Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to nonpeptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

Adjuvant and immunomodulatory potential of Natural Killer T (NKT) activation by NKTT320

Invariant natural killer T-lymphocytes (iNKT) are a unique subset of immunomodulatory innate T-cells with an invariant TCRα chain recognizing glycolipids presented on the MHC class-I-like CD1d molecule. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate innate and adaptive immunity, and modulate inflammation. While iNKT activation by glycolipid agonists are being explored as an adjuvant, their use depends on CD1d-restricted antigen presentation. Here, we report the effects of iNKT activation by a novel humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. A single dose of NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple pro-inflammatory and chemotactic plasma analytes within 24 hours in cynomolgus macaques. Flow cytometry and RNA-Seq confirmed downstream effects of NKTT320 on multiple immune cell subsets. Inflammatory response, JAK/STAT and PI3K/AKT pathway genes were enriched along with upregulation of the inflammation-modulating genes CMKLR1, ARG2 and NLRP12. Finally, NKTT320 induced iNKT trafficking to adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, properties that support its future application as an immunotherapeutic and vaccine adjuvant.

Thymic iNKT single cell analyses unmask the common developmental program of mouse innate T cells

Most T lymphocytes leave the thymus as naïve cells with limited functionality. However, unique populations of innate-like T cells differentiate into functionally distinct effector subsets during their development in the thymus. Here, we profiled >10,000 differentiating thymic invariant natural killer T (iNKT) cells using single-cell RNA sequencing to produce a comprehensive transcriptional landscape that highlights their maturation, function, and fate decisions at homeostasis. Our results reveal transcriptional profiles that are broadly shared between iNKT and mucosal-associated invariant T (MAIT) cells, illustrating a common core developmental program. We further unmask a mutual requirement for Hivep3, a zinc finger transcription factor and adapter protein. Hivep3 is expressed in early precursors and regulates the post-selection proliferative burst, differentiation and functions of iNKT cells. Altogether, our results highlight the common requirements for the development of innate-like T cells with a focus on how Hivep3 impacts the maturation of these lymphocytes.

Functional alteration of innate T cells in critically ill Covid-19 patients

Covid-19 can induce lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors in Covid-19-driven ARDS are poorly understood. Here, we dynamically analyzed the biology of innate T cells, a heterogeneous class (MAIT, γδT and iNKT cells) of T lymphocytes, presenting potent anti-infective and regulatory functions. Patients presented a compartmentalized lung inflammation paralleled with a limited systemic inflammation. Circulating innate T cells of critically ill Covid-19 patients presented a profound and persistent phenotypic and functional alteration. Highly activated innate T cells were detected in airways of patients suggesting a recruitment to the inflamed site and a potential contribution in the regulation of the local inflammation. Finally, the expression of the CD69 activation marker on blood iNKT and MAIT cells at inclusion was predictive of disease severity. Thus, patients present an altered innate T cell biology that may account for the dysregulated immune response observed in Covid-19-related acute respiratory distress syndrome.

A Delayed Innate T Cell Reconstitution Is Associated with Epstein-Barr Virus Reactivation after Haploidentical Hematopoietic Stem Cell Transplantation Using Anti-Thymoglobulin and High-Dose Post-Transplant Cyclophosphamide

Background: Allogeneic hematopoietic stem cell transplantation (allo-HCT) using a haploidentical donor (haplo-HCT) using post-transplant high-dose cyclophosphamide (PTCy) is increasingly used for patients lacking a matched related or unrelated donor. We recently noticed a relatively high incidence of infectious complications, especially Epstein-Barr virus (EBV) reactivation after haplo-HCT. However, the mechanisms underlying the increased incidence of this complication in the haplo-HCT setting are unknown. We hypothesized that the use of PTCy may be associated with a deficit in innate-like effector T cells essential for preventing EBV reactivation.This study aimed to analyze immune reconstitution following haplo-HCT using peripheral blood stem cells (PBSC) grafts and to evaluate the correlations with EBV reactivation and outcomes. Patients and Methods: One hundred and twenty-three consecutive patients who underwent allo-HCT for hematological malignancies between 2013 and 2016 were included in this single-center retrospective study. All patients received G-CSF mobilized PBSC grafts and ATG 2.5-5 mg/Kg total dose. All patients received a combination of cyclosporine A and mycophenolate mofetil for GvHD prophylaxis, except for patients with a matched related donor (MRD) who received cyclosporine A alone and patients with an haploidentical donor who received PTCy (50mg/kg/d at d3+/-d5). α/β T cells (CD3+, CD4+ and CD8+), γ/δ T cells (pan γ/δ and Vδ2+), mucosal-associated T cells wich express a highly restricted TCR comprising a semi-variant Vα7.2-Jα33 (MAIT), invariant NK T cells, NK cells, B cells, Tregs, monocytes subsets and dendritic cells (myeloid DC, plasmacytoid DC and Slan-DC) were analyzed by multi-color flow cytometry at months (M) 1, 3, 6 and 12 following allo-HCT. Clinical data [acute GvHD (aGvHD), relapse incidence, chronic GvHD (cGvHD), viral reactivations, bacterial and fungal infections, non-relapse mortality (NRM), progression-free survival (PFS), refined GvHD-free and progression-free survival (GPFS), overall survival (OS)] were assessed together with sequential quantitative evaluation of immune subsets. Results: Median age was 55 (range, 17-70) years, with 32 male patients (26%) receiving a graft from a female donor. Diagnoses were myeloid malignancies (66%) or lymphoid malignancies (34%). Thirty-three patients (27%) received a graft from a MRD, 65 from an unrelated donor (MUD, 53%), and 25 from a haplo-identical donor (20%). Thirty patients (24%) with refractory disease received a sequential conditioning regimen while the remaining (n=93, 76%) received a RIC/RTC regimen based on fludarabine, busulfan +/- thiotepa. At d180, the cumulative incidences (CIs) of grade II-IV and grade III-IV aGvHD were 34% and 5%, respectively. The 2 years CIs of cGvHD, extensive cGvHD and relapse were 23%, 8% and 17%, respectively. At 2 years, NRM was 7%, PFS was 77%, GPFS was 66% and OS was 83%. The rate of EBV reactivation was significantly increased in haplo-HCT recipients as compared to fully-matched donor recipients (respectively, 68% versus 26%, P< .001). At one month after allo-HCT, the median counts of all immune cells subsets (except monocytes) was significantly lower in haplo-HCT recipients as compared to MRD or MUD recipients. At 3 months, α/β T cells, iNK T cells, NK cells, B cells, Tregs and Slan-DC reached similar median counts in haplo-HCT recipients as compared to MRD or MUD recipients. In contrast, Vδ2+ T cells and MAIT cells median counts remained significantly lower at 3, 6 and 12 months in haplo-HCT recipients compared to MRD or MUD recipients (at M1, M3, M6, M12, the median Vδ2+ T cells counts were 0.05/µL, 0.24/µL, 1.38/µL and 2.97/µL, and MAIT cells were 0.07/µL, 0.70/µL, 1.00/µL and 1.21/µL, respectively). Lower Vδ2+ T-cells and MAIT cells counts at one month was associated with a significantly increased CI of EBV reactivation (respectively, P=.04 and P<.0001) in landmark study. Conclusion: Immunological reconstitution of innate T cells is significantly delayed after haplo-HCT and low-dose ATG and PTCy. This prolonged deficiency is associated with an increased risk of EBV reactivation. Development of new strategies for innate-T cells expansion are necessary after haplo-HCT. Disclosures Mohty: MaaT Pharma: Consultancy, Honoraria.