Characterization of unique B-cell populations in the circulation of people living with HIV prior to non-Hodgkin lymphoma diagnosis

Abstract

People living with HIV (PLWH) are at higher risk of developing lymphoma. In this study, we performed cytometry by time-of-flight (CyTOF) on peripheral blood mononuclear cells of cART-naïve HIV+ individuals and cART-naïve HIV+ individuals prior to AIDS-associated non-Hodgkin lymphoma (pre-NHL) diagnosis. Participants were enrolled in the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Uniform Manifold Approximation and Projection (UMAP) and unsupervised clustering analysis were performed to identify differences in the expression of B-cell activation markers and/or oncogenic markers associated with lymphomagenesis. CD10⁺CD27^- B cells, CD20⁺CD27^- B cells, and B-cell populations with aberrant features (CD20⁺CD27⁺CXCR4⁺CD71⁺ B cells and CD20⁺CXCR4⁺cMYC⁺ B cells) were significantly elevated in HIV+ cART-naïve compared to HIV-negative samples. CD20⁺CD27⁺CD24⁺CXCR4⁺CXCR5⁺ B cells, CD20⁺CD27⁺CD10⁺CD24⁺CXCR4⁺cMYC⁺ B cells, and a cluster of CD20⁺CXCR4^hiCD27^-CD24⁺CXCR5⁺CD40⁺CD4⁺AICDA⁺ B cells were significantly elevated in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. A potentially clonal cluster of CD20⁺CXCR4⁺CXCR5⁺cMYC⁺AICDA⁺ B cells and a cluster of germinal center B-cell-like cells (CD19^-CD20⁺CXCR4⁺Bcl-6⁺PD-L1⁺cMYC⁺) were also found in the circulation of HIV+ pre-NHL (cART-naïve) samples. Moreover, significantly elevated clusters of CD19⁺CD24^hiCD38^hi cMYC⁺ AICDA⁺ B regulatory cells were identified in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. The present study identifies unique B-cell subsets in PLWH with potential pre-malignant features that may contribute to the development of pre-tumor B cells in PLWH and that may play a role in lymphomagenesis.

Keywords: B regulatory cells; B-cells; HIV+ cART-naïve; HIV+ pre-NHL (cART-naïve); mass cytometry.

Figure 1

Unsupervised clustering of CD19⁺ B cells identifies 60 metaclusters in HIV-negative, HIV+ cART-naïve, and HIV+ pre-NHL (cART-naïve) samples. (A) Quantification and comparison in the percentage of CD19⁺ B cells (CD3^-CD14^-CD11b^-) from total viable cells in PBMCs isolated from HIV-negative (n = 10), HIV⁺ cART-naïve (n = 20), and HIV⁺ pre-NHL (cART-naïve) (n = 10) (Mann–Whitney, unpaired non-parametric test, p > 0.05). (B) Contour plots of UMAPs for each group. UMAP plots were generated from an equal subsampling of 75,000 CD19⁺ B cells (CD3^-CD14^-CD11b^-) for each group after manual gating ( Supplementary Figures 1A, B ). (C) Concatenated metacluster data [metacluster (MC) 1 to 60] shown as UMAP plots for each group; HIV-negative (n = 10), HIV+ cART-naïve (n = 20), and HIV+ pre-NHL (cART-naïve) (n = 10).

Figure 2

CD10⁺CD27^- B cells (MC02), CD20⁺CD27⁺CXCR4⁺CD71⁺ B cells (MC36 and MC49), CD20⁺CXCR4⁺cMYC⁺ B cells (MC50), and CD20⁺CD24⁺CXCR4⁺CXCR5⁺ B cells (MC48) are significantly elevated in HIV+ cART-naïve compared with HIV-negative. (A) Volcano plot showing log FC (log fold change) and adjusted p-values (p < 0.05) for statistically significant differences in B-cell metaclusters (MC) for HIV-negative and HIV+ cART-naïve samples. A total of 25 significant metaclusters are shown above the −log10 (p-value of 0.05) threshold (dotted line): 7 MCs for HIV-negative and 18 MCs for HIV+ cART-naïve. Data are for concatenated files of HIV-negative (n = 10) and HIV+ cART-naïve (n = 20). (B) Box plots showing cell counts within each significant metacluster shown in (A). (C) Heatmap of median marker expression values in metaclusters identified in (A). Concatenated data are summarized in a heatmap for selected metaclusters elevated in HIV+ cART-naïve compared to HIV-negative (MC02, MC36, MC49, MC48, MC50, and MC42).

Figure 3

CD27⁺IgM⁺ B cells (MC10), CD20⁺CXCR4⁺ B cells (MC37 and MC39), CD20⁺CD27⁺CD24⁺CXCR4⁺cMYC⁺ B cells (MC47), and CD20⁺CD24⁺CXCR4⁺CXCR5⁺ B cells (MC44 and MC48) are significantly elevated in HIV+ pre-NHL (cART-naïve). (A) Volcano plot showing log FC (log fold change) and adjusted p-values (p < 0.05) for statistically significant differences in B-cell metaclusters for HIV+ cART-naïve and HIV+ pre-NHL (cART-naïve). A total of 16 significant metaclusters are shown above the −log10 (p-value of 0.05) threshold (dotted line): 10 MCs for HIV+ cART-naïve and 6 MCs for HIV+ pre-NHL (cART-naïve). Data are for concatenated files of HIV+ (n = 20) and HIV+ Pre-NHL (n = 10). (B) Box plots showing cell counts in each significant metacluster shown in (A). (C) Concatenated data summarized in a heatmap with median marker expression values for metaclusters elevated in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve (MC10, MC37, MC48, MC47, MC39, and MC44). (D) Heatmap of median marker expression values for the metaclusters elevated in HIV+ cART-naïve samples (MC29, MC49, MC14, MC21, MC08, MC28, and MC31).

Figure 4

CD10⁺CXCR4⁺ B cells (MC11, MC47), CD20⁺CD27^- B cells (MC35), CD20⁺CXCR4⁺ B cells (MC45), CD20⁺CD24⁺CXCR4⁺CXCR5⁺ B cells (MC48), and CD20⁺CD86⁺ B cells (MC50) are significantly elevated in HIV+ pre-NHL (cART-naïve) compared with HIV-negative. (A) Volcano plot showing log FC (log fold change) and adjusted p-values (p < 0.05) for statistically significant differences in B-cell metaclusters for HIV-negative and HIV+ pre-NHL (cART-naïve). A total of 34 significant metaclusters are shown above the −log10 (p-value of 0.05) threshold (dotted line):13 MCs for HIV-negative and 21 MCs for HIV+ pre-NHL (cART-naïve). Data are for concatenated files of HIV-negative (n = 10) and HIV+ pre-NHL (cART-naïve) (n = 10). (B) Box plots showing cell counts for each significant metacluster shown in (A). (C) Concatenated data summarized in a heatmap with median marker expression values in select metaclusters elevated in HIV+ pre-NHL (cART-naïve) (MC11, MC35, MC42, MC47, MC48, MC50, MC01, and MC45).

Figure 5

Elevated levels of CD20⁺CXCR4^hi B cells expressing CD24 and/or AICDA are observed in HIV+ pre-NHL (cART-naïve). (A) Unsupervised clustering of CD19⁺CD20⁺CXCR4^hi B cells provide 38 metaclusters in HIV+ cART-naïve and HIV+ pre-NHL (cART-naïve), and contour plots are shown. UMAP plots were generated from an equal subsampling of 12,500 CD19⁺CD20⁺CXCR4^hi B cells for each group. UMAP plots are for concatenated files of HIV+ cART-naïve (n = 20) and HIV+ pre-NHL (cART-naïve) (n = 10). (B) Volcano plot showing significantly elevated metaclusters in HIV+ cART-naïve (MC28, MC25, MC19, and MC15) and HIV+ pre-NHL (cART-naïve) (MC23 and MC36). Significant metaclusters are shown above the −log10 (p-value of 0.05) threshold (dotted line). (C) Box plots showing cell counts for the two significantly elevated metaclusters in HIV+ pre-NHL (cART-naïve) (MC23 and MC36) compared to those metaclusters in HIV+ cART-naïve samples. (D) Clustered heatmap with median marker expression values of metaclusters 36 and 23.

Figure 6

Bregs are significantly elevated in HIV+ pre-NHL (cART-naïve) and have unique pre-lymphoma phenotypes. (A) CD24^hiCD38^hi cells were manually gated from CD19⁺ B cells (CD3^-CD14^-CD11b^-) from PBMCS of HIV+ cART-naïve (n = 20) and HIV+ pre-NHL (cART-naïve) (n = 10) samples. Shown is a representative plot of Bregs for each group with percent values. (B) Counts of manually gated Bregs in HIV+ cART-naïve (n = 20) and HIV+ pre-NHL (cART-naïve) (n = 10) (Mann–Whitney, unpaired non-parametric test, ***p < 0.0005). (C) Counts of PD-L1+, AICDA+, IL-10+, CD71+, cMYC+, Bcl-6+, FoxP3 +, IgM+, or IgMhi Bregs in HIV+ cART-naïve and HIV+ pre-NHL (cART-naïve) samples (Mann–Whitney, unpaired non-parametric test, *p < 0.05; **p<0.005; ***p < 0.0005; ****p<0.00005). (D) Unsupervised clustering of Bregs provided 25 metaclusters in HIV+ cART-naïve and HIV+ pre-NHL (cART-naïve). UMAPs were generated from an equal subsampling of 5,000 Bregs for each group. Contour plots are shown. (E) Volcano plot with significantly elevated metaclusters in HIV+ cART-naïve (MC17, MC21, and MC23) and HIV+ pre-NHL (cART-naïve) (MC20, MC04, and MC03) [shown above the −log10 (p-value of 0.05) threshold, dotted line]. (F) Heatmap of median marker expression values of significantly elevated metaclusters in HIV+ pre-NHL (cART-naïve) (MC20, MC04, and MC03). (G) Heatmap of median marker expression values of significantly elevated metaclusters in HIV+ cART-naïve samples (MC17, MC21, and MC23).