Study design and analysis of CD4+CD8+ DP T cells
We isolated PBMCs from five HCs, five IRs, and three INRs, then performed scRNA-seq and transcriptomic analysis (Fig. 1A). After filtering raw data, the distribution of single cells and their related gene counts were annotated (Fig. 1B). From scRNA-seq data, we found the percentage of CD4+CD8+ DP T cells to PBMCs was lower in INRs than that in IRs and HCs (p > 0.05) (Fig. 1C). Additionally, a significantly decreased proportion of CD4+CD8+ DP T cells to total T cells in INRs compared to HCs (p < 0.05) (Fig. 1D), suggesting a reduced proportion of CD4+CD8+ DP T cells during incomplete immune reconstitution after HIV infection.
Decreased proportion of CD4+CD8+ DP T cells in INRs
To further verify the percentage of CD4+CD8+ DP T cells to total T cells, flow cytometry was performed. There was a decreasing percentage of DP T cells to total T cells in INRs compared to IRs (p < 0.05) (Fig. 2A, B), which was consistent with scRNA-seq analysis (Fig. 1D). Further, we also detected the percentage of CD4+CD8+ double-negative (DN) T cells and CD8+ SP T cells to total T cells. Compared to HCs and INRs, a slight reduction of DN T cells was observed in IRs (p > 0.05) (Fig. 2A, C). Additionally, there was a significant increase of CD8+ SP T cells in both INRs and IRs compared to HCs (p < 0.05) (Fig. 2A, D). The scRNA-seq and flow cytometry results revealed decreased CD4+CD8+ DP T cells in INRs consistently. These data highlight the potential role of DP T cells in mediating immune reconstitution after HIV infection.
Identification of three subtypes of CD4+CD8+ DP T cells
Because incomplete immune reconstitution principally decreased the percentage of CD4+CD8+ DP T cells, then we focused on the heterogeneous character of CD4+CD8+ DP T cell subsets. Based on the expression of cell marker genes, eight CD4+CD8+ DP T cells clusters were identified and named clusters 1 to 8 (Fig. 3A). CD3D, CD4, CD40LG, CD45, CD8A, CD8B, GZMB and GZMK were highly expressed in all eight clusters (Fig. 3B). We further analyzed the proportion of each DP T cluster to total DP T cells, it showed that the cluster 1 and cluster 2 were the predominant subsets (Fig. 3C). In INRs, the proportion of cluster 1 slightly decreased, and the percentage of cluster 2 increased inversely.
Moreover, we further compared the distinctive characteristics of eight clusters at the transcriptional level. Every cluster had specific transcriptomic features. For instance, cluster 1 highly expressed MACF1, PTPRC, LNPEP, and so on. Cluster 2 highly expressed PRF1, GZMH, CCL5 and S100A4 (Fig. 3D). According to the level of CD4, CD8A, and CD8B expressed in each cluster, we divided these 8 clusters into 3 subgroups, CD4highCD8low, CD4lowCD8high, and CD4lowCD8low, respectively (Fig. 3D, Fig. S1). That is, cluster 1 was the CD4highCD8low subtype, cluster 2 belonged to the CD4lowCD8high subtype, and clusters 3 to 8 were considered as CD4lowCD8low subtype (Fig. 3D). To clarify the heterogeneity of the three subtypes, then we also performed transcriptome analysis. In the CD4highCD8low subtype, the expression of predominant genes was much higher than the other two subtypes, CD4lowCD8high and CD4lowCD8low showed the opposite tendency (Fig. 3E). Notably, the CD4highCD8low subtype harbored a much higher expression of cytokine genes, such as IL2RA, IL2RB, IL4R, IL10, IL17RE, IL23R and IL26. Additionally, genes PRF1, GZMK, KLRK1, KLRD1, KLRF1 and KLRC1 related to cytotoxicity, were also highly expressed in the CD4highCD8low subtype (Fig. 3E). In summary, these data suggested that the CD4highCD8low subtype, in other words, cluster 1, was clearly distinguished from other DP T subpopulations, and highlighted the potential role of CD4highCD8low subtype on the HIV-related immunodeficiency.
Differentially expressed genes and related functions of CD4highCD8low DP T cells
To detect which genes of CD4highCD8low DP T cells were differentially expressed in INRs, we did pairwise comparisons among INRs, IRs and HCs, and noted significantly different genes (Fig. 4A). Subsequently, we performed a Venn diagram to reveal 47 differentially expressed genes (DEGs) closely associated with INRs (Fig. 4B), enumerated in Figure 4C. Compared to HCs, 17 genes were higher expressed, and MT-ND6, MAML2, TCF7, and LEF1 were much lower in INRs. However, except LINCO2384, KIR3DL1 and KLRC4 were highly expressed in INRs, while the other 22 DEGs were lower compared to IRs (Fig. 4C). Lower expressed genes from INRs, including TCF7, LEF1, CCR7, CD4 and CD27 are all primarily involved in the immune system process and cell differentiation. Furthermore, GO analysis revealed that these 47 DEGs common to INR donors were enriched in pathways involved in the regulation of cell adhesion, T-cell activation and differentiation (Fig. 4D).
We further analyzed the landscape of function of CD4highCD8low DP T cells in INRs using GSEA. Compared to HCs, genes that related to the pathway of cell fate specification, natural killer (NK) cell-mediated cytotoxicity and response to type I interferon were highly expressed in INRs. Compared to INRs, genes that related to the pathways of the viral protein interaction with cytokine and cytokine receptor, and the establishment of protein localization to endoplasmic reticulum were highly expressed in IRs (Fig. 4E). Taken together, the dysfunction of CD4highCD8low DP T cells' fate specification and cell differentiation could contribute to the immunological deficiency of INRs.
Differentiation of CD4hig CD8low DP T cells
Next, we examined the differentiation trajectories of CD4highCD8low DP T cells, naïve CD4+ SP T cells, and naïve CD8+ SP T cells, as well as compared the differences among IRs, INRs and HCs (Fig. 5A). In the cell trajectory, we identified two nodes indicating the direction of cellular differentiation branches, named node 1 and node 2. We showed that activated CD4highCD8low DP T cells were on the high differentiation statuses, which were distributed on the terminal of the trajectories (Fig. 5B). Naïve CD4+ and naïve CD8+ SP T cells were on the low differentiation statuses, which were distributed around node 1. After HIV infection, the number of Naïve CD4+ SP T cells and naïve CD8+ SP T cells around node 2 was much less than HCs. In HCs, CD4highCD8low DP T cells, naïve CD4+ SP T cells and naïve CD8+ SP T cells exhibited partially merged trajectories, CD4highCD8low DP T cells, naïve CD4+ SP T cells subtype 1 and naïve CD8+ SP T cells subtype 2 exhibited partially merged trajectories in IRs, whereas INRs did not exhibit merged trajectories in all three cell populations (Fig. 5C-G). These results suggested a distinct origin of CD4highCD8low DP T cells among different groups, which indicated that CD4highCD8low DP T cells with various origins may have multiple effects on immune reconstitution after HIV infection.
To investigate the functional heterogeneity of CD4highCD8low DP T cells with different differentiation trajectories, we analyzed the transcriptome of CD4highCD8low DP T cells in different statuses. Consistent with Fig. 5B, the CD4highCD8low DP T cells, naïve CD4+ SP T and naïve CD8+ SP T cells in both IRs and INRs were mainly distributed in state 1, state 3 and state 4 of the end of differentiation trajectories, respectively (Fig. 6A). HCs group possessed most three cell subtypes in state 2, while INRs had much fewer cells than HCs and IRs (Fig. 6A). Compared to state 3, CD4highCD8low DP T cells in state 4 had a decreased level of GZMA/B/H, LAG3, IL2RB, GNLY, NKG7 and KLRD1 (Fig. 6B and Fig. S2), which were related to the function of cytotoxicity. The decreased cell number in state 4 was identified in the INRs compared to the HCs and IRs (Fig. 5B). These results suggested that CD4highCD8low DP T cells with low cytotoxicity were reduced in INRs. Furthermore, CD4highCD8low DP T cells in state 3 were increased in IRs and INRs, suggesting CD4highCD8low DP T cells with high cytotoxicity increased after HIV infection.
In summary, these results demonstrated that the distinct origin of CD4highCD8low DP T cells had various functions, and different statuses of CD4highCD8low DP T cells had multiple roles in immune reconstitution after HIV infection.
Functional pathways of differentiation-signature genes
Subsequently, 81 differentiation-related signature genes that were differently expressed in CD4highCD8low DP T cells between state 3 and state 4 were used for GO and KEGG analysis in the Metascape database. The results showed that biological processes enriched in programmed cell death, T cell differentiation, and NK cell-mediated cytotoxicity (Fig. 7), which was consistent with the results in Fig. 4. We further analyzed CD4highCD8low DP T cell-related immune functions, such as cytokine production, regulation of lymphocyte activation and proliferation. The results showed distinctive gene profiles of CD4highCD8low DP T cells in INRs that were different from IRs (heatmaps in Fig. 7). It suggests that these differentiated genes had a significant role in regulating the immune reconstitution of HIV-infected patients.