Human peripheral γδT cell phenotypic and functional shift in response to stimulation with E.coli

It is known that γδT cells provide immune-surveillance and host defense against infection and cancer. Our understanding of γδT cell functionality and antimicrobial immunity, however, remains poor. Limited data suggests that γδT cells can phagocytose particles and act as professional antigen-presenting cells (pAPCs). In order to examine γδT cell bacterial interactions, an ex vivo co-culture model of human peripheral blood mononuclear cell (PBMC) responses to Escherichia coli was employed. Following PBMC stimulation with E.coli, Vγ9Vδ2 cells underwent rapid T cell receptor (TCR)-dependent proliferation and functional transition from cytotoxic, inflammatory cytokine immunity, to cell expansion with diminished cytokine but increased costimulatory molecule expression, and capacity for professional phagocytosis. Phagocytosis was augmented by IgG opsonization, and inhibited by TCR-blockade, suggesting a licensing interaction involving the TCR and FcγR. Vγ9Vδ2 cells displayed potent cytotoxicity through TCR-dependent and independent mechanisms. Vγ9Vδ2 cell cytokine responses and cytotoxicity further presented with variable sensitivity to blocking of host butyrophilin 3A (BTN3A), in response to both, self and non-self, stimuli. We conclude that i) Vγ9Vδ2 T cells transition from early inflammatory cytotoxic killers to myeloid-like pAPCs in response to infectious stimuli, and that ii) Vγ9Vδ2 T cell recognition of targets is predominantly governed by TCR recognition of self stress markers in a BTN3A-dependent manner

Preclinical platforms to study therapeutic efficacy of human γδT‐cells for oncology indications

In this commentary, we discuss recent advances in the study of γδT cell-based immunotherapeutics. As an allo-compatible cell therapy chassis without clear functional homologs in mice, γδT cells represent a challenge and an opportunity for preclinical modelling. We discuss some of the techniques and approaches that can be used to demonstrate and characterise γδT cell behaviour in biomimetic systems

A Simple and Robust Single-Step Method for CAR-Vδ1 γδT Cell Expansion and Transduction for Cancer Immunotherapy

The γδT cell subset of peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR)-expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αβ-TCR-expressing counterparts. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 cell product despite pre-clinical promise for oncology indications stretching back to the 1980s. A contributing factor to the paucity of clinical progress with Vδ1 cells is the lack of robust, consistent and GMP-compatible expansion protocols. Herein we describe a reproducible one-step, clinically translatable protocol for Vδ1-γδT cell expansion from peripheral blood mononuclear cells (PBMCs), that is further compatible with high-efficiency gene engineering for immunotherapy purposes. Briefly, αβTCR- and CD56-depleted PBMC stimulation with known-in-the-art T cell stimulators, anti-CD3 mAb (clone: OKT-3) and IL-15, leads to robust Vδ1 cell expansion of high purity and innate-like anti-tumor efficacy. These Vδ1 cells can be virally transduced to express chimeric antigen receptors (CARs) using standard techniques, and the CAR-Vδ1 exhibit antigen-specific persistence, cytotoxicity and produce IFN-γ. Practicable, GMP-compatible engineered Vδ1 cell expansion methods will be crucial to the wide-spread clinical testing of these cells for oncology indications

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells

Background aims: The targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues. / Methods: To explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs. / Results: A lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity. / Conclusions: These data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target

Short-wave infrared imaging enables high-contrast fluorescence-guided surgery in neuroblastoma

Fluorescence-guided surgery is set to play a pivotal role in the intraoperative management of pediatric tumors. Short-wave infrared imaging (SWIR) has advantages over conventional near-infrared I (NIR-I) imaging with reduced tissue scattering and autofluorescence. Here, two NIR-I dyes (IRDye800CW and IR12), with long tails emitting in the SWIR range, were conjugated with a clinical-grade anti-GD2 monoclonal antibody (Dinutuximab-beta) to compare NIR-I and SWIR imaging for neuroblastoma surgery. A first-of-its-kind multispectral NIR-I/SWIR fluorescence imaging device was constructed to allow an objective comparison between the two imaging windows. Conjugates were first characterized in vitro. Tissue-mimicking phantoms, imaging specimens of known geometric and material composition, were used to assess the sensitivity and depth penetration of the NIR-I/SWIR device, showing a minimum detectable volume of ~0.9 mm3 and depth penetration up to 3 mm. In vivo, fluorescence imaging using the NIR-I/SWIR device showed a high tumor-to-background ratio (TBR) for both dyes, with anti-GD2-IR800 being significantly brighter than anti-GD2-IR12. Crucially, the system enabled higher TBR at SWIR wavelengths than at NIR-I wavelengths, verifying SWIR imaging enables high-contrast delineation of tumor margins. This work demonstrates that by combining the high-specificity of anti-GD2 antibodies with the availability and translatability of existing NIR-I dyes, along with the advantages of SWIR in terms of depth and tumor signal-to-background ratio, GD2-targeted NIR-I/SWIR-guided surgery could improve the treatment of neuroblastoma patients, warranting investigation in future clinical trials

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies

Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.National Institutes of Health (U.S.