Balfour Avuyonke, Masina Nomawethu, Queiroz Artur T L, Andrade Bruno B, Fukutani Eduardo R, Bekiswa Abulele, Lewinsohn David, Lewinsohn Deborah, Goliath Rene, Wilkinson Katalin A, Schutz Charlotte, Meintjes Graeme, Shey Muki S
Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Centre for Infectious Diseases Research in Africa (CIDRI-Africa), Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
bioRxiv. 2025 Mar 28:2025.03.27.643001. doi: 10.1101/2025.03.27.643001.
Evidence exists that some individuals resist ("Resisters") (Mtb) infection despite sustained exposure, but the protective mechanisms involved are not fully understood. We investigated immune responses induced by stimulation of peripheral blood mononuclear cells (PBMC) with live Mtb in Resisters compared to those with latent TB infection (LTBI).
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.
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.
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.
有证据表明，一些个体尽管持续暴露于结核分枝杆菌（Mtb），但仍能抵抗感染（“抵抗者”），但其中涉及的保护机制尚未完全了解。我们研究了与潜伏性结核感染（LTBI）个体相比，抵抗者外周血单个核细胞（PBMC）受到活Mtb刺激后诱导的免疫反应。
对在高结核暴露医疗设施中工作超过5年的未感染HIV的医护人员，使用干扰素-γ释放试验（IGRA）和结核菌素皮肤试验（TST）进行Mtb致敏筛查。我们确定了抵抗者（TST<10mm且IGRA<0.35 IU/mL，n = 129）和LTBI个体（TST≥10mm且IGRA≥0.35 IU/mL，n = 145）。我们选择了一组“极端抵抗者”（TST = 0mm且IGRA<0.2 IU/mL；n = 26）和“极端LTBI”个体（TST≥15mm且IGRA≥1 IU/mL；n = 24）进行这些分析。采集血液，分离PBMC并与活H37Rv Mtb一起培养18小时。收集上清液，用于通过Luminex测量65种分泌的分析物。我们还使用流式细胞术评估了这些参与者中CD4 T细胞和单核细胞的细胞相关细胞因子表达。
使用R语言中的特征选择，我们确定了一组4种细胞因子：IL-17A、MCP-1、IL-8和MDC，它们共同将极端抵抗者与极端LTBI个体区分开来，曲线下面积（AUC）为0.67（0.54 - 0.85）。仅关注极端抵抗者，并使用层次聚类，该组参与者分为两个主要聚类（抵抗者_c1和抵抗者_c2）。进一步分析发现，与抵抗者_c1相比，抵抗者_c2中有37种细胞因子显著升高，15种细胞因子降低。一组5种细胞因子（TRAIL、MIP-1β、Fractalkine、GRO-α和IL-1α）共同将这两个聚类区分开来，AUC为1（1 - 1）。两个聚类之间CD4 T细胞和单核细胞对Mtb的反应没有显著差异。
Mtb特异性免疫反应将极端抵抗者分为两个不同的聚类，这些个体对Mtb感染具有明显的抵抗力，并且可能有助于区分真正具有抵抗力的个体与感染但具有IGRA和TST未检测到的替代免疫反应的个体。
