LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma.

Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.

Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

T Cell Recruitment to the Intestinal Stem Cell Compartment Drives Immune-Mediated Intestinal Damage after Allogeneic Transplantation.

The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage.

PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

Blockade of CCR5 and CXCR3 attenuates murine acute graft-versus-host disease through modulating donor-derived T-cell distribution and function.

Lymphocyte trafficking via chemokine receptors such as C-C chemokine receptor 5 (CCR5) and CXCR3 plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Our previous studies showed that the addition of CCR5 or CXCR3 antagonists could only slightly alleviate the development of aGVHD. Given the specificity of T lymphocytes bearing CXCR3 and CCR5, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD. A mouse model of aGVHD was established to assess the efficacy of CCR5 and/or CXCR3 blockade on the development of aGVHD. The distribution of lymphocytes was calculated by quantification of immunostaining cells. The immunomodulatory effect on T cells was assessed by evaluating T-cell proliferation, viability, and differentiation. Using the murine allogeneic hematopoietic stem cell transplantation model, we demonstrated that blockade of both CCR5 and CXCR3 could efficiently alleviate the development of aGVHD. Further investigation on the immune mechanisms for this prophylactic effect showed that more T cells were detained into secondary lymphoid organs (SLOs), which may lead to reduced infiltration of T cells into GVHD target organs. Our study also showed that T cells detained in SLOs dampened the activation, suppressed the polarization toward T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells, and induced the production of Treg cells. These data suggest that concurrent blockade of CCR5 and CXCR3 attenuates murine aGVHD through modulating donor-derived T-cell distribution and function, and this might be applicable for aGVHD prophylaxis in clinical settings.

Allogeneic hematopoietic stem cell transplantation: lessons learned by a molecular immunologist/transplant patient.

Much has been learned about the genes and pathways that contribute to a diverse array of hematopoietic malignancies and other hematopoietic diseases. However, for many of these diseases, an allogeneic hematopoietic stem cell (HSC) transplant remains the preferred treatment option. This opinion article provides the perspective of a molecular immunologist who became a transplant patient after many years studying basic mechanisms of blood cell development. Among many lessons learned were the magnitude of racial and ethnic disparities in donor registries, the substantial improvement in outcomes over time that were due to the collective impact of numerous advances, the benefits and limitations of genetic and clinical data, and the remarkably intricate balance between promoting graft-versus-disease activity of donor cells while suppressing graft-versus-host disease (GVHD).

Effect of allogeneic hematopoietic stem cell transplantation for chronic granulomatous disease in children: A multicentre, retrospective cohort study in China.

Chronic granulomatous disease (CGD) in children is a rare primary immunodeficiency disorder that can lead to life-threatening infections and inflammatory complications. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is increasingly being used to treat severe CGD in children. We conducted a multicenter retrospective analysis of children with CGD who were treated with allo-HSCT at four pediatric hematopoietic stem cell transplant centers in China from September 2005 to December 2019. The study included a total of 171 patients (169 males and 2 females). The median age at the time of transplantation was 6.1 (0-16.4) years. Among them, 154 patients had X-linked recessive inheritance caused by CYBB gene mutations, 12 patients were autosomal recessive, 1 patient had DNAH11 and HYDIN gene mutations, and 4 patients had no gene mutations. The median follow-up period was 36.3 (1.9-79) months. All participating patients were applied to myeloablative conditioning (MAC) regimens. The rates of OS, EFS, and GEFS within three years were 87.5%, 85.3%, and 75.2%, respectively. The total graft failure and the total mortality rate were 5.3% and 11.1%. The cumulative incidence of acute GVHD was 53.8% and the incidence of chronic GVHD was 12.9%, The incidence of chronic GVHD was higher for patients who received unrelated donor cord blood stem cell transplantation (UD-CB) (P = 0.001). Chronic GVHD and coinfections are the risk factors for OS and EFS in patients with CGD after receiving allo-HSCT. UD-CB is a risk factor for EFS and the presence of pneumonia before transplantation is a risk factor for OS. In conclusion, through this study, we have demonstrated that allo-HSCT has excellent efficacy in the treatment of CGD in children, especially, RD-haplo is associated with a lower rate of graft failure incidence and mortality than the treatment modalities of other donor type. Therefore, allo-HSCT is strongly recommended when a well-matched donor is available. If a well-matched donor is not available, the HLA-mismatched donor should be carefully evaluated, and the conditioning regimen modified accordingly.

Low- versus standard-dose post-transplant cyclophosphamide as GVHD prophylaxis for haploidentical transplantation.

Haploidentical haematopoietic cell transplantation (haplo-HCT) using post-transplant cyclophosphamide (PTCY) as graft-versus-host disease (GVHD) prophylaxis is the standard of care for various haematological malignancies. The original PTCY dose after haplo-HCT was 100 mg/kg, but no dose-finding studies have been performed to identify the optimal dose. We performed a retrospective analysis to compare standard-dose PTCY (100 mg/kg) with reduced-dose PTCY (80 mg/kg): 969 in the standard-dose group and 538 in the reduced-dose group. As there was a significant difference between the two groups regarding patient and transplant characteristics, we performed propensity score (PS) matching. After PS matching, 425 patients in each group were included. The probabilities of 2-year OS were 55.9% in the standard-dose group and 47.0% in the reduced-dose group (p = 0.36). The cumulative incidences of 2-year non-relapse mortality were 21.3% in the standard-dose group and 20.5% in the reduced-dose group (p = 0.55). There was no significant difference in the incidence of acute (grade II-IV 29.2% [95% CI, 24.9-33.6] vs. 25.3% [95% CI, 21.3-29.6]; grade III-IV 7.3% [95% CI, 5.1-10.0] vs. 6.6% [95% CI, 4.5-9.3]) or chronic GVHD. In conclusion, reduced- and standard-dose PTCY were comparable in terms of major clinical outcomes.

HLA peptide-binding pocket diversity modulates immunological complications after cord blood transplant in acute leukaemia.

Pocket motifs and their amino acid positions of HLA molecules are known to govern antigen presentation to effector cells. Our objective was to analyse their influence on the risk of graft-versus-host disease (GVHD) and relapse after umbilical cord blood transplant (UCBT). The transplant characteristics of 849 patients with acute leukaemia were obtained from the Eurocord/EBMT database. Higher acute (a) GVHD was associated with homozygosity of UCB HLA-C amino acid positions 77 and 80 (NN/KK) (p = 0.008). Severe aGVHD was associated with HLA-A pocket B YSAVMENVHY motif (p = 0.002) and NN and RR genotypes of the HLA-C amino acid positions 77 and 156 (p = 0.006 and p = 0.002). Such risk was also increased in case of recipient and UCB mismatches in P4 (p < 0.0001) and P9 (p = 0.003) pockets of HLA-DQB1 alleles. For chronic GVHD, the pocket B YYAVMEISNY motif of the HLA-B*15:01 allele and the absence of mismatch between recipient and UCB in the P6 pocket of HLA-DRB1 were associated with a lower risk (p = 0.0007 and p = 0.0004). In relapse, both UCB pocket B YFAVMENVHY belonging to HLA-A*32:01 and recipient pocket B YDSVGENYQY motif of the HLA-C*07:01 allele were associated with higher risk (p = 0.0026 and p = 0.015). We provide clues on HLA-mediated cellular interactions and their role in the development of GVHD and relapse.

Increased donor inhibitory KIR are associated with reduced GVHD and improved survival following HLA-matched unrelated donor HCT in paediatric acute leukaemia.

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated effects after haematopoietic stem cell transplantation (HCT). Previous work has shown that accounting for known KIR-KIRL interactions may identify donors with optimal NK cell-mediated alloreactivity in the adult transplant setting. Paediatric acute leukaemia patients were retrospectively analysed, and KIR-KIRL combinations and maximal inhibitory KIR ligand (IM-KIR) scores were determined. Clinical outcomes were examined using a series of graphs depicting clinical events and endpoints. The graph methodology demonstrated that prognostic variables significant in the occurrence of specific clinical endpoints remained significant for relevant downstream events. KIR-KIRL combinations were significantly predictive for reduced grade 3-4 aGVHD likelihood, in patients transplanted with increased inhibitory KIR gene content and IM-KIR = 5 scores. Improvements were also observed in associated outcomes for both ALL and AML patients, including relapse-free survival, GRFS and overall survival. This study demonstrates that NK cell KIR HLA interactions may be relevant to the paediatric acute leukaemia transplant setting. Reduction in aGVHD suggests KIR effects may extend beyond NK cells. Moving forward clinical trials utilizing donors with a higher iKIR should be considered for URD HCT in paediatric recipients with acute leukaemia to optimize clinical outcomes.

Mini-dose methotrexate combined with methylprednisolone for the initial treatment of acute GVHD: a multicentre, randomized trial.

BACKGROUND: There is an urgent unmet need for effective initial treatment for acute graft-versus-host disease (aGVHD) adding to the standard first-line therapy with corticosteroids after allogeneic haematopoietic stem cell transplantation (allo-HSCT). METHODS: We performed a multicentre, open-label, randomized, phase 3 study. Eligible patients (aged 15 years or older, had received allo-HSCT for a haematological malignancy, developed aGVHD, and received no previous therapies for aGVHD) were randomly assigned (1:1) to receive either 5 mg/m2 MTX on Days 1, 3, or 8 and then combined with corticosteroids or corticosteroids alone weekly. RESULTS: The primary endpoint was the overall response rate (ORR) on Day 10. A total of 157 patients were randomly assigned to receive either MTX plus corticosteroids (n = 78; MTX group) or corticosteroids alone (n = 79; control group). The Day 10 ORR was 97% for the MTX group and 81% for the control group (p = .005). Among patients with mild aGVHD, the Day 10 ORR was 100% for the MTX group and 86% for the control group (p = .001). The 1-year estimated failure-free survival was 69% for the MTX group and 41% for the control group (p = .002). There were no differences in treatment-related adverse events between the two groups. CONCLUSIONS: In conclusion, mini-dose MTX combined with corticosteroids can significantly improve the ORR in patients with aGVHD and is well tolerated, although it did not achieve the prespecified 20% improvement with the addition of MTX. TRIAL REGISTRATION: The trial was registered with clinicaltrials.gov (NCT04960644).

Immunological dynamic characteristics in acute myeloid leukemia predict the long-term outcomes and graft-versus host-disease occurrences post-transplantation.

To investigate the relationship between immune dynamic and graft-versus-host-disease (GVHD) risk, 111 initial diagnostic acute myeloid leukemia patients were reviewed. The flow cytometry data of 12 major lymphocyte subsets in bone marrow (BM) from 60 transplant patients at four different time points were analyzed. Additionally, 90 immune subsets in peripheral blood (PB) of 11 post-transplantation on day 100 were reviewed. Our results demonstrated that transplant patients had longer OS compared to non-transplant patients (P < 0.001). Among transplant patients, those who developed GVHD showed longer OS than those without GVHD (P < 0.05). URD donors and CMV-negative status donors were associated with improved OS in transplant patients (P < 0.05). Importantly, we observed a decreased Th/Tc ratio in BM at initial diagnostic in patients with GVHD compared to those without GVHD (P = 0.034). Receiver operating characteristic analysis indicated that a low Th/Tc ratio predicted an increased risk of GVHD with a sensitivity of 44.44% and specificity of 87.50%. Moreover, an increased T/NK ratio in BM of post-induction chemotherapy was found to be associated with GVHD, with a sensitivity of 75.76% and specificity of 65.22%. Additionally, we observed a decreased percentage of NK1 (CD56-CD16+NK) in PB on day 100 post-transplantation in the GVHD group (P < 0.05). These three indicators exhibit promising potential as specific and useful biomarkers for predicting GVHD. These findings provide valuable insights for the early identification and management of GVHD risk, thereby facilitating the possibility of improving patient outcomes.

The role of interleukin-2 in graft-versus-host disease pathogenesis, prevention and therapy.

Graft-versus-host disease (GVHD) is a significant complication following allogeneic hematopoietic cell transplantation (allo-HCT), posing substantial risks to patient survival. In the late follow-up phase of transplanted patients, GVHD is also a major cause of morbidity and disability, mostly due to low response to first-line steroids and the lack of effective standard therapies in the second line. This review provides a description of GVHD pathogenesis, with a focus on the central role of Interleukin-2 (IL-2). IL-2 is one of the critical mediators in the complex pathogenesis of GVHD, contributing to the intricate balance between regulatory T cells (Tregs) and effector T cells (Teffs). Due to this pivotal role, several studies investigate the potential of IL-2 as a therapeutic option for GVHD management. We discuss the outcomes of low-dose IL-2 therapies and their impact on Treg proliferation and steroid dependency reduction. Additionally, the effects of combining IL-2 with other treatments, such as extracorporeal photopheresis (ECP) and Treg-enriched lymphocyte infusions, are highlighted. Novel approaches, including modified IL-2 complexes and IL-2 receptor blockade, are explored for their potential in selectively enhancing Treg function and limiting Teff activation. The evolving understanding of IL-2's pivotal role in immune regulation presents promising prospects for applying treatment and prevention strategies for GVHD.

Post-transplantation cyclophosphamide and cyclosporine A versus methotrexate and cyclosporine A for graft-versus-host disease prophylaxis after allogeneic peripheral stem cell transplantation in adult acute myeloid leukemia patients.

BACKGROUND: Different prophylactic protocols are available for preventing graft-versus-host disease (GVHD) after matched sibling donor (MSD) allogeneic hematopoietic cell transplantation (allo-HCT). This study aimed to compare the effectiveness of post-transplantation cyclophosphamide plus cyclosporine A (PT-CY/CSA) versus methotrexate plus CSA (MTX/CSA) as GVHD prophylaxis protocols in adult acute myeloid leukemia (AML) patients who received peripheral blood stem cells (PBSC) from fully matched donors. METHODS: The 1-year outcomes of 89 patients treated with PT-CY/CSA and 90 patients treated with MTX/CSA who had MSD allo-HCT for AML using unmanipulated mobilized PBSC were examined and compared. RESULTS: The cumulative incidence of acute GVHD at 100 days was considerably lower in the PT-CY/CSA group (4% vs 19.3%, p = 0.002), however there were no statistically significant difference in the cumulative incidence of chronic GVHD at 1-year (19.6% vs 37.4%, p = 0.053). Significant delays in neutrophil and platelet engraftments were reported in the PT-CY/CSA group (17 vs 12 days) and (13 vs 12 days), respectively (p < 0.001). The cumulative incidences of relapse (19.1% vs 13.7%, p = 0.470), overall survival (79.1% vs 77.3%, p = 0.986), non-relapse mortality (16.5% vs 16.8%, p = 0.837), and the GVHD and relapse-free survival (GRFS) (53.7% vs 46.6%, p = 0.478) did not differ statistically at 1-year. CONCLUSION: PT-CY/CSA demonstrated a significant decrease in the rate of acute GVHD. However, it was associated with engraftment delay.

Recent updates on allogeneic CAR-T cells in hematological malignancies.

CAR-T cell therapy is known as an effective therapy in patients with hematological malignancies. Since 2017, several autologous CAR-T cell (auto-CAR-T) drugs have been approved by the US Food and Drug Administration (FDA) for the treatment of some kinds of relapsed/refractory hematological malignancies. However, some patients fail to respond to these drugs due to high manufacturing time, batch-to-batch variation, poor quality and insufficient quantity of primary T cells, and their insufficient expansion and function. CAR-T cells prepared from allogeneic sources (allo-CAR-Ts) can be an alternative option to overcome these obstacles. Recently, several allo-CAR-Ts have entered into the early clinical trials. Despite their promising preclinical and clinical results, there are two main barriers, including graft-versus-host disease (GvHD) and allo-rejection that may decline the safety and efficacy of allo-CAR-Ts in the clinic. The successful development of these products depends on the starter cell source, the gene editing method, and the ability to escape immune rejection and prevent GvHD. Here, we summarize the gene editing technologies and the potential of various cell sources for developing allo-CAR-Ts and highlight their advantages for the treatment of hematological malignancies. We also describe preclinical and clinical data focusing on allo-CAR-T therapy in blood malignancies and discuss challenges and future perspectives of allo-CAR-Ts for therapeutic applications.

International Society for Cell and Gene Therapy Clinical Translation Committee recommendations on mesenchymal stromal cells in graft-versus-host disease: easy manufacturing is faced with standardizing and commercialization challenges.

Mesenchymal stromal cells (MSCs) have been used in multiple clinical trials for steroid-refractory moderate-severe (grade II-IV) acute graft-versus-host disease (aGVHD) across the world over the last two decades. Despite very promising results in a variety of trials, it failed to get widespread approval by regulatory agencies such as the U.S. Food and Drug Administration and the European Medicines Agency. What lessons can we learn from this for future studies on MSCs and other cell therapy products? Broad heterogeneity among published trials using MSCs in aGVHD was likely the core problem. We propose a standardized approach in regards to donor-related factors, MSCs-related characteristics, as well as clinical trial design, to limit heterogeneity in trials for aGVHD and to fulfill the requirements of regulatory agencies. This approach may be expanded beyond MSCs to other Cell and Gene therapy products and trials in other diseases.

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity.

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Source of hematopoietic progenitor cells determines their capacity to generate innate lymphoid cells ex vivo.

BACKGROUND AIMS: The success of allogeneic hematopoietic cell transplantation (HCT) as therapy for hematologic conditions is negatively impacted by the occurrence of graft-versus-host disease (GVHD). Tissue damage, caused, for example, by chemotherapy and radiotherapy, is a key factor in GVHD pathogenesis. Innate lymphoid cells (ILCs) are important mediators of tissue repair and homeostasis. The presence of ILCs before, and enhanced ILC reconstitution after, allogeneic HCT is associated with a reduced risk to develop mucositis and GVHD. However, ILC reconstitution after allogeneic HCT is slow and often incomplete. A way to replenish the pool of ILC relies on the differentiation of hematopoietic progenitor cells (HPCs) into ILC. METHODS: We developed an ex vivo stromal cell-containing culture system to study the capacity of HPCs to differentiate into all mature helper ILC subsets. RESULTS: ILC development depended on the source of HPCs. ILCs developed at high frequencies from umbilical cord blood- and fetal liver-derived HPC and at low frequencies when HPCs were obtained from allogeneic or autologous adult HCT grafts or healthy adult bone marrow. Although all helper ILC subsets could be generated from adult HPC sources, development of tissue protective ILC2 and NKp44+ ILC3 was notoriously difficult. CONCLUSIONS: Our data suggest that slow ILC recovery after allogeneic HCT may be related to an intrinsic incapability of adult HPC to develop into ILC.