High parameter flow cytometry for analysis of MAIT cells and other rare cell populations in human blood and tissues

Abstract

Approximately 70 years after flow cytometry’s inception, technical advances are continually being made to pave the way for biological insights. In the current thesis, we first describe a new metric called ‘unmixing dependent spread’ that encapsulates how individual fluorochromes have increased variance when interpreted by a complex spectral unmixing matrix. Using this newly described metric among other best practice procedures, we demonstrate the first use of a 50-color flow cytometry panel for in depth phenotyping of the T cell and APC compartment. As human samples can be limited or small in size as is the case with resected tumor biopsies, analysis approaches that can provide broad cell type identification with in-depth phenotyping are paramount to extrapolate the most information from a patient cohort. We next demonstrate the use of high parameter flow cytometry with low input human samples for remote immune monitoring where our findings have applications for decentralized clinical and research studies. Often transportation to a clinic is a barrier to individuals to participate in centralized studies, therefore we asked whether individuals using an at-home blood collection device (ABCD) could draw their blood at home and mail it in to the lab for analysis without loss of quality. Remarkably, we found that T cell numbers, subsets, and phenotypes remained stable when mailed to the lab. We further discuss the applications of ABCDs and identify areas of improvement for remote immune monitoring by flow cytometry. Last, we use multiple high parameter flow cytometry panels to interrogate the functional outcomes of mucosal associated invariant T (MAIT) cells in response antigenic, inflammatory, and regulatory signals. Because MAIT cell antigen is made by both commensal and pathogenic bacteria, it is unclear how TCR stimulus integrates with other signals to inform their effector function so that they do not contribute to of target inflammation. Notable among our findings, we show that a population of Granzyme B producing MAIT cells remains constant when activated in the presence of any quantity of TGF-β, a potent regulatory cytokine, while inflammatory cytokines TNF⍺ and IFNγ are greatly reduced. This data suggests that MAIT cells prioritize cytotoxicity over cell signaling, which may be a mechanism to facilitate the control of infection in a cell contact dependent manner instead of propagating inflammation by activating and recruiting additional immune cells. We further discuss the relevancy of our findings in context of using MAIT cells for cell-based therapies.