T cells with tumour-specific reactivity. Moreover, the assessment of TCR clonality has enabled a molecular approach to track the trajectories, clonal dynamics and phenotypic changes of antitumour T cells over the course of immunotherapeutic intervention. Here, we review the current knowledge on the cellular states and antigen specificities of antitumour T cells and examine how fine characterization of T cell dynamics in patients has provided meaningful insights into the mechanisms underlying effective cancer immunotherapy. We highlight those T cell subsets associated with productive T cell responses and discuss how diverse immunotherapies might leverage the pre-existing tumour-reactive T cell pool or instruct de novo generation of antitumour specificities. Future studies aimed at elucidating the factors associated with the elicitation of productive antitumour T cell immunity are anticipated to instruct the design of more efficacious treatment strategies.
BackgroundMutation-associated neoantigen (MANA)-specific T cells play a key role in tumor control and response to immune checkpoint inhibition (ICI).1 2 However, the majority of tumor-infiltrating ...lymphocytes (TIL) are not specific for the tumor.3 Herein, we developed and validated MANAscore, a bioinformatic scoring algorithm based on the transcriptional programs of MANA-specific T cells to isolate antitumor T cells from bystander T cells in lung cancer and melanoma.MethodsCombined single-cell (sc) RNA-seq/TCR-seq was performed on TIL obtained from 15 resectable non-small cell lung cancer (NSCLC) patients receiving neoadjuvant anti-PD-1 (NCT02259621). MANA-specific clonotypes were identified by coculturing autologous T cells with predicted MANA, and were validated by cloning the full TCR alpha and beta chain as previously described.1 Using the TCRβ CDR3 as a barcode, antigen-specific T cells were linked with their intratumoral sc expression profile. Using the first two patients enrolled in the clinical trial as a discovery cohort, MANAscore was developed to identify gene programs that best distinguish MANA-specific vs viral-specific T cells from NSCLC. Prediction performance was assessed in independent patients from the NSCLC and melanoma cohort.2 Seven MANAscore< sup >hi TCRs were cloned and queried for reactivity to peptide libraries of putative MANA derived from whole-exome sequencing of the respective tumor. Association of MANAscorehi clones with response to ICIs among all patients was assessed.ResultsA total of 890 MANA- and 542 viral-specific T cells were identified in sc TIL from six NSCLC patients. MANA- and viral-specific TIL presented with unique transcriptional profiles. Particularly, MANA-specific CD8 TIL expressed a partially activated cytolytic program with co-expression of multiple immune checkpoints and upregulated transcriptional regulators of T cell dysfunction. MANAscore showed high prediction accuracy and outperformed CD39 in identifying tumor-reactive T cells in independent NSCLC patients, as well as in an external validation cohort of melanoma patients (3936 MANA-specific T cells and 626 viral-specific T cells from 4 patients). Of seven MANAscore< sup >hi clones tested for reactivity, three were confirmed as MANA-specific. The pseudobulk expression profile of MANAscore< sup >hi clones showed a significant correlation with response to ICI, which is not observed in total CD8+ TIL.ConclusionsMANA-specific TIL demonstrated a distinct gene signature that enabled us to identify de novo antitumor TIL in NSCLC and melanoma. MANAscore may serve as a useful tool in facilitating mechanistic studies of ICI response and resistance.Trial RegistrationNCT01970358,NCT02259621ReferencesSimoni, Yannick, et al. “Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates.” Nature 557.7706 (2018):575–579.Caushi, Justina X, et al. “Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.” Nature (2021):1–7.Oliveira, Giacomo, et al. “Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.” Nature (2021):1–7.Ethics ApprovalThe melanoma clinical trial was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB) (NCT01970358). The NSCLC clinical trial was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU) and Memorial Sloan Kettering Cancer Center (NCT02259621)ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal
Despite the considerable progress in understanding the molecular bases of acute myeloid leukemia (AML), new tools to link disease biology to the unpredictable patient clinical course are still ...needed. Herein, high-throughput metabolomics, combined with the other "-omics" disciplines, holds promise in identifying disease-specific and clinically relevant features.In this study, we took advantage of nuclear magnetic resonance (NMR) to trace AML-associated metabolic trajectory employing two complementary strategies. On the one hand, we performed a prospective observational clinical trial to identify metabolic changes associated with blast clearance during the first two cycles of intensive chemotherapy in nine adult patients. On the other hand, to reduce the intrinsic variability associated with human samples and AML genetic heterogeneity, we analyzed the metabolic changes in the plasma of immunocompromised mice upon engraftment of primary human AML blasts.Combining the two longitudinal approaches, we narrowed our screen to seven common metabolites, for which we observed a mirror-like trajectory in mice and humans, tracing AML progression and remission, respectively. We interpreted this set of metabolites as a dynamic fingerprint of AML evolution.Overall, these NMR-based metabolomic data, to be consolidated in larger cohorts and integrated in more comprehensive system biology approaches, hold promise for providing valuable and non-redundant information on the systemic effects of leukemia.
The use of posttransplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (HSCT), allowing safe ...infusion of unmanipulated T cell–replete grafts. PT-Cy selectively eliminates proliferating alloreactive T cells, but whether and how it affects natural killer (NK) cells and their alloreactivity is largely unknown. Here we characterized NK cell dynamics in 17 patients who received unmanipulated haploidentical grafts, containing high numbers of mature NK cells, according to PT-Cy–based protocols in 2 independent centers. In both series, we documented robust proliferation of donor-derived NK cells immediately after HSCT. After infusion of Cy, a marked reduction of proliferating NK cells was evident, suggesting selective purging of dividing cells. Supporting this hypothesis, proliferating NK cells did not express aldehyde dehydrogenase and were killed by Cy in vitro. After ablation of mature NK cells, starting from day 15 after HSCT and favored by the high levels of interleukin-15 present in patients' sera, immature NK cells (CD62L+NKG2A+KIR−) became highly prevalent, possibly directly stemming from infused hematopoietic stem cells. Importantly, also putatively alloreactive single KIR+ NK cells were eliminated by PT-Cy and were thus decreased in numbers and antileukemic potential at day 30 after HSCT. As a consequence, in an extended series of 99 haplo-HSCT with PT-Cy, we found no significant difference in progression-free survival between patients with or without predicted NK alloreactivity (42% vs 52% at 1 year, P = NS). Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are lost upon PT-Cy administration, blunting NK cell alloreactivity in this transplantation setting.
•Posttransplantation cyclophosphamide eliminates most mature donor NK cells infused with the graft, including alloreactive NK cells.•High levels of serum interleukin-15 early after HSCT provide a favorable environment for adoptive infusion of mature donor NK cells.
Long-living memory stem T cells (TSCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. ...Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8+ TSCM lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L+CCR7+CD45RA+CD45R0+IL-7Rα+CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, TSCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived TSCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified TSCM are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.
•Identification of the biologic requirements for memory stem T cell (TSCM) generation and expansion from naive precursors ex vivo.•Differentiation, expansion, and genetic manipulation of human TSCM for cancer adoptive cellular therapy.
Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses
; however, the relationship between phenotypic characteristics and TCR ...properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8
T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.
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