Two distinct cytokine response clusters identified in healthcare workers with apparent resistance to infection with Mycobacterium tuberculosis despite sustained occupational exposure

Abstract

Introduction Evidence exists that some individuals resist (“Resisters”) Mycobacterium tuberculosis (Mtb) infection despite sustained exposure, but the protective mechanisms involved are not fully understood. We investigated immune responses induced by ex vivo stimulation of peripheral blood mononuclear cells (PBMC) with live Mtb in Resisters compared to those with latent TB infection (LTBI).

Methods HIV-uninfected healthcare workers working in high TB exposure healthcare facilities for over 5 years were screened for Mtb sensitization using the interferon-gamma release assay (IGRA) and the tuberculin skin test (TST). We identified Resisters (TST<10mm and IGRA<0.35 IU/mL, n=129) and those with LTBI (TST≥10mm and IGRA≥0.35 IU/mL n=145). We selected a subset of ‘extreme resisters’ (TST=0mm and IGRA<0.2 IU/mL; n=26) and ‘extreme LTBI’ (TST≥15mm and IGRA≥1 IU/mL; n=24) for these analyses. Blood was collected and PBMC isolated and cultured with live H37Rv Mtb for 18 hours. Supernatants were collected and used for measuring 65 secreted analytes by Luminex. We also evaluated cell-associated cytokine expression by CD4 T cells and monocytes in these participants using flow cytometry.

Results Using the feature selection in R, we identified a set of 4 cytokines: IL-17A, MCP-1, IL-8, and MDC, which collectively classified extreme Resisters from extreme LTBI with an area-under-the-curve (AUC) of 0.67 (0.54-0.85). Focusing only on the extreme Resisters, and using hierarchical clustering, participants in this group segregated into two main clusters (Resister_c1 and Resister_c2). Further analyses identified 37 cytokines that were significantly higher and 15 cytokines that were lower in Resister_c2 compared with Resister_c1. A set of 5 cytokines (TRAIL, MIP-1β, Fractalkine, GRO-α and IL-1α) collectively classified these two clusters with an AUC of 1 (1-1). CD4 T cell and monocyte responses to Mtb did not significantly differ between the two clusters.

Conclusions Mtb-specific immune responses segregate extreme Resisters into two distinct clusters in individuals with apparent resistance to Mtb infection and may allow discrimination of individuals with true resistance from those with infection but have alternative immune responses not detected by IGRA and TST.