Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

Analysis of the T-cell repertoire and transcriptome identifies mechanisms of regulatory T-cell suppression of GVHD.

CD4+FOXP3+ regulatory T cells (Tregs) have demonstrated efficacy in the prevention and treatment of graft-versus-host disease (GVHD). Preclinical and clinical studies indicate that Tregs are able to protect from GVHD without interfering with the graft-versus-tumor (GVT) effect of hematopoietic cell transplantation (HCT), although the underlying molecular mechanisms are largely unknown. To elucidate Treg suppressive function during in vivo suppression of acute GVHD, we performed paired T-cell receptor (TCRα and ΤCRß genes) repertoire sequencing and RNA sequencing analysis on conventional T cells (Tcons) and Tregs before and after transplantation in a major histocompatibility complex -mismatched mouse model of HCT. We show that both Tregs and Tcons underwent clonal restriction, and Tregs did not interfere with the activation of alloreactive Tcon clones and the breadth of their TCR repertoire but markedly suppressed their expansion. Transcriptomic analysis revealed that Tregs predominantly affected the transcriptome of CD4 Tcons and, to a lesser extent, that of CD8 Tcons, thus modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes, inducing a switch from glycolysis to oxidative phosphorylation. Finally, Tregs did not interfere with the induction of gene sets involved in the GVT effect. Our results shed light onto the mechanisms of acute GVHD suppression by Tregs and will support the clinical translation of this immunoregulatory approach.

Prevention of acute GVHD using an orthogonal IL-2/IL-2Rß system to selectively expand regulatory T cells in vivo.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow (BM) failure syndromes. Graft-versus-host disease (GVHD) remains a leading cause of morbidity posttransplant. Regulatory T cell (Treg) therapies are efficacious in ameliorating GVHD but limited by variable suppressive capacities and the need for a high therapeutic dose. Here, we sought to expand Treg in vivo by expressing an orthogonal interleukin 2 receptor ß (oIL-2Rß) that would selectively interact with oIL-2 cytokine and not wild-type (WT) IL-2. To test whether the orthogonal system would preferentially drive donor Treg expansion, we used a murine major histocompatibility complex-disparate GVHD model of lethally irradiated BALB/c mice given T cell-depleted BM from C57BL/6 (B6) mice alone or together with B6Foxp3+GFP+ Treg or oIL-2Rß-transduced Treg at low cell numbers that typically do not control GVHD with WT Treg. On day 2, B6 activated T cells (Tcons) were injected to induce GVHD. Recipients were treated with phosphate-buffered saline (PBS) or oIL-2 daily for 14 days, then 3 times weekly for an additional 14 days. Mice treated with oIL-2Rß Treg and oIL-2 compared with those treated with PBS had enhanced GVHD survival, in vivo selective expansion of Tregs, and greater suppression of Tcon expansion in secondary lymphoid organs and intestines. Importantly, oIL-2Rß Treg maintained graft-versus-tumor (GVT) responses in 2 distinct tumor models (A20 and MLL-AF9). These data demonstrate a novel approach to enhance the efficacy of Treg therapy in allo-HSCT using an oIL-2/oIL-2Rß system that allows for selective in vivo expansion of Treg leading to GVHD protection and GVT maintenance.

How to integrate CD19 specific chimeric antigen receptor T cells with other CD19 targeting agents in diffuse large B-cell lymphoma?

About one third of patients with diffuse large B-cell lymphoma (DLBCL) have a relapsing/refractory (R/R) disease after first line chemo-immunotherapy, with particularly poor outcomes observed in patients with primary refractory disease and early relapse. CD19 specific chimeric antigen receptor (CAR) T cell therapy is a game changer that results in durable and complete response rates in almost half of the patients with R/R DLBCL. Other emerging CD19-targeting therapies include monoclonal antibodies, bispecific antibodies and targeting antibody-drug conjugates, which also show encouraging results. However, the timing and sequencing of different anti-CD19-targeting agents and how they might interfere with subsequent CAR T cell treatment is still unclear. In this review, we summarize the results of the pivotal clinical trials as well as evidence from real-world series of the use of different CD19-targeting approved agents. We discuss the effect of various therapies on CD19 expression and its implications for treatment sequencing.

Invariant natural killer T-cell subsets have diverse graft-versus-host-disease-preventing and antitumor effects.

Invariant natural killer T (iNKT) cells are a T-cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic graft-versus-host-disease (GVHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2, and iNKT17 sublineages, which differ transcriptomically and epigenomically and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GVHD is currently unknown. In this work, we generated highly purified murine iNKT sublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We show that iNKT2 and iNKT17, but not iNKT1, cells efficiently suppress T-cell activation in vitro and mitigate murine acute GVHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we report for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of iNKT cell therapies to the clinic for cancer immunotherapy as well as for the prevention and treatment of GVHD.

IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor.

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rß during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rß(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.

NK Cell Functional Impairment after Allogeneic Hematopoietic Stem Cell Transplantation Is Associated with Reduced Levels of T-bet and Eomesodermin.

NK cells play a major role in protection against tumor recurrence and infection after allogeneic hematopoietic stem cell transplantation (HSCT). It has been shown that NK cell function after HSCT is impaired, but underlying molecular mechanisms are not well-known. In this report we show that the level of T-bet and Eomesodermin (Eomes), two T-box transcription factors regulating lymphocyte effector functions, is strongly reduced in NK cells from HSCT recipients compared with healthy control subjects. Reduction of T-bet and Eomes expression appeared early and persisted for years after HSCT, affecting all peripheral blood NK cells independently of their differentiation status. Reduced T-bet levels in NK cells from allogeneic HSCT recipients significantly correlated with reduced perforin expression. Acute, but not chronic, graft-versus-host disease, as well as CMV reactivation, was associated with further downregulation of T-bet expression in NK cells. Lower levels of T-bet expression in NK cells were associated with less favorable outcome after HSCT as a result of increased nonrelapse mortality. Collectively, our results provide a possible molecular explanation for the previously reported functional exhaustion of NK cells after allogeneic HSCT and suggest an impact of the NK transcriptional machinery status on HSCT outcome.

[Reactive hemophagocytic syndrome]. / Syndrome hémophagocytaire réactionnel.

The reactive hemophagocytic syndrome comes from an overstimulation of the immune system which causes a cytokine storm. This is a life-threatening condition caracterised by a febrile cytopenia, hepatosplenomegaly and multi-organ failure. The diagnosis is not easy and the HScore can be useful, looking at hyperferritinemia, hypertriglyceridemia, and hypofibrinogenemia. The evidence of hemophagocytosis in the bone marrow is not necessary nor sufficient to make the diagnosis but is part of the workup. The underlying cause has to be actively sought, typically an infectious, malignant or autoimmune disorder. This syndrome should be supported in conjunction with the hematologist, and initiation of a treatment is a medical emergency.

Le syndrome hémophagocytaire réactionnel est la conséquence d'une activation incontrôlée du système immunitaire, responsable d'une tempête cytokinique. La présentation clinique est celle d'une cytopénie fébrile avec hépato-splénomégalie et atteinte multiviscérale pouvant rapidement engager le pronostic vital. Le diagnostic est difficile et peut être facilité par le HScore, comprenant la recherche d'une hyperferritinémie, d'une hypertriglycéridémie et d'une hypofibrinogénémie. La mise en évidence d'hémophagocytose médullaire n'est ni nécessaire ni suffisante pour poser le diagnostic mais peut le conforter. L'étiologie sous-jacente, généralement infectieuse, néoplasique et/ou auto-immune est à rechercher. Ce syndrome doit être pris en charge conjointement avec l'hématologue, et l'initiation d'un traitement est une urgence médicale.

High eomesodermin expression among CD57+ CD8+ T cells identifies a CD8+ T cell subset associated with viral control during chronic human immunodeficiency virus infection.

During HIV infection, increased CD57 expression among CD8(+) T cells has been associated with immune senescence and defective immune responses. Interestingly, CD57-expressing CD8(+) T cells exhibit a dual profile, being simultaneously highly cytotoxic (terminally differentiated effectors) and poorly proliferative (replicative senescent). Recent publications point toward a positive role of CD57-expressing CD8(+) T cell subsets, presumably due to their high cytolytic activity. We further investigated the phenotype of CD57-expressing CD8(+) T cells in healthy donors and during HIV infection combining CD57 expression to Eomesodermin (EOMES), a T box transcription factor which determines, coordinately with T-bet, effector and memory CD8(+) T cell differentiation. We defined in healthy donors two functionally distinct CD57-expressing CD8(+) T cell subsets exhibiting different levels of EOMES expression: EOMES(hi) CD57(+) and EOMES(int) CD57(+) CD8(+) T cells. EOMES(hi) CD57(+) cells exhibited low cytotoxic activity but preserved proliferative capacity and interleukin 7 (IL-7) receptor expression, whereas EOMES(int) CD57(+) cells exhibited obvious cytotoxic functions and a more terminally differentiated phenotype. We next performed a similar analysis in different contexts of HIV infection: primary infected patients, long-term viremic patients, aviremic patients treated with antiretroviral therapy, and HIV controllers; we demonstrated a higher percentage of CD57-expressing cells in all HIV-infected patients regardless of virological status. When heterogeneity in EOMES expression among CD57 cells was taken into account, we detected significantly higher proportions of EOMES(hi) CD57(+) cells among HIV-specific and nonspecific CD8(+) T cells from HIV controllers than in aviremic antiretroviral-treated patients and viremic patients. Importantly, such a peculiar non-terminally differentiated EOMES(hi) CD57(+) phenotypic profile was associated with viral control. Importance: This study demonstrates that functional heterogeneity exists among CD57-expressing CD8 T cells, which include both terminally differentiated, highly cytotoxic EOMES(int) CD57(+) CD8(+) T cells and less differentiated EOMES(hi) CD57(+) CD8 T cells, which do not exhibit immediate cytotoxic functions but present high proliferative capacity. Interestingly, HIV controllers present a high proportion of EOMES(hi) CD57 cells among CD57-expressing HIV-specific CD8 T cells compared to both long-term viremic and aviremic antiretroviral therapy (ART)-treated patients, suggesting a beneficial role for this cell subset in viral control.

Granulocyte-Macrophage Colony-Stimulating Factor in Allogenic Hematopoietic Stem Cell Transplantation: From Graft-versus-Host Disease to the Graft-versus-Tumor Effect.

Allogenic hematopoietic stem cell transplantation (allo-HSCT) is a widely used treatment for a broad range of hematologic malignancies because of its graft-versus-tumor (GVT) effect. Unfortunately, allo-HSCT is still associated with morbidity and mortality related to relapse and transplantation complications, namely graft-versus-host-disease (GVHD). In an era of therapies specifically targeting molecular pathways, transcription factors, and cytokines, a better understanding of GVHD physiopathology is essential for the development of new therapeutic approaches. In this review, we outline the current knowledge of the role of granulocyte- macrophage colony-stimulating factor (GM-CSF) in allo-HSCT. We first discuss the biology of GM-CSF and its signaling pathways, with a focus on the main producing cells, T cells. We discuss recent preclinical studies pointing to a pivotal role of GM-CSF in GVHD, in particular gastrointestinal GVHD. We then summarize the potential role of GM-CSF in the GVT effect, discussing some potential strategies for exploiting GM-CSF in the context of allo-HSCT.

Molecular Imaging of Acute Graft-Versus-Host Disease.

Noninvasive molecular imaging of acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation has great potential to detect GvHD at the early stages, aid in grading of the disease, monitor treatment response, and guide therapeutic decisions. Although the specificity of currently available tracers appears insufficient for clinical GvHD diagnosis, recently, several preclinical studies have identified promising new imaging agents targeting one or more biologic processes involved in GvHD pathogenesis, ranging from T-cell activation to tissue damage. In this review, we summarize the different approaches reported to date for noninvasive detection of GvHD using molecular imaging with a specific focus on the use of PET. We discuss possible applications of molecular imaging for the detection of GvHD in the clinical setting, as well as some of the predictable challenges that are faced during clinical translation of these approaches.

Severe anaphylaxis after chimeric antigen receptor T-cell injection: a case report.

Anaphylactic reactions at the time of chimeric antigen receptor T (CAR-T) cell infusion are adverse events that have not been reported in pivotal clinical trials or in real-world series. We report the case of patient with severe anaphylaxis with cardiac arrest after tisagenlecleucel injection for Diffuse Large B cell Lymphoma, who recovered after resuscitation and intensive care treatment; we also conducted a Food and Drug Administration Adverse Event Reporting System database analysis and found several cases of severe anaphlyaxis after CAR-T cell injection. Although not reported in pivotal CAR-T cell studies, anaphylaxis can occur after CAR-T cell injection, highlighting the need to include anaphylaxis as a possible side effect in future studies.

Remission of relapsed/refractory classical Hodgkin lymphoma induced by brentuximab vedotin and pembrolizumab combination after allogeneic hematopoietic stem cell transplantation: a case report.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with highly chemorefractory Hodgkin lymphoma (HL). The CD30-targeting antibody-drug conjugate Brentuximab-Vedotin (BV) and programmed cell death protein-1 (PD-1) blocking agents have demonstrated clinical activity with durable responses in relapsed/refractory (r/r) HL. However, patients with a history of allo-HSCT were frequently excluded from clinical trials due to concerns about the risk of graft-versus-host disease (GVHD). We report the clinical history of a patient with refractory classical HL who underwent two allo-HSCTs (first from matched unrelated and second from haploidentical donor) after relapsing on BV and nivolumab and for whom durable remission was finally obtained using BV-pembrolizumab combination for relapse after haploidentical HSCT. Such treatment was associated with the onset of GVHD after only two cycles which led to treatment discontinuation. However, the side effects were rapidly controlled, and after 2 years of follow-up, the patient is still in remission. Our data support the feasibility and efficacy of combining PD-1 blockade with BV to enhance the graft-versus-lymphoma effect after allo-HSCT.

Single-cell transcriptomics reveal different maturation stages and sublineage commitment of human thymic invariant natural killer T cells.

Invariant natural killer T cells are a rare, heterogeneous T-cell subset with cytotoxic and immunomodulatory properties. During thymic development, murine invariant natural killer T cells go through different maturation stages differentiating into distinct sublineages, namely, invariant natural killer T1, 2, and 17 cells. Recent reports indicate that invariant natural killer T2 cells display immature properties and give rise to other subsets, whereas invariant natural killer T1 cells seem to be terminally differentiated. Whether human invariant natural killer T cells follow a similar differentiation model is still unknown. To define the maturation stages and assess the sublineage commitment of human invariant natural killer T cells during thymic development, in this study, we performed single-cell RNA sequencing analysis on human Vα24+Vß11+ invariant natural killer T cells isolated from thymocytes. We show that these invariant natural killer T cells displayed heterogeneity, and our unsupervised analysis identified 5 clusters representing different maturation stages, from an immature profile with high expression of genes important for invariant natural killer T cell development and proliferation to a mature, fully differentiated profile with high levels of cytotoxic effector molecules. Evaluation of expression of sublineage-defining gene sets revealed mainly cells with an invariant natural killer T2 signature in the most immature cluster, whereas the more differentiated ones displayed an invariant natural killer T1 signature. Combined analysis with a publicly available single-cell RNA sequencing data set of human invariant natural killer T cells from peripheral blood suggested that the 2 main subsets exist both in thymus and in the periphery, while a third more immature one was restricted to the thymus. Our data point to the existence of different maturation stages of human thymic invariant natural killer T cells and provide evidence for sublineage commitment of invariant natural killer T cells in the human thymus.

Donor lymphocyte infusion after allogeneic haematopoietic cell transplantation for haematological malignancies: basic considerations and best practice recommendations from the EBMT.

Since the early description of three patients with relapsed leukaemia after allogeneic haematopoietic cell transplantation (HCT) who obtained complete remission after donor lymphocyte infusions (DLIs), the added value of this procedure to induce or maintain graft-versus-leukaemia immunity has been undisputed. For more than 30 years, DLIs have become common practice as prophylactic, pre-emptive, or therapeutic immunotherapy. However, as with many aspects of allogeneic HCT, centres have developed their own routines and practices, and many questions related to the optimal applications and toxicity, or to the immunobiology of DLI induced tumour-immunity, remain. As a part of the Practice Harmonization and Guidelines Committee and the Cellular Therapy and Immunobiology Working Party of the European Society for Blood and Marrow Transplantation effort, a panel of experts with clinical and translational knowledge in transplantation immunology and cellular therapy met during a 2-day workshop in September, 2023, in Lille, France, and developed a set of consensus-based recommendations for the application of unmanipulated DLI after allogeneic HCT for haematological malignancies. Given the absence of prospective data in the majority of publications, these recommendations are mostly based on retrospective studies and expert consensus.

Targeting TGFß-activated kinase-1 activation in microglia reduces CAR T immune effector cell-associated neurotoxicity syndrome.

Cancer immunotherapy with chimeric antigen receptor (CAR) T cells can cause immune effector cell-associated neurotoxicity syndrome (ICANS). However, the molecular mechanisms leading to ICANS are not well understood. Here we examined the role of microglia using mouse models and cohorts of individuals with ICANS. CD19-directed CAR (CAR19) T cell transfer in B cell lymphoma-bearing mice caused microglia activation and neurocognitive deficits. The TGFß-activated kinase-1 (TAK1)-NF-κB-p38 MAPK pathway was activated in microglia after CAR19 T cell transfer. Pharmacological TAK1 inhibition or genetic Tak1 deletion in microglia using Cx3cr1CreER:Tak1fl/fl mice resulted in reduced microglia activation and improved neurocognitive activity. TAK1 inhibition allowed for potent CAR19-induced antilymphoma effects. Individuals with ICANS exhibited microglia activation in vivo when studied by translocator protein positron emission tomography, and imaging mass cytometry revealed a shift from resting to activated microglia. In summary, we prove a role for microglia in ICANS pathophysiology, identify the TAK1-NF-κB-p38 MAPK axis as a pathogenic signaling pathway and provide a rationale to test TAK1 inhibition in a clinical trial for ICANS prevention after CAR19 T cell-based cancer immunotherapy.

Early and long-lasting alteration of effector CD45RA(-)Foxp3(high) regulatory T-cell homeostasis during HIV infection.

Regulatory T-cell (Treg) quantification in human immunodeficiency virus (HIV) infection remains ill defined because of the lack of reliable specific markers to identify human Tregs and the diversity of clinical stages of HIV infection. Using a recently described Treg identification strategy based on CD45RA and Foxp3 expression, we performed an extensive quantification of total, naive (CD45RA(+)Foxp3(low)), and effector (CD45RA(-)Foxp3(high)) Tregs in different contexts of HIV infection: primary HIV infection, long-term viremic patients, aviremic patients treated with highly active antiretroviral therapy, and HIV controllers. We showed that although total Treg percentages were mildly affected by HIV infection, Treg absolute numbers were significantly reduced in all groups studied. We demonstrated that although naive Treg numbers were essentially preserved, effector Tregs were consistently affected during HIV infection. Finally, we demonstrated that effector but not total or naive Treg numbers were negatively correlated with the magnitude of HIV-specific CD8 T-cell responses.