Implications of accumulation of clonally expanded and senescent CD4⁺GNLY⁺ T cells in immunological non-responders of HIV-1 infection

ABSTRACT

Increased CD4⁺GNLY⁺ T cells have been confirmed to be inversely associated with CD4⁺ T cell count in immunological non-responders (INRs), however, the underlying mechanisms are unknown. This study aimed to elucidate the characteristics of CD4⁺GNLY⁺ T cells and their relationship with immune restoration. Single-cell RNA sequencing, single-cell TCR sequencing, and flow cytometry were used to analyze the frequency, phenotypes, and function of CD4⁺GNLY⁺ T cells. Moreover, Enzyme linked immunosorbent assay was performed to detect plasma cytokines production in patients. CD4⁺GNLY⁺ T cells were found to be highly clonally expanded, characterized by higher levels of cytotoxicity, senescence, P24, and HIV-1 DNA than CD4⁺GNLY⁻ T cells. Additionally, the frequency of CD4⁺GNLY⁺ T cells increased after ART, and further increased in INRs, and were positively associated with the antiretroviral therapy duration in INR. Furthermore, increased IL-15 levels in INRs positively correlated with the frequency and senescence of CD4⁺GNLY⁺ T cells, suggesting that CD4⁺GNLY⁺ T cells may provide new insights for understanding the poor immune reconstitution of INRs. In conclusion, increased, highly clonally expanded, and senescent CD4⁺GNLY⁺ T cells may contribute to poor immune reconstitution in HIV-1 infection.

GRAPHICAL ABSTRACT

Introduction

Since the discovery of human immunodeficiency virus type 1 (HIV-1) in 1983, approximately 38 million people have been infected worldwide [1,2]. Although most patients with HIV-1 infection received antiretroviral therapy (ART) and achieved immune reconstitution after ART, improving the outcome of HIV-1 infection remains a challenge because latent HIV-1 reservoirs are long-lived and > 20% of patients with HIV-1 infection developed poor immune restoration even after efficient ART [3,4]. Therefore, understanding of the immune mechanisms associated with persistent HIV-1 reservoir and poor immune reconstitution is critically important.

CD4⁺ cytotoxic T lymphocytes (CD4⁺ CTLs) are characterized by their ability to lyse target cells mainly through granular exocytosis, in an MHC class-II-restricted fashion [5,6]. In general, CD4⁺ CTLs are present in low numbers, comprising approximately 2% of the total CD4⁺ T cells in healthy individuals [7]; however, their frequency increases under pathological conditions such as autoimmune disorders disease [5,8], hematological malignancy [9], and virus infection [5]. During HIV-1 infection, CD4⁺ CTLs are clonally expanded and enriched with HIV-1 RNA, potentially contributing to HIV-1 persistence [10–12]. The underlying mechanisms are complex, including evasion of CD8⁺ T killing by granzyme B (GZMB) resistance and promoting longevity of cells by high expression of anti-apoptotic proteins.

CD4⁺ CTLs are heterogeneous in their secretion of cytotoxic granule proteins, including perforin, GZMB, and granulysin (GNLY). An increased frequency of CD4⁺KLRG1⁺ T cells with cytotoxic features is associated with disease relapse in Graves’ orbitopathy [8]. SLAMP7⁺CD4⁺ T cells are associated with good progression in melanoma [13]. Furthermore, the frequency of CD4⁺CD107a⁺ T cells is decreased in chronic hepatitis B virus infection, and hepatocellular carcinoma [14]. In our previous study, high levels of CD4⁺GNLY⁺ T cells were associated with poor immune restoration in patients with HIV-1 infection. However, the mechanism underlying the CD4⁺GNLY⁺ T cell-mediated failure of immune reconstitution is yet to be elucidated.

In this study, we aimed to analyze the frequency, phenotypes, and functional characteristics of CD4⁺GNLY⁺ T cells, and the correlations between the frequencies of CD4⁺GNLY⁺ T cells and CD4⁺ T cell counts, CD4/CD8 ratio, and ART duration in different stages of chronic HIV-1 infection. Finally, cytokines that were associated with the increase of CD4⁺GNLY⁺ T cells were analyzed. Overall, our study provided preliminary insights into the characteristics of CD4⁺GNLY⁺ T cells in patients with HIV-1 infection.

Methods

Study participants

A total of 60 patients with HIV-1 infection were recruited from the Fifth Medical Center of Chinese PLA General Hospital, between January 2021 and November 2023. The chronic HIV-1 infected patients were divided into the treatment-naive (TN) patients and ART groups. Twenty patients who had a plasma HIV-1 RNA level > 1000 copies/ml without ART [15] were grouped as TN, 20 patients who had received ART for >2 years and had CD4⁺ T cell count > 350 cells/mm³ were grouped as immunological responders (IRs) [16], and 20 patients who had received ART for >2 years and had CD4⁺ T cell count < 250 cell/mm³ were grouped as INRs [17]. HIV-1 RNA levels were undetectable in all patients who underwent ART. Patients with opportunistic infections combined with tumours or viral infections (including hepatitis viruses type A–E) were excluded. The clinical characteristics of the enrolled patients are listed in Table 1. No statistically significant differences were observed in characteristics among TN patients, IR, and INR groups at enrollment. A flowchart of the study design is shown in Supplementary Figure 1. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Fifth Medical Center of Chinese PLA General Hospital (Number: 2016164D). All the participants provided informed consent.

Table 1.

Characteristics of patients with HIV-1 infection in the study.

	TN	ART
		IR	INR
Total, n	20	20	20
Age (years)	34 (21–57)	34 (23–49)	38 (29–56)
Gender, n Male Female	20 0	20 0	20 0
CD4+ T cell count (cells/ul)	292 (106–659)	650 (354–1177)	173 (102–230)
CD8+ T cell count (cells/ul)	1030 (375–1659)	778.5 (379–1287)	512 (276–1239)
CD4/CD8 ratio	0.28 (0.11–0.93)	0.87 (0.37–1.54)	0.28 (0.16–0.65)
HIV-1 RNA (log10 copies/ml)	4.67 (3.50–6.51)	ND	ND
Time on ART (months)	0	41.5 (24–60)	43 (25–73)
Mode of HIV transmission, n
MSM	19	15	17
Others	1	5	3
ART regimens, n
3TC/TDF/EFV	NA	18	17
Others	NA	2	3
Nadir CD4+ T cell count (cells/ul)	292 (106–659)	332 (59–697)	14 (3–39)

Note:^:Data were expressed as counts for categorical variables and as the median and range for continuous variables. TN, treatment-naive; ART, antiretroviral therapy; IR, immunological responder; INR, immunological non-responder; MSM, men who have sex with men; NA, not applicable; ND, not determined.

Single-cell RNA sequencing (scRNA-seq) analyses and single-cell TCR sequencing (scTCR-seq) analyses

Raw scRNA-seq data (10× Genomics) and scTCR-seq data from CD4⁺ T cells purified from peripheral blood mononuclear cells (PBMCs) were processed as previously reported [16]. Sequencing data has been uploaded to the Genome Sequence Archive in Beijing Institute of Genomics, Chinese Academy of Science with accession number HRA0000190. The scRNA-seq data were processed using the Cell Ranger (v.6.0.1) pipeline against the GRCh38 human reference genome to generate gene expression matrices. Quality control was performed using the Seurat package (v.4.0.5) in R software (v.4.1.0). The criteria for cell retention were the same as a previous report [18]. The scrublet package (v.0.2.3) was used to remove potential doublets with doublets scores > 0.4 and an expected doublet rate of 6%. Seurat’s Normalize Data function was applied to normalize and log-transform the filtered gene-cell expression matrix, which was then processed for dimension reduction, batch effect correction, and unsupervised clustering. For CD4⁺ T cells, the resolution was set to 0.15, and the dimensions of reduction were set to 1–30. The method for integrating multiple datasets (each for one person) can be found in a previously published articles [18]. All clusters were manually annotated according to their hallmark genes. The bubble heat map was used to show the expression distribution of selected canonical cell markers in different clusters. T cell antigen receptor (TCR) V(D)J sequencing and analysis were performed in the same manner as previously described [16]. Each unique TCR α-chain (TRA) and TCR β-chain (TRB) pair was defined as a clonotype.

Flow cytometric analysis

PBMCs were freshly isolated from the peripheral blood of patients. PBMCs were labelled with fluorescently conjugated antibodies for detecting surface or intracellular protein, according to the manufacturer’s instructions [18]. Briefly, PBMCs were stained with Fixable Viability Stain 575v (live/dead) and surface antibodies at 4 ℃ for 30 min. After washing, cells were permeabilized and fixed at 4 ℃ for 30 min and then stained with intracellular antibodies at 4 ℃ for 30 min. Finally, cells were fixed in 1% paraformaldehyde. For gating CD4⁺GNLY⁺ T cells, anti-CD3, anti-CD8, anti-CD4, anti-CD158e1 (KIR3DL1), anti-TCRγδ, and anti-GNLY were selected. Furthermore, anti-CD45RA and anti-CCR7 antibodies were used to detect the memory markers. The cytotoxic markers of CD4⁺GNLY⁺ T cells were treated with the following antibodies: anti-T-bet, anti-EOMES, anti-perforin, anti-GZMB, anti-CX3CR1, and anti-KLRG1. Senescence markers were detected using anti-CD28, anti-CD27, anti-CD57, and anti-CD49d antibodies. The activity of Senescence-associated β-galactosidase (SA-β-gal) was detected by the SA-β-gal activity assay kit (35302, CST). To detect CXCR4 and CCR5, both are HIV-1-infected co-receptors, anti-CXCR4 and anti-CCR5 antibodies, were used. Anti-CD122, anti-CD212, and anti-CD218a antibody were used to detect CD122 (an IL-15 receptor), CD212 (an IL-12 receptor), and CD218a (an IL-18 receptor) respectively. Detailed information on the antibodies is provided in Supplementary Table 1. Flow cytometric analysis was performed using a BD FACSymphony A5 flow cytometer (BD Biosciences). Data were analyzed using NovoExpress software (Acea Bioscience, Inc.) and FlowJo software (FlowJo).

HIV-1 reactivation assay

Cryopreserved PBMCs (4 × 10⁶) were thawed, washed, and resuspended in complete medium. PBMCs were then stimulated in 162 nM phorbol 12-myristate 13-acetate (PMA, P1585, sigma), 1 µg/ml ionomycin (Iono, 10634, sigma), and 2 uM Golgistop (554724, BD Biosciences) for 24 h at 37 ℃, with 5% CO₂, and then stained for flow cytometry. Briefly, PBMCs were stained with Fixable Viability Stain 575v (live/dead), anti-CD3, anti-CD8, anti-CD158e1(KIR3DL1), and anti-TCRγδ antibodies at 4 ℃ for 30 min. After washing with media, PBMCs were permeabilized and fixated using 200 µl of BD Cytofix/Cytoperm Fixation/Permeabilization Kit at 4 ℃ for 30 min. PBMCs were washed with permeabilization buffer and then stained with a combination of anti-GNLY and anti-HIV-1 P24 antibodies at 4 ℃ for 30 min. Detailed information on the antibodies is provided in Supplementary Table 1.

HIV-1 DNA quantification

PBMCs were stained with flow cytometry antibodies at room temperature for 30 min and then sorted into CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells using a MA900 flow cytometer (SONY) (gate strategy and sorted cell GNLY purity are shown in Supplementary Figure 2). Total cellular DNA was extracted from sorted CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells using Qiagen QIAsymphony DNA Mini Kits (Qiagen). HIV-1 DNA levels were quantified using a fluorescence-based real-time SUPBIO HIV-1 Quantitative Detection Kit (SUPBIO) and a QuantStudio Dx Real-Time PCR Instrument (Biosystems) [19]. To quantify cell numbers, the actin gene was simultaneously assessed under the same PCR conditions.

Plasma cytokine detection

The plasma concentrations of IL-15 and IL-12 were detected using an IL-15 ELISA Kit (Cat: D1500) and IL-12 ELISA Kit (Cat: ab46035), respectively, according to the manufacturer’s instructions (R&D Systems, USA and Abcam, UK, respectively).

Statistical analysis

All statistical data were calculated and analyzed using IBM SPSS Statistics version 25. The results are presented as medians with ranges (minimum–maximum). The two groups were compared using the unpaired nonparametric Mann–Whitney U test. Paired data were compared using the Wilcoxon matched-pairs signed-rank test. Correlations between variables were assessed using Spearman’s correlation coefficient (r). Statistical significance was set at P < 0.05.

Results

Significant accumulation of clonally expanded CD4⁺GNLY⁺ T cells in patients with HIV-1 infection

By dimensionality reduction and clustering analysis, seven major CD4⁺ T cell subsets were identified according to the expression of canonical gene markers, including naïve cells (CCR7⁺TCF7⁺LEF1⁺), effector memory cells (TNFRSF4⁺GATA3⁺S100A4⁺), regulatory T cells (Treg) (FOXP3⁺), CD4⁺GZMK⁺ T cells (GZMK⁺), CD4⁺proliferating (MKI67⁺PCNA⁺) T cells, mucosal-associated invariant T (MAIT) (TRAV1-2⁺SLC4A10⁺) cells, and CD4⁺GNLY⁺ T cells (GNLY⁺) (Figure 1(A, B)). We found that CD4⁺GNLY⁺ T cells displayed significantly higher clonal expansion than other CD4⁺ T cell subsets in the scTCR-seq analysis (Figure 1(C)), and the frequency of CD4⁺GNLY⁺ T cells increased with ART (Figure 1(D)). Notably, we found that the majority of the CD4⁺GNLY⁺ T cells were T_EM cells characterized by the negative expressions of CD45RA and CCR7, followed by terminally differentiated effectors memory T (T_EMRA) cells (P < 0.0001 for T_EM and P = 0.0009 for T_EMRA) (Figure 1(E–G)), highlighting that CD4⁺GNLY⁺ T cells were antigen experienced and expanded after HIV-1 infection.

Figure 1.

CD4⁺GNLY⁺ T cells showed a significant clonally expanded effector memory T cell (T_EM) phenotype in patients with HIV-1 infection. (A) Populations for CD4⁺ T cells. The uniform manifold approximation and projection (UMAP) projection of 37,960 single cells, showing the formation of seven clusters with the respective labels. (B) Dot plots showing the expression levels of selected canonical cell markers in the seven clusters of CD4⁺ T cells. The size of the circle indicates the percentage of cells expressing pathway-associated genes under each condition. The colour of the circle represents the expression levels of pathway-associated genes under each condition, and the colour blue means a relatively high expression level, and white means a relatively low expression level. (C) UMAP plots showing T cell antigen receptor (TCR) clonotype counts in patients with HIV-1 infection. (D) Pool data showing the frequencies of CD4⁺GNLY⁺ T cells in CD4⁺ T cell population in treatment-naïve (TN) and antiretroviral therapy (ART) patients (n = 20 and 40, respectively). (E) Representative dot plot of memory phenotypes of circulating CD4⁺GNLY⁺ T cells from one patient. Values in the quadrant indicate the percentage of corresponding subset. (F) Pool data indicating the distribution of memory phenotypes of CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells (n = 7 for TN patients, n = 8 for ART patients). (G) Pie charts showing the proportions of memory phenotypes of CD4 ⁺GNLY^- T and CD4⁺GNLY⁺ T cells in patients with HIV-1 infection (n = 15). P values are shown. TN, treatment-naïve; ART, antiretroviral therapy; T_EM, effector memory T cell; T_EMRA, terminally differentiated effector memory T cells.

CD4⁺GNLY⁺ T cells have cytotoxic characteristics

Interestingly, we found that CD4⁺GNLY⁺ T cells expressed higher levels of cytotoxic genes including GZMA, GZMB, GZMH, PRF1, CX3CR1, TBX21, EOMES, NKG7, KLRD1, KLRG1, and KLRK1 (Figure 2(A)) and higher levels of cytotoxic proteins including KLRG1, CX3CR1, perforin, GZMB, EOMES, and T-bet (all P < 0.0001, Figure 2(B–G)) when compared to CD4⁺GNLY⁻ T cells, suggesting that CD4⁺GNLY⁺ T cells were cytotoxic CD4⁺ T cells. Furthermore, although the expression of EOMES increased after ART (P = 0.0055, Figure 2(F)), it cannot be ignored that CX3CR1, EOMES, and T-bet were significantly reduced in INRs compared to IRs (P = 0.0185 for CX3CR1, P = 0.0175 for EOMES, and P = 0.0147 for T-bet, Figure 2(C,F,G)), and the GZMB also showed a decreasing trend (Figure 2(E)), which highlight the cytotoxicity of clonally expanded CD4⁺GNLY⁺ T cells, and the cytotoxicity is weakened in INRs compared to IRs even after ART.

Figure 2.

CD4⁺GNLY⁺ T cells displayed a dominant cytotoxic phenotype in patients with HIV-1 infection. (A) Dot plot showing expression of selected cytotoxic genes in CD4⁺GNLY⁻ T cells and CD4⁺GNLY⁺ T cells from TN and ART patients. The size of the circle indicates the percentage of cells expressing cytotoxic genes under each condition. The colour of the circle represents the expression levels of cytotoxic genes under each condition, and the colour green/orange represents a relatively high expression level, respectively, and white means a relatively low expression level. (B–G) Pool data showing the expression levels of KLRG1, CX3CR1, perforin, GZMB, EOMES, and T-bet of CD4⁺GNLY⁻ T cells and CD4⁺GNLY⁺ T cells from patients with HIV-1 infection across the three groups (n = 10 for each group). Orange dashed box represents ART groups, including IRs and INRs. Pink dashed box represents all groups, including TN patients, IRs, and INRs. P values are shown. TN, treatment-naïve; ART, antiretroviral therapy; IR, immunological responder; INR, immunological non-responder.

CD4⁺GNLY⁺ T cells have senescent characteristics

Persistent HIV-1 stimulation leads to chronic immune activation, contributing to the development of T cell cellular senescence [20]. In the scRNA-seq analysis, we found that CD4⁺GNLY⁺ T cells expressed higher levels of HLA-DRA and HLA-DRB1 when compared with CD4⁺GNLY⁻ T cells (Figure 3(A)), suggesting that CD4⁺GNLY⁺ T cells were stimulated. Besides, we also found that the expression of senescent genes, including B3GAT1 (encoding CD57), ITGA4 (encoding CD49d), PDCD1 (encoding PD-1), HAVCR2 (encoding Tim-3), and KLRG1 increased, whereas the expression of CD27 and CD28, co-stimulatory molecules providing second signal decreased (Figure 3(A)), and the anti-apoptosis genes, including BCL2L1, BCL2L2, MTRNR2L3, RFFL, and SERPINB9 were increased (Figure 3(B)). Furthermore, the golden standard marker for cellular senescence, SA-β-gal, was much higher in CD4⁺GNLY⁺ T cells than in CD4⁺GNLY⁻ T cells (Figure 3(C)). In the flow cytometric analysis, we found that the CD27 and CD28 decreased, and the CD57 and CD49d increased, especially in those CD4⁺GNLY⁺ T cells than CD4⁺GNLY⁻ T cells (all P < 0.0001, Figure 3(D–G)). Additionally, after efficient ART, the expression of CD28 increased (P = 0.0439, Figure 3(E)), and the expression of CD57 decreased (P = 0.0276, Figure 3(F)). Furthermore, CD49d was increased in INRs compared to IRs (P = 0.0185, Figure 3(G)), highlighting that the senescence of CD4⁺GNLY⁺ T cells in INRs, even after long-term ART.

Figure 3.

CD4⁺GNLY⁺ T cells displayed a dominant senescent phenotype in patients with HIV-1 infection. (A) Dot plot showing expression of selected activation and senescence genes of CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from TN and ART patients. The size of the circle indicates the percentage of cells expressing activation and senescence genes under each condition. The colour of the circle represents the expression levels of activation and senescent genes under each condition, and the colour green/orange represents a relatively high expressions level in CD4⁺GNLY⁻ T cells and CD4⁺GNLY⁺ T cells, respectively, and white means a relatively low expression level. (B) Heat map showing expression of anti-apoptosis genes of CD4⁺GNLY⁻ T cells and CD4⁺GNLY⁺ T cells in TN and ART patients. The colour orange represents a relatively high expression level and green means a relatively low expression level. (C) Representative mean fluorescence intensity (MFI) image of senescence-associated β galactosidase (SA-β-Gal) activity in control, circulating CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from a patient. Values above indicate the MFI of SA-β-Gal under each condition. (D – G) Pool data showing the expression levels of CD27, CD28, CD57, and CD49d on CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from patients with HIV-1 infection across the three groups (n = 10 for each group). Orange dashed box represents ART groups, including IR and INR. Pink dashed box represents all groups, including TN patients, IRs, and INRs. P values are shown. TN, treatment-naïve; ART, antiretroviral therapy; SA-β-Gal, senescence-associated β galactosidase; IR, immunological responder; INR, immunological non-responder.

CD4⁺GNLY⁺ T cells are associated with HIV-1 persistence

Notably, we found that CD4⁺GNLY⁺ T cells, but not CD4⁺GNLY⁻ T cells, expressed high levels of the HIV-1 long-term persistence related genes, including HIV-1 receptors and transcript indicators (CCR5, CXCR4, and PRDM1), helper T cell 1 (Th1) – related indicators (STAT1, CXCR3, IL2, and IFNG), naïve/memory indicators (CCR7, SELL, and S100A4), immune checkpoint indicators (PDCD1, TIGIT, and LAG3), resting/activating indicators (HLA-DRB1, CD69, CD27, and ITGA4), and cytokines receptors (IL2RA and IL2RG) (Figure 4(A)). Additionally, the expression of CXCR4 and CCR5 levels that facilitate HIV-1 entry were simultaneously detected. The expression of CXCR4 on CD4⁺GNLY⁺ T cells increased in IRs (P = 0.0232, Figure 4(B)), and the expression of CCR5 levels on CD4⁺GNLY⁺ T cells increased compared to the CD4⁺GNLY⁻ T cells in all of the patients (all P < 0.01, respectively, Figure 4(C)). In addition, the expression of CCR5 on CD4⁺GNLY⁺ T cells decreased in IRs compared to TN patients (P < 0.0001, Figure 4(C)). Meanwhile, there was no correlation of frequency of circulating CD4⁺GNLY⁺ T cells between plasma HIV-1 RNA levels in TN (data not shown), indicating that the high expression of CCR5 is an inherent characteristic of CD4⁺GNLY⁺ T cells. Furthermore, to identify the function of CD4⁺GNLY⁺ T cells, CD3 ⁺KIR3DL1^-TCRγδ^-CD8⁻ cells were firstly selected as purified CD4⁺ T cells, and co-expressed CD57 [21], KLRG1 [8], and CX3CR1 [22] were selected to substitute and improve the purity of GNLY (Supplementary Figure 2). We found that CD4⁺GNLY⁺ T cells were enriched with HIV-1 DNA (P = 0.0344, Figure 4(D)) and expressed high levels of P24 (P = 0.0078, Figure 4(E)).

Figure 4.

CD4⁺GNLY⁺ T cells showed higher concentrations of HIV-1 DNA in ART than in CD4⁺GNLY⁻ T cells. (A) Dot plot showing expression of HIV-1 reservoir-associated genes of CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from TN and ART patients. The size of the circle indicates the percentage of cells expressing HIV-1 reservoir-associated genes under each condition. The colour of the circle represents the expression levels of HIV-1 reservoir associated genes under each condition, and the colour green/orange means a relatively high expression level respectively, and white means a relatively low expression level. (B-C) Pool data showing the expression levels of CXCR4 and CCR5 on CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from patients with HIV-1 infection across the three groups. Green lines represent CD4⁺GNLY⁻ T cells and orange lines represent CD4⁺GNLY⁺ T cells (n = 10 for each group). (D) The data showing the quantification of HIV-1 DNA in CD4⁺GNLY⁻ T cells and CD4⁺GNLY⁺ T cells from ART patients (n = 7 for each group). (E) Pool data showing the expression levels of P24 on CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from ART patients after being treated with medium or PMA (162 nM) plus Iono(1ug/ml) for 24 h (n = 8 for each group). The colour blue represents P24 expression level in control group under each subset, and red represents P24 expression level in PMA and Iono group under each subset. P values are shown. TN, treatment-naïve; ART, antiretroviral therapy; Th1, helper T cell 1; IR, immunological responder; INR, immunological non-responder; PMA, phorbol 12-myristate 13-acetate; Iono, ionomycin.

Increased CD4⁺GNLY⁺ T cells are associated with ART duration in INRs

Considering the increase in CD4⁺GNLY⁺ T cells in patients with HIV-1 infection, the association between CD4⁺GNLY⁺ T cells and CD4⁺ T cell counts and CD4/CD8 ratio were analyzed across different stages. We found that the frequency of CD4⁺GNLY⁺ T cells was higher in INRs than in IRs (P < 0.0001, Figure 5(A)). The frequency of CD4⁺GNLY⁺ T cells was slightly negatively correlated with CD4⁺ T cell counts (P = 0.07, r = −0.41, Figure 5(B)) and significantly correlated with CD4/CD8 ratio (P = 0.03, r = −0.48, Figure 5(C)) in IRs. On the contrary, the frequency of CD4⁺GNLY⁺ T cells has no relationship with CD4⁺ T cell counts (P = 0.60, r = −0.13, Figure 5(E)) and CD4/CD8 ratio in INRs (P = 0.81, r = −0.18, Figure 5(F)). Importantly, we found that the frequency of CD4⁺GNLY⁺ T cells has no significant correlation with ART duration in IRs (P = 0.07, r = 0.41, Figure 5(D)), while it positively correlated with ART duration in INRs (P < 0.01, r = 0.61, Figure 5(G)). These findings implied that the increase of CD4⁺GNLY⁺ T cells is associated with poor immune reconstitution.

Figure 5.

CD4⁺GNLY⁺ T cells were Increased and associated with ART duration in INRs. (A) Pool data showing the frequencies of CD4⁺GNLY⁺ T cells in the CD4⁺ T cells population in IRs and INRs (n = 20 for each group). (B – G) Correlations between the frequencies of CD4⁺GNLY⁺ T cells and CD4⁺ T cell counts, CD4/CD8 ratio, and ART duration were calculated in IRs or INRs, respectively (n = 20 for each graph). Green plots represent IR group, and yellow plots represent INR group. P values are shown. TN, treatment-naïve; ART, antiretroviral therapy; IR, immunological responder; INR, immunological non-responder.

Increased IL-15 levels were associated with high expression of CD4⁺GNLY⁺ T cells in INRs

Cytokines produced during HIV-1 infection may be associated with a high frequency of CD4⁺GNLY⁺ T cells. Using scRNA-seq analysis, we found that the CD4⁺GNLY⁺ T cells expressed higher levels of cytokine receptor genes including IL2RB, IL2RG, IL12RB1, IL15RA, and IL-18R1 than CD4⁺GNLY⁻ T cells (Figure 6(A)). The expression of CD122, which represents the ligand for IL-15 and IL-2, increased in INRs compared to IRs (P = 0.0355, Figure 6(B)). Whereas, the expression of CD212 (IL-12 receptor) and CD218a (IL-18 receptor) showed no obvious differences between IRs and INRs (Figure 6(C,D)). Furthermore, plasma IL-15 levels, but not IL-12 levels (data not shown), were higher in INRs than in IRs (P = 0.0002, Figure 6(E)), and the frequency of CD4⁺GNLY⁺ T cells was positively associated with IL-15 levels (P < 0.01, r = 0.45, Figure 6(F)). Furthermore, we found that plasma IL-15 levels were positively associated with CD49d, which is a senescence marker (P = 0.02, r = 0.61, Figure 6(G)). These findings indicated that increased plasma IL-15 levels, may be associated with the accumulation and senescence of CD4⁺GNLY⁺ T cells in INRs.

Figure 6.

Frequency of CD4⁺GNLY⁺ T cells increased significantly with elevated IL-15 levels in ART patients. (A) Dot plot showing expression of cytokine receptors-associated related genes of CD4⁺GNLY⁻ T and CD4⁺GNLY⁺ T cells from IRs and INRs. The size of the circle indicates the percentage of cells expressing cytokine receptor-associated genes under each condition. The colour of the circle represents the expression levels of cytokine receptor-associated genes under each condition, and the colour green/orange means a relatively high expression level respectively, and white means a relatively low expression level. (B–D) Pool data showing the MFI of CD122 (IL-15 receptor), CD212 (IL-12 receptor), and CD218a (IL-18 receptor) on CD4⁺GNLY⁺ T cells in IRs and INRs (n = 10 for each group). (E) Fold change values showing the plasma IL-15 levels in IRs and INRs. Fold change values are defined as the ratio of each value/median value of TN patients (n = 20 for each group). (F) A significant correlation was observed between the frequencies of CD4⁺GNLY⁺ T cells and plasma IL-15 levels in ART patients (n = 20 for each group). Green plots represent IR group, and yellow plots represent INR group. (G) A significant correlation was observed between the plasma IL-15 levels and expression levels of CD49d on CD4⁺GNLY⁺ T cells in ART patients (n = 7 for IR group, n = 8 for INR group). Green plots represent IR group, and yellow plots represent INR group. P values are shown. MFI, median fluorescence intensity; TN, treatment-naïve; ART, antiretroviral therapy; IR, immunological responder; INR, immunological non-responder.

Discussion

HIV-1 infection results in progressive loss of CD4⁺ T cells and immunological disorders. The dichotomous roles of CD4⁺ CTLs in HIV-1 control and persistence during chronic HIV-1 infection remain unclear. Here, we identified one type of CD4⁺ CTLs, coined as CD4⁺GNLY⁺ T cells, which were characterized by high clonal expansion, dominant senescence, and HIV-1 DNA enrichment. Notably, an increase in these cells was associated with poor immune reconstitution in INRs. Furthermore, high levels of IL-15 production were attributed to the enlargement and senescence of these cells in INRs. Understanding the phenotype and function of CD4⁺GNLY⁺ T cells may be beneficial for elucidating the immune mechanisms that lead to poor immune restoration during HIV-1 infection.

Initially, CD4⁺ CTLs were identified as artificial owing to their properties of exhaustion and highly clonal expansion in vitro. However, eventually, CD4⁺ CTLs were discovered to possess cytotoxic activity in vivo. CD4⁺ CTLs are heterogeneous according to the secretion of cytotoxic granule proteins, and include perforin ⁺CD4⁺ T, CD4 ⁺GZMB⁺ T, and CD4⁺ GNLY⁺ T cells. In our previous study, we found that increased CD4+ Effector-GNLY T cells frequency are associated with poor outcomes in INRs. This allowed for a comprehensive examination of the characteristics of CD4⁺GNLY⁺ T cells in this study.

Previous studies have delineated the cytotoxicity of CD4⁺GNLY⁺ T cells. However, in this study, we demonstrated that the CD4⁺GNLY⁺ T cells were highly clonally expanded, and mainly constituted by T_EM cells. The reasons for the expansion of CD4⁺GNLY⁺ T cells may be associated with repeated antigen stimulation [23], homeostasis expansion [23], and integration of HIV-1 genes into cancer-related genes [24]. The senescent phenotype may contribute to the longevity of these cells, and high anti-apoptosis genes expression may confer CD4⁺GNLY⁺ T cells with resistance to death. Although the expansion of CD4⁺GNLY⁺ T cells with ART excludes HIV-1 antigen – induced cell proliferation, other mechanisms remain to be elucidated. Moreover, INRs cause severe destruction of the intestinal barrier, and bacterial translocation contributes to the proliferation of GNLY⁺ T cells, which is also powered by antibiotic function [25]. In addition, CD4⁺GNLY⁺ T cells express high levels of SERPINB9, which significantly antagonizes GZMB production by CD8⁺ T cells [10]. Therefore, the reason for the persistence and proliferation of CD4⁺GNLY⁺ T cells may be complex and requires further investigation.

HIV-1 replication and latency have been observed in CD4⁺ T cells, ever after effective ART; the cells harbouring HIV-1 can produce infectious HIV-1 viruses if ART is discontinued [26]. Our study reveals that CD4⁺GNLY⁺ T cells may contribute to HIV-1 persistence. First, CD4⁺GNLY⁺ T cells are more susceptible to HIV-1 infection because of increased expression of CCR5 than CD4⁺GNLY⁻ T cells. Second, CD4⁺GNLY⁺ T cells contained more integrated HIV-1, owing to the higher HIV-1 DNA content and the ability to reproduce HIV-1 after stimulation. Third, intracellular HIV-1 P24 in CD4⁺GNLY⁺ T cells was increased in comparison with that in their counterparts after stimulation in patients who had received ART for >2 years, implying the characteristics of the HIV-1 reservoir of CD4⁺GNLY⁺ T cells. Finally, CD4⁺GNLY⁺ T cells were similar to cells harbouring HIV-1 persistence, such as significant clonal expansion [27], memory [10] and Th1 phenotype [28] (TBX21), dominant cytotoxicity (GZMA, GZMB, GZMH) [10,11], higher immune checkpoint genes expression (PDCD1, TIGIT, LAG3) [29], and difficult activation [30] (lack of CD27, CD28). In addition, compared to CCR5⁺CD4⁺GNLY⁻ T cells, CCR5⁺CD4⁺GNLY⁺ T cells were significantly more prevalent in all three patient groups, with lower frequency in the IRs versus INRs. Considering no correlation between the frequency of CCR5⁺CD4⁺GNLY⁺ T cells and plasma HIV-1 RNA levels in TN (data not shown), HIV-1 was precluded in the contribution of the expansion of CCR5⁺CD4⁺GNLY⁺ T cells. Moreover, it cannot be ignored that the expression of CCR5 is also regulated by the inflammatory and senescent microenvironments, which deteriorate in HIV-1 infected patients, especially in TN patients and INRs [31–34]. The reasons for the fluctuation of CCR5⁺CD4⁺GNLY⁺ T cells may be complicated across the three groups, and need further investigation.

Cytokines increased in INRs may be associated with a high frequency of CD4⁺GNLY⁺ T cells. Therefore, cytokines receptors genes were detected and found that IL2RB, IL2RG, IL12RB1, IL15RA, and IL18R1 showed higher expression in CD4⁺GNLY⁺ T cells than CD4⁺GNLY⁻ T cells. IL-2 is necessary for the survival of lymphocytes, but the IL-2 special receptors gene IL2RA didn’t increased in CD4⁺GNLY⁺ T cells. Thus, the contribution of IL-2 in the accumulation of CD4⁺GNLY⁺ T cells may be precluded. Additionally, IL-12 receptor CD212 and IL-18 receptor CD218a on CD4⁺GNLY⁺ T cells have no statistically significance in INRs compared to IRs. Meanwhile, considering the lack of a variation in plasma IL-12 levels [35] and IL-18 [35,36] between IRs and INRs, the significances of IL-12 and IL-18 were precluded in the augment of CD4⁺GNLY⁺ T cells. IL-15 was indicated in the promotion of HIV-1-infected CCR5⁺CD4⁺ T cells [37] and was associated with high susceptibility of memory CD4⁺ T cells to simian immunodeficiency virus (SIV) infection [38]. IL-15 receptor consists of three subunits, including CD215 (coded by IL15RA), CD122 (coded by IL2RB), and CD132 (coded by IL2RG). CD132 was shared by multiple cytokines receptors without high specificity. CD215 generally existed in the cytoplasm of antigen-presenting cells (such as dendritic cells) [39] and naïve T cells [40], and was short of signalling functions [41,42]. CD122 is generally presented on the cell membrane of effector and memory cells, with moderate affinity for IL-15. Therefore, CD122 rather than CD215 may be better to reflect the reaction level of CD4 GNLY T cells to IL-15. In our study, we found that CD4⁺GNLY⁺ T cells expressed higher IL-15 receptor CD122 in INRs but not IRs, in agreement with the increased plasma IL-15 levels in INRs but not IRs, and the level of IL-15 was positively related to the frequency of CD4⁺GNLY⁺ T cells and senescent marker CD49d. Taken together, IL-15 may be associated with the augment and senescence of CD4⁺GNLY⁺ T cells via some uncertain mechanisms.

This study has several limitations, including a restricted sample size, which challenges the robustness of its findings. Furthermore, the sorting of CD4⁺GNLY⁺ T cells was conducted by different substitute markers, such as CD3, CD8, KIR3DL1, TCRγδ, KLRG1, CD57, and CX3CR1, which may influence the rigour of the in vitro experiment. Most importantly, the contribution of IL-15 in the proliferation and senescence of CD4+GNLY+ T cells, especially in INRs, necessitates mechanistic validation.

Overall, our study indicated that highly clonally expanded and senescent CD4⁺GNLY⁺ T cells were enriched with HIV-1 DNA. The frequency of CD4⁺GNLY⁺ T cells increased with ART duration in INRs. Additionally, increased IL-15 levels may contribute to the persistence of CD4⁺GNLY⁺ T cells in INRs. Research focusing on CD4⁺GNLY⁺ T cells may provide a better understanding of the poor immune restoration observed in INRs.

Funding Statement

This work was supported by grants from National Key Research and Development Program of China [2022YFC2304403; 2023YFC2306800], the National Natural Science Foundation of China [82171732 and 82271781], and National Clinical Research Center for Infectious Diseases [NCRC-ID202109].

Disclosure statement

No potential conflict of interest was reported by the author(s).