Splice it up: Atypical transcripts to boost leukemia immunotherapy.

Neoantigens are prime targets for cancer immunotherapy, but their identification in low mutational burden malignancies remains challenging. In this issue of Immunity, Ehx et al. show that atypical transcripts, and particularly retained introns, expand the spectrum of leukemia immunotherapy targets.

Landscape of helper and regulatory antitumour CD4+ T cells in melanoma.

Within the tumour microenvironment, CD4+ T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules1,2, but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4+ T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4+ T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4+ T regulatory (TReg) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4+ TReg clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4+ T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4+ TReg cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; 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.

Mechanisms of response and resistance to combined decitabine and ipilimumab for advanced myeloid disease.

The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: dynamics and clinical implications.

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.

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

PD-L1, LAG3, and HLA-DR are increasingly expressed during smoldering myeloma progression.

Symptomatic multiple myeloma (MM) is a plasma cell neoplasm that represents the final stage of a continuum of clinical conditions that start from monoclonal gammopathy of unknown significance (MGUS), then transits in the more advance, but still asymptomatic, smoldering MM (SMM), with a final evolution in symptomatic MM. To investigate SMM microenvironment modifications, we studied 16 patients diagnosed at our hospital. Eight of them (group A) developed MM within 2 years from diagnosis while the others (group B) had stable SMM. Samples were bone marrow biopsies at diagnosis and after 2 years (± 4 months) and were analyzed by immunohistochemical analysis. Firstly, we found a significant increase in both CD4+ cells (11 vs 17%, p < 0.01) and CD8+ cells (15 vs 18%, p < 0.01) between diagnosis and at follow-up samples (whole cohort). This was associated to an increase in the CD4+/CD8+ ratio (0.74 vs 0.93, p < 0.01). Secondly, we discovered an increased expression of T cell inhibitory molecules during SMM evolution. In fact, plasma cell PD-L1 and microenvironment cell LAG3 expression increased from 1 to 12% (p = 0.03) and 4 to 10% (p = 0.04), respectively, from diagnosis to follow-up. Also, plasma cells and microenvironment cells HLA-DR expression augmented during SMM evolution from 7 to 10% (p = 0.04) and 29 to 39% (p = 0.01), respectively. When comparing group A vs group B, we found an increased CD68-KP1+ cell infiltration in favor of group B at diagnosis (23 vs 28%, p = 0.01) and a greater plasma cell infiltration at follow-up (50 vs 26%, p < 0.01). Our findings suggest how immune escape mechanisms appear earlier during multiple myeloma evolution, and that LAG3 could be a possible immunologic target in this setting.

Adoptive immunotherapy with genetically modified lymphocytes in allogeneic stem cell transplantation.

Hematopoietic stem cell transplantation from a healthy donor (allo-HSCT) represents the most potent form of cellular adoptive immunotherapy to treat malignancies. In allo-HSCT, donor T cells are double edge-swords: highly potent against residual tumor cells, but potentially highly toxic, and responsible for graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies coupled with current knowledge on cancer immunology have generated a wide range of approaches aimed at fostering the immunological response to cancer cells, while avoiding or controlling GVHD. In this review, we discuss cell and gene therapy approaches currently tested in preclinical models and in clinical trials.

Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation.

Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.

Rituximab for indolent lymphomas before and after allogeneic hematopoietic stem cell transplantation.

PURPOSE OF REVIEW: The most substantial advancement in the treatment of indolent B-cell non-Hodgkin lymphoma (NHL), since the advent of combination chemotherapy, has been the introduction of the monoclonal anti-CD20 antibody rituximab. However, the optimal schedule, timing, and duration of rituximab therapy remain controversial. RECENT FINDINGS: Since its initially reported single-agent activity in 1997, the role of rituximab has greatly expanded and it is now ubiquitously integrated in all treatment phases of indolent NHL. Yet, several questions remain to be addressed: should asymptomatic patients be treated at diagnosis with single-agent rituximab or still kept in watchful waiting, what are the optimal first-line treatments to combine with rituximab, what is the role of maintenance therapy, and is there a benefit in incorporating rituximab in autologous and allogeneic stem cell transplantation schemes for these diseases? Recent and ongoing clinical trials tackling these relevant issues will be presented and critically discussed in this article. SUMMARY: Excellent outcomes are reported with rituximab therapy in indolent NHL, both early and late in the disease course. Continued study of this most valuable therapeutic agent is warranted to set the optimal treatment approach leading to cure the majority of patients.

Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells.

Haploidentical hematopoietic stem cell transplantation (HSCT) performed using bone marrow (BM) grafts and post-transplantation cyclophosphamide (PTCy) has gained much interest for the excellent toxicity profile after both reduced-intensity and myeloablative conditioning. We investigated, in a cohort of 40 high-risk hematological patients, the feasibility of peripheral blood stem cells grafts after a treosulfan-melphalan myeloablative conditioning, followed by a PTCy and sirolimus-based graft-versus-host disease (GVHD) prophylaxis (Sir-PTCy). Donor engraftment occurred in all patients, with full donor chimerism achieved by day 30. Post-HSCT recovery of lymphocyte subsets was broad and fast, with a median time to CD4 > 200/µL of 41 days. Cumulative incidences of grade II to IV and III-IV acute GVHD were 15% and 7.5%, respectively, and were associated with a significant early increase in circulating regulatory T cells at day 15 after HSCT, with values < 5% being predictive of subsequent GVHD occurrence. The 1-year cumulative incidence of chronic GVHD was 20%. Nonrelapse mortality (NRM) at 100 days and 1 year were 12% and 17%, respectively. With a median follow-up for living patients of 15 months, the estimated 1-year overall and disease-free survival (DFS) was 56% and 48%, respectively. Outcomes were more favorable in patients who underwent transplantation in complete remission (1-year DFS 71%) versus patients who underwent transplantation with active disease (DFS, 34%; P = .01). Overall, myeloablative haploidentical HSCT with peripheral blood stem cells (PBSC) and Sir-PTCy is a feasible treatment option: the low rates of GVHD and NRM as well as the favorable immune reconstitution profile pave the way for a prospective comparative trial comparing BM and PBSC in this specific transplantation setting.

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.

Long-living memory stem T cells (T(SCM)) 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(+) T(SCM) 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, T(SCM) 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 T(SCM) 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 T(SCM) 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 T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

Integrative genotyping of cancer and immune phenotypes by long-read sequencing.

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.

Enhancing CAR-T cell functionality in a patient-specific manner.

Patient responses to autologous CD19 chimeric antigen receptor (CAR) T-cell therapies are limited by insufficient and inconsistent cellular functionality. Here, we show that controlling the precise level of stimulation during T-cell activation to accommodate individual differences in the donor cells will dictate the functional attributes of CAR-T cell products. The functionality of CAR-T cell products, consisting of a diverse set of blood samples derived from healthy donors, acute lymphoblastic leukemia (ALL), and chronic lymphocytic lymphoma (CLL) patient samples, representing a range of patient health status, is tested upon culturing on artificial antigen-presenting cell scaffolds to deliver T-cell stimulatory ligands (anti-CD3/anti-CD28) at highly defined densities. A clear relationship is observed between the dose of stimulation, the phenotype of the T-cell blood sample prior to T-cell activation, and the functionality of the resulting CAR-T cell products. We present a model, based on this dataset, that predicts the precise stimulation needed to manufacture a desired CAR-T cell product, given the input T-cell attributes in the initial blood sample. These findings demonstrate a simple approach to enhance CAR-T functionality by personalizing the level of stimulation during T-cell activation to enable flexible manufacturing of more consistent and potent CAR-T cells.

60 Years Young: The Evolving Role of Allogeneic Hematopoietic Stem Cell Transplantation in Cancer Immunotherapy.

The year 2020 marked the 30th anniversary of the Nobel Prize in Medicine awarded to E. Donnall Thomas for the development of allogeneic hematopoietic stem cell transplantation (allo-HSCT) to treat hematologic malignancies and other blood disorders. Dr. Thomas, "father of bone marrow transplantation," first developed and reported this technique in 1957, and in the ensuing decades, this seminal study has impacted fundamental work in hematology and cancer research, including advances in hematopoiesis, stem cell biology, tumor immunology, and T-cell biology. As the first example of cancer immunotherapy, understanding the mechanisms of antitumor biology associated with allo-HSCT has given rise to many of the principles used today in the development and implementation of novel transformative immunotherapies. Here we review the historical basis underpinning the development of allo-HSCT as well as advances in knowledge obtained by defining mechanisms of allo-HSCT activity. We review how these principles have been translated to novel immunotherapies currently utilized in clinical practice and describe potential future applications for allo-HSCT in cancer research and development of novel therapeutic strategies.

Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy.

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.

CD4+ Memory Stem T Cells Recognizing Citrullinated Epitopes Are Expanded in Patients With Rheumatoid Arthritis and Sensitive to Tumor Necrosis Factor Blockade.

OBJECTIVE: Memory stem T (Tscm) cells are long-lived, self-renewing T cells that play a relevant role in immunologic memory. This study was undertaken to investigate whether Tscm cells accumulate in rheumatoid arthritis (RA). METHODS: The polarization and differentiation profiles of circulating T cells were assessed by flow cytometry. Antigen-specific T cells were characterized by staining with major histocompatibility complex class II tetramers. The T cell receptor (TCR) repertoire was analyzed by high-throughput sequencing using an unbiased RNA-based approach in CD4+ T cell subpopulations sorted by fluorescence-activated cell sorting. RESULTS: We analyzed the dynamics of circulating Tscm cells (identified as CD45RA+CD62L+CD95+ T cells) by flow cytometry in 27 RA patients, 16 of whom were also studied during treatment with the anti-tumor necrosis factor (anti-TNF) agent etanercept. Age-matched healthy donors were used as controls. CD4+ Tscm cells were selectively and significantly expanded in RA patients in terms of frequency and absolute numbers, and significantly contracted upon anti-TNF treatment. Expanded CD4+ Tscm cells displayed a prevalent Th17 phenotype and a skewed TCR repertoire in RA patients, with the 10 most abundant clones representing up to 53.7% of the detected sequences. CD4+ lymphocytes specific for a citrullinated vimentin (Cit-vimentin) epitope were expanded in RA patients with active disease. Tscm cells accounted for a large fraction of Cit-vimentin-specific CD4+ cells. CONCLUSION: Our results indicate that Tscm cells, including expanded clones specific for relevant autoantigens, accumulate in RA patients not exposed to biologic agents, and might be involved in the natural history of the disease. Further analysis of Tscm cell dynamics in autoimmune disorders may have implications for the design and efficacy assessment of innovative therapies.

Real-life feasibility of salvage allogeneic transplantation in peripheral T-cell lymphomas.

Relapsed/refractory Peripheral T-cell Lymphomas are characterized by a poor prognosis, especially for patients who are not candidates for allogeneic hematopoietic stem-cell transplantation. We conducted a retrospective analysis on 73 consecutive patients affected by relapsed/refractory T-Cell lymphomas who were considered eligible for allogeneic transplant. All patients were referred at our center from 2001 to 2017. With a median follow-up of 40 months (range 9-192 months), 4-year second-line failure-free survival and overall survival were 14% (CI95%:7-24) and 34% (CI95%:22-46). Extranodal disease at relapse (HR 2.25, CI95%: 1.11-4.56, p = 0.02) and first-line failure-free survival < 12 months (HR 3.37, CI95%: 1.67-6.88, p < 0.01) had a negative prognostic impact on survival. Only 45 out of 73 patients (62%) received allogeneic transplant. For the 28 (38%) patients who did not proceed to transplant, disease progression was the main reason for ineligibility. Median survival from time of transplant was 31 months (range 4-185 months). A first-line failure-free survival < 12 months had a negative prognostic impact also for allografted patients (2-year survival 45% vs 73%, p = 0.03) identifying a very high-risk population which requires novel treatments pre and post-transplant to obtain a long-term disease control.