Impairment of antigen-presenting function of peripheral γδ T cells in patients with sepsis

Impairment of antigen-presenting functions is a key mechanism contributing to sepsis-induced immunosuppression. Recently, γδ T cells have been demonstrated as professional antigen-presenting cells (APCs); however, their role in sepsis remains unknown. In this in vitro study, the APC function of human peripheral γδ T cells was assessed using samples collected from 42 patients with sepsis and 27 age-matched healthy controls. The APC-related markers HLA-DR, CD27, CD80, and CCR7 on fresh γδ T cells were significantly higher in patients with sepsis compared with matched controls; however, they responded poorly to 4-hydroxy-3-methyl-2-butenyl pyrophosphate (HMBPP) stimulation, characterised by the deactivation of these APC markers and impaired proliferation. Furthermore, the adhesion function of γδ T cells, essential for antigen presentation, was greatly reduced in patients with sepsis; for instance, in co-cultures with green fluorescent protein-expressing Escherichia coli, HMBPP-activated γδT cells from healthy individuals adhered to E. coli efficiently, whereas no such phenomenon was observed with respect to γδT cells from patients with sepsis. In line with these results, in co-cultures with isolated CD4 + αβ T cells, HMBPP-activated γδT cells of healthy individuals promoted the efficient proliferation of CD4 + αβ T cells, whereas γδT cells from patients with sepsis did not do so. In conclusion, our findings show that the antigen-presenting function of γδ T cells is severely impaired in patients with sepsis and the mechanisms behind need further study.

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2− γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

P06.01 αβ-T cells engineered to express γδ-T cell receptors can kill neuroblastoma organoids independent of MHC-I expression

BackgroundCurrently ~50% of patients with the diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αβ-T cells engineered to express a defined γδ-T cell receptor, which are thought to recognize and kill target cells independent of MHC-I. In this pilot project we have tested the potential efficacy of TEG002 therapy as a novel treatment for neuroblastoma, with tumor organoids.Materials and MethodsEffector cells were created from healthy donor peripheral blood T cells. The TEG002 cells were engineered by transducing αβ-T cells with a defined Vγ9Vδ2-T cell receptor. Both the untransduced αβ-T cells and the endogenous Vγ9Vδ2-T cells from the same healthy donor were used as controls in all experiments. Activation and killing of TEG002 was tested in a co-culture setting with neuroblastoma organoids. Supernatant of the co-culture was collected at 24 hours for IFNγ ELISA to measure activation of TEG002. The dynamics of cytotoxicity were analyzed over time from 0 till 72 hours, using the live-cell imaging system IncuCyte from Sartorius®. Killing was quantified using a Caspase3/7 Green dye and the IncuCyte software. Transcriptional profiling of the neuroblastoma organoids was done by RNA sequencing and MHC-I expression of the neuroblastoma organoids was determined by flow cytometry.ResultsWe showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling of the neuroblastoma organoids showed that this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independently of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αβ-T cells or endogenous γδ-T cells.ConclusionsWe demonstrated that 50% of tested neuroblastoma organoids can effectively activate TEG002 and that killing of the organoids is independent of MHC-I expression. Hence, this pilot study identified TEG002 as a promising novel cellular product for immunotherapy for a subset of neuroblastoma tumors, warranting further investigations into its clinical application.Disclosure InformationJ.G.M. Strijker: None. E. Drent: A. Employment (full or part-time); Significant; Gadeta BV. J.J.F. van der Hoek: None. R. Pscheid: A. Employment (full or part-time); Significant; Gadeta BV. B. Koopmans: None. K. Ober: None. S.R. van Hooff: None. W.M. Kholosy: None. C. Coomans: A. Employment (full or part-time); Significant; Gadeta BV. A. Bisso: A. Employment (full or part-time); Significant; Gadeta BV. M. van Loenen: A. Employment (full or part-time); Significant; Gadeta BV. J.J. Molenaar: None. J. Wienke: None.

Clonal expansion of CD8+ T cells reflects graft-versus-leukemia activity and precedes durable remission following DLI

Donor lymphocyte infusion (DLI) is a standard of care for relapse of AML after allogeneic hematopoietic stem cell transplantation (aHSCT). Currently it is poorly understood how and when CD8+ αβ T cells exert graft-versus-leukemia (GvL) activity after DLI. Also, there is no reliable biomarker to monitor GvL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αβ T cell clones in DLI-patients. In this prospective clinical study of 29 patients, we performed deep T cell receptor β (TRB) sequencing of sorted CD8+ αβ T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GvL, longitudinal analyses revealed a preferential expansion of distinct CD8+ TRB clones (n=14). This did not occur in samples of patients without signs of GvL (n=11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36 months study follow-up. Furthermore, absence of clonal outgrowth of the CD8+ TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+ TRB diversity at day 15 after DLI (n=13) had a lower relapse incidence (P=0.0040) compared to patients without clonal expansion (n=6). In conclusion, CD8+ TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.

αβ-T Cells Engineered to Express γδ-T Cell Receptors Can Kill Neuroblastoma Organoids Independent of MHC-I Expression

Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αβ-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αβ-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients.

Increased double-negative αβ+ T-cells reveal adult-onset autoimmune lymphoproliferative syndrome in a patient with IgG4-related disease

Not available.

αβ/γδ T cell lineage outcome is regulated by intrathymic cell localization and environmental signals

αβ and γδ T cells are two distinct sublineages that develop in the vertebrate thymus. Thus far, their differentiation from a common progenitor is mostly understood to be regulated by intrinsic mechanisms. However, the proportion of αβ/γδ T cells varies in different vertebrate taxa. How this process is regulated in species that tend to produce a high frequency of γδ T cells is unstudied. Using an in vivo teleost model, the medaka, we report that progenitors first enter a thymic niche where their development into γδ T cells is favored. Translocation from this niche, mediated by chemokine receptor Ccr9b, is a prerequisite for their differentiation into αβ T cells. On the other hand, the thymic niche also generates opposing gradients of the cytokine interleukin-7 and chemokine Ccl25a, and, together, they influence the lineage outcome. We propose a previously unknown mechanism that determines the proportion of αβ/γδ lineages within species.

High Th2 cytokine levels and upper airway inflammation in human inherited T-bet deficiency

We have described a child suffering from Mendelian susceptibility to mycobacterial disease (MSMD) due to autosomal recessive, complete T-bet deficiency, which impairs IFN-γ production by innate and innate-like adaptive, but not mycobacterial-reactive purely adaptive, lymphocytes. Here, we explore the persistent upper airway inflammation (UAI) and blood eosinophilia of this patient. Unlike wild-type (WT) T-bet, the mutant form of T-bet from this patient did not inhibit the production of Th2 cytokines, including IL-4, IL-5, IL-9, and IL-13, when overexpressed in T helper 2 (Th2) cells. Moreover, Herpesvirus saimiri–immortalized T cells from the patient produced abnormally large amounts of Th2 cytokines, and the patient had markedly high plasma IL-5 and IL-13 concentrations. Finally, the patient’s CD4+ αβ T cells produced most of the Th2 cytokines in response to chronic stimulation, regardless of their antigen specificities, a phenotype reversed by the expression of WT T-bet. T-bet deficiency thus underlies the excessive production of Th2 cytokines, particularly IL-5 and IL-13, by CD4+ αβ T cells, causing blood eosinophilia and UAI. The MSMD of this patient results from defective IFN-γ production by innate and innate-like adaptive lymphocytes, whereas the UAI and eosinophilia result from excessive Th2 cytokine production by adaptive CD4+ αβ T lymphocytes.

CD1a autoreactivity: When size does matter

CD1a-autoreactive T cells represent a significant proportion of circulating αβ T cells in humans and appear to be enriched in the skin. How their autoreactivity is regulated remains unclear. In this issue of JEM, Cotton et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202699) show that CD1a molecules do not randomly survey cellular lipids but instead capture certain lipid classes that broadly interfere with the binding of autoreactive T cell antigen receptors to the target CD1a. These findings provide new potential therapeutic avenues for manipulating CD1a autoreactive T cell responses.