In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic property of fetal hematopoietic stem and precursor cells (HSPCs) caused by high expression of the RNA-binding protein Lin28b. In conclusion, our data indicate that the human fetal thymus generates, in an HSPC/Lin28b-dependent manner, invariant γδ T cells with programmed effector functions.

Phosphoantigen-reactive Vγ9Vδ2 T cells represent the main innate human γδ T cell subset and dominate the fetal and adult peripheral blood γδ T cell repertoire. It has been hypothesized that adult blood Vγ9Vδ2 T cells find their origin in the fetus like it is established for mouse innate γδ T cells. In order to address this issue, we analyzed the complementarity-determining-region-3 (CDR3) of the T cell receptor (TCR) of human blood and thymic Vγ9Vδ2 T cells from fetal until adult life. We first identified key differences in the CDR3 repertoire of fetal and adult blood Vγ9Vδ2 T cells, including in CDR3 features important for phosphoantigen-reactivity. Next, we showed that most of these key adult CDR3 features were already present in the postnatal thymus and were further enhanced upon selection in vitro by the microbial-derived phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). Finally, we demonstrated that the generation of a fetal-type or adult-type Vγ9Vδ2 CDR3 repertoire is determined by the fetal and post-natal nature of the hematopoietic-stemand-precursor-cell (HSPC). Thus, our data indicate that fetal blood Vγ9Vδ2 T cells find their origin in the fetal thymus while adult blood Vγ9Vδ2 T cells are generated to a large degree independently after birth.

Acute myeloid leukemia (AML) remains a major clinical challenge due to frequent chemotherapy resistance and deadly relapses. We are exploring the immunotherapeutic potential of peripheral blood V1 + T-cells, which associate with improved long-term survival of stem-cell transplant recipients, but have never been applied as adoptive cell therapy. Using our recently developed clinical-grade protocol for expansion and differentiation of "Delta One T" (DOT) cells, we found them to be highly cytotoxic against AML primary samples and cell lines, including cells selected for resistance to standard chemotherapy. Interestingly, unlike chemotherapy, DOT-cell targeting did not select for outgrowth of specific AML lineages, suggesting a broad recognition domain, which was also consistent with the striking polyclonality of the DOTcell TCR repertoire. Moreover, whereas AML reactivity was only slightly impaired upon anti-V1 + TCR antibody blockade, it was strongly dependent on expression of the NKp30 ligand, B7-H6. In contrast, DOT-cells did not show any reactivity against normal leukocytes, including CD33 + or CD123 + myeloid cells. Importantly, adoptive transfer of DOT-cells in vivo markedly reduced AML load in the blood and target organs of multiple human AML xenograft models; and significantly prolonged host survival, without any noticeable toxicity, thus providing the proof-of-concept for DOT-cell application in AML treatment.

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

<p>Supplementary Figures 1-5 and tables 1-3</p>