Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well-established lymphocyte markers of memory and activation. Comparable frequencies of both common and rare cell subpopulations were observed with fluorescence and mass cytometry using biaxial gating. The unsupervised high-dimensional analysis tool viSNE was then used to analyze data sets generated from both mass and fluorescence cytometry. viSNE analysis effectively characterized PBMC using eight features per cell and identified similar frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of unsupervised analysis programs and extended multiparameter cytometry will be indispensable tools for detecting perturbations in protein expression in both health and disease.

Background Influenza related morbidity and mortality disproportionately impacts older adults. The serologic response to vaccine is diminished in older adults; however, high dose inactivated influenza vaccine (HD IIV) has shown improved rates of seroconversion compared to standard dose (SD IIV). We hypothesize this may be due to the superior ability of high dose vaccine to activate T follicular helper (Tfh) cells and provide B cell dependent T cell help. Methods We measured peripheral Tfh (pTfh) activation in 50 community dwelling adults 65 years or older who were randomly assigned to receive either the HD IIV or SD IIV. Results The HD vaccination elicited significantly higher levels of ICOS expression on pTfh cells, at day 7 compared to SD vaccination (p= 0.02). The magnitude of the increase in ICOS+ pTfh cells from baseline to day 7 was predictive of seroconversion for both influenza A and B vaccination. Conclusion Strong Tfh activation in response to influenza vaccination forecasts successful seroconversion in older adults, and HD IIV elicits greater Tfh activation than SD IIV. Future vaccine studies should focus on ways to further optimize the Tfh response.

Infection with Human Immunodeficiency Virus Type 1 (HIV-1) induces defects of both cellular and humoral immune responses. Impaired CD4+ T cell help and B cell dysfunction may partially explain the low frequency of broadly neutralizing antibodies in HIV-infected individuals. To understand the extent of B cell dysfunction during HIV infection, we assessed the level of B cell activation at baseline and after stimulation with a variety of antigens. Increased levels of viremia were associated with higher baseline expression of the activation marker CD86 on B cells and with decreased ability of B cells to increase expression of CD86 after in vitro stimulation with inactivated HIV-1. In a series of cell isolation experiments B cell responses to antigen were enhanced in the presence of autologous CD4+ T cells. HIV infected individuals had a higher frequency of PD-1 expression on B cells compared to HIV- subjects and PD-1 blockade improved B cell responsiveness to HIV antigen, suggesting that inhibitory molecule expression during HIV-1 infection may contribute to some of the observed B cell defects. Our findings demonstrate that during chronic HIV infection, B cells are activated and lose full capacity to respond to antigen, but suppression of inhibitory pressures as well as a robust CD4+ T cell response may help preserve B cell function.

Peripheral CD4+CXCR5+PD-1+ T cells are a putative circulating counterpart to germinal center T follicular helper (TFH) cells. They show both phenotypic and functional similarities to TFH cells, which provide necessary help for the differentiation of B cells to antibody-secreting plasmablasts. In this study we evaluated the frequency, phenotypes, and responses of peripheral TFH-like (pTFH) cells to superantigen and recall antigen stimulation in 10 healthy and 34 chronically infected treatment-naïve HIV-1+ individuals. There was no difference in the frequency of pTFH cells between HIV+ and HIV− individuals. Surface expression of ICOS, but not CD40L, was higher on pTFH cells at baseline in HIV+ individuals. Compared to HIV− individuals, pTFH cells from HIV+ individuals had decreased maximal expression of ICOS and CD40L in response to in vitro superantigen stimulation. This decreased response did not correlate with viral control, CD4+ T cell count, duration of infection, or the degree of neutralizing antibody breadth. Despite a decreased maximal response, however, pTFH responses to HIV gag and tetanus toxoid recall antigens were preserved.

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