Establishing a successful immune response requires cell-cell interactions, where the nature of antigen presentation dictates functional outcomes. Methods to study these interactions, however, suffer from limited throughput and a lack of control over cell pairing. Here we describe a microfluidic platform that achieves high-throughput deterministic pairing of lymphocytes with a defined contact time, thereby allowing accurate assessment of early activation events for each pair in controlled microenvironments. More importantly, the platform allows the capture of dynamic processes and static parameters from both partners simultaneously, thus enabling pairwise-correlated multiparametric profiling of lymphocyte interactions over hundreds of pairs in a single experiment. Using our platform, we characterized early activation dynamics of CD8 T cells (OT-1 and TRP1 transnuclear (TN)) and investigated the extent of heterogeneity in T-cell activation and the correlation of multiple readouts. The results establish our platform as a promising tool for quantitative investigation of lymphocyte interactions.

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection 1. However, the outcome of interactions between a flu hemagglutinin (HA)-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer (SCNT) we generated mice that harbor B cells with a BCR specific for the HA of A/WSN/33 (FluBI mice). Their B cells secrete an IgG2b that neutralizes infectious virus. While B cells from FluBI and control mice bind equivalent amounts of virus through interactions of HA with surface-disposed sialic acids, the A/WSN/33 virus infects only the HA-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with HA, causing both disruption of antibody secretion and FluBI B cell death within 18 hours. 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, while 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 prior to the initiation of an effective adaptive response.

Despite the benefits of chimeric antigen receptor (CAR)–T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3ζ, we performed similar analyses on γδT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3ζ but containing DAP10 stimulatory domains. These CCR-γδT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-γδT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies.

iNKT cell functional subsets are defined by key transcription factors and output of cytokines such as IL-4, IFNγ, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice, cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα, paired with unique Vβ7 or Vβ8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, as well as cytokine profiles and found that although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity.

γδT cells provide immune-surveillance and host defense against infection and cancer. Surprisingly, functional details of γδT cell antimicrobial immunity to infection remain largely unexplored. Limited data suggests that γδT cells can phagocytose particles and act as professional antigen-presenting cells (pAPC). These potential functions, however, remain controversial. To better understand γδT cell-bacterial interactions, an ex vivo co-culture model of human peripheral blood mononuclear cell (PBMC) responses to Escherichia coli was employed. 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. We conclude that γδT cells transition from early inflammatory cytotoxic killers to myeloid-like APC in response to infectious stimuli.