γ9δ2 T-Cell Expansion and Phenotypic Profile Are Reflected in the CDR3δ Repertoire of Healthy Adults.

γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire-phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics.

The Role of γδ T Cells as a Line of Defense in Viral Infections after Allogeneic Stem Cell Transplantation: Opportunities and Challenges.

In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αß T cells, we describe that by delaying αß T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.

γ9δ2T cell diversity and the receptor interface with tumor cells.

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

Cellular immunotherapy on primary multiple myeloma expanded in a 3D bone marrow niche model.

Bone marrow niches support multiple myeloma, providing signals and cell-cell interactions essential for disease progression. A 3D bone marrow niche model was developed, in which supportive multipotent mesenchymal stromal cells and their osteogenic derivatives were co-cultured with endothelial progenitor cells. These co-cultured cells formed networks within the 3D culture, facilitating the survival and proliferation of primary CD138+ myeloma cells for up to 28 days. During this culture, no genetic drift was observed within the genomic profile of the primary myeloma cells, indicating a stable outgrowth of the cultured CD138+ population. The 3D bone marrow niche model enabled testing of a novel class of engineered immune cells, so called TEGs (αßT cells engineered to express a defined γδTCR) on primary myeloma cells. TEGs were engineered and tested from both healthy donors and myeloma patients. The added TEGs were capable of migrating through the 3D culture, exerting a killing response towards the primary myeloma cells in 6 out of 8 donor samples after both 24 and 48 hours. Such a killing response was not observed when adding mock transduced T cells. No differences were observed comparing allogeneic and autologous therapy. The supporting stromal microenvironment was unaffected in all conditions after 48 hours. When adding TEG therapy, the 3D model surpassed 2D models in many aspects by enabling analyses of specific homing, and both on- and off-target effects, preparing the ground for the clinical testing of TEGs. The model allows studying novel immunotherapies, therapy resistance mechanisms and possible side-effects for this incurable disease.

RhoB Mediates Phosphoantigen Recognition by Vγ9Vδ2 T Cell Receptor.

Human Vγ9Vδ2 T cells respond to tumor cells by sensing elevated levels of phosphorylated intermediates of the dysregulated mevalonate pathway, which is translated into activating signals by the ubiquitously expressed butyrophilin A1 (BTN3A1) through yet unknown mechanisms. Here, we developed an unbiased, genome-wide screening method that identified RhoB as a critical mediator of Vγ9Vδ2 TCR activation in tumor cells. Our results show that Vγ9Vδ2 TCR activation is modulated by the GTPase activity of RhoB and its redistribution to BTN3A1. This is associated with cytoskeletal changes that directly stabilize BTN3A1 in the membrane, and the subsequent dissociation of RhoB from BTN3A1. Furthermore, phosphoantigen accumulation induces a conformational change in BTN3A1, rendering its extracellular domains recognizable by Vγ9Vδ2 TCRs. These complementary events provide further evidence for inside-out signaling as an essential step in the recognition of tumor cells by a Vγ9Vδ2 TCR.