Cluster of differentiation 8 (CD8 T) lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV) replication. CD8 T cells may be divided into two subpopulations on the basis of CD8 molecule protein expression level: one population of T cells exhibits low expression of the CD8 receptor (CD8low),[1,2] and the other population of T cells bears relatively high CD8 protein expression (CD8high). The number of CD8low T cells has been reported to be increased in viral infections, such as HIV-1,[3,4] hepatitis B virus (HBV), hepatitis C virus (HCV), and bacterial infections; in progressive forms of multiple sclerosis; and after kidney or liver transplantation. CD3+CD8low T cells in asthmatic children were increased compared with those in nonasthmatic children, partially accounting for the role played by CD3+CD8low T cells in asthma. Indeed, the emergence of circulating CD8low T cells population has been associated with poor disease outcomes in chronic HIV infection. In acute HIV-1 infection (AHI), the frequency of CD8low T cells directly correlated with viral load and clinical predictors of more rapid disease progression. Evidence has shown that CD8 is downregulated in lymphoid and mucosal tissues in early and chronic simian immunodeficiency virus (SIV) infection and even on SIV-specific cytotoxic lymphocytes, which exhibited lower capacity for proliferation and cytokine responses, especially in lymphoid tissues.
After HIV-1 enters individuals, a large number of CD4+ T cells can be infected. At this stage, anti-HIV immunity has not yet been established, so HIV replication and transmission remain uncontrolled to a large extent. Then, as HIV-1 viremia reaches peak levels until the adaptive immune response is initiated, particular HIV-1-specific CD8 T-cell responses. During acute HIV-1 infection, the number of CD8 T cells increases significantly at the time of peak viremia, while that of CD4 T cells decreases sharply. After peak viremia, the CD8 T-cell count decreases, and the CD4 T-cell count increases, and the levels never return to a normal range. Indeed, in addition to the dynamic alteration in the quantity of CD8 T cells after the HIV-1 infection of individuals, CD8 T cells exhibit a profound cascade of immunological events, leading to increased immune activation, cell turnover, and cell differentiation and causing the number of naïve cells to decease and that of highly differentiated cells to increase; all of these effects induce the exhaustion, immunosenescence, and sustained cell apoptosis of CD8+ T cells.
In murine T-cell receptor (TCR) transgenic models, CD8low T cells derived from a proliferative compartment exhibit a short half-life, as well as low proliferative capacity. CD8low T cells are characterized by an activation phenotype and are functionally compromised in mice. Nonetheless, little is known about the immunological events in the CD8low T-cell population, including changes in the number of CD8 T cells or their immune activation, inhibitory molecule expression or HIV-1-specific responses induced by acute and chronic HIV-1 infection. Our findings in this study lead to additional insights into the changing roles played by CD8low T cells post HIV infection.
This study and all the relevant experiments were approved by the Beijing Youan Hospital Research Ethics Committee (No. 2020-063), and written informed consent was obtained from each participant in accordance with the Declaration of Helsinki. All participants provided written informed consent for the collection of information and for clinical samples to be stored and used for research. The methods used conformed to approved guidelines and regulations.
The subjects in this study were recruited from the Beijing PRIMO clinical cohort, a prospective study cohort of HIV-negative men who have had sex with men established to identify cases of acute HIV-1 infection at Beijing Youan Hospital, Beijing, China, starting in October 2006. The enrolled subjects were monitored every 2 months for HIV antibodies, HIV ribonucleic acid (RNA) levels, and clinical signs of acute/early infection, as previously described. Peripheral whole-blood collection was performed at weeks 1, 2, 4, 8, and 12 and then every 3 months after seroconversion identification, and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. The subjects for whom HIV antibodies were either unidentified or indeterminate but for whom a nucleic acid amplification test was positive were defined to be acutely infected with HIV. Alternatively, acute HIV infection was estimated to have been established at the midpoint between the last seronegative and the first seropositive test. The date of acquisition of HIV infection was defined as follows: (1) HIV infection was determined to be 14 days before a sample was first found to be positive for HIV RNA but negative for HIV antibody; (2) when western blotting was indeterminate, HIV infection was estimated to have occurred 30 days prior to the index measurement or subject enrollment; and (3) subjects who were negative for the anti-HIV antibody and negative for HIV-1 RNA followed by seropositivity and RNA positivity with a time between tests <2 months were esmitated to be infected on the midpoint date.[16-18]
Nineteen individuals from the Beijing PRIMO cohort who did not receive antiretroviral treatment during the first year of HIV-1 infection were enrolled in this study, and among these patients, 17 men visited the hospital in months 1, 3, and 12, while 2 of these patients visited the hospital twice, at months 3 and 12. In addition, 20 individuals who had been infected with HIV-1 for at least 2 years, considered to have chronic infection (CHI) and had not received antiretroviral treatment were also enrolled in this study. Twenty-three healthy donors (HD) who were age matched and HIV-1-negative were enrolled as controls.
CD4 T-cell count and viral load measurement
Routine blood CD4 T-cell counts (cells/μl) were measured via four-color flow cytometry detection of human CD45+, CD3+, CD4+, and CD8+ T-cell markers (BD Biosciences, San Jose, CA, USA) in peripheral whole-blood samples obtained from each patient and added to FACS-lysing solution (BD Biosciences) according to the manufacturer's instructions. Plasma HIV-1 viral load (copies/mL of plasma) was quantified by real-time PCR (Abbott Molecular Inc., Des Plaines, IL, USA). This assay has a sensitivity for viral RNA detection of 40 copies/mL of plasma. The viral load set point at the very early stage of HIV-1 infection was calculated and reported in our previous study.
Intracellular cytokines and cell degranulation substances were detected by staining and flow cytometry
PBMCs were thawed and incubated overnight in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone), 50 IU/mL penicillin–streptomycin (HyClone), and 2 mmol L-glutamine (HyClone). They were stimulated with 2 μg/mL pooled HIV-1 Gag peptide pool as described in our previous study; 1 μg/mL anti-CD28/CD49d antibodies (Cat. 347690; BD Biosciences); and anti-CD107a-PE antibody (clone H4A3, BioLegend). After 1 h of incubation, 3 μg/mL brefeldin A and 2 μmol/L monensin (eBioscience™, diluted 1:1000) were added to the cells, which were incubated for an additional 5 h. PBMCs were stimulated with RPMI medium 1640 and without peptide as negative control. Positive control cells were stimulated with 20 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA) and 1 μmol/mL ionomycin (Sigma) and cultured for 6 h at 37°C. The PBMCs were stained with fluorescence dye-conjugated human monoclonal antibodies (mAbs), namely, CD3-Percp-Cy5.5 (clone HIT3a; BioLegend, San Diego, CA, USA), CD8-Alexa Fluor 700 (clone SK1, BioLegend), CD38-Brilliant Violet 711™ (clone HIT2, BioLegend), HLA-DR-Brilliant Violet 650™ (clone L243, BioLegend), TIGIT-PE/Dazzle™ 594 (clone A15153G, BioLegend), and PD-1-Brilliant Violet 605™ (clone EH12.1, BD). The PBMCs were washed and then fixed and permeabilized with BD FACS™ permeabilizing solution (Cat. No. 340457), and intracellular staining was performed for interferon gamma (IFN-γ)- phycoerythrin (PE) and tumor neucrosis factor (TNF)-α-PE-Cy7 for 30 min at 4°C. Isotype control monoclonal antibodies (mAbs) were purchased from the companies from which the corresponding mAbs had been obtained. The cells were then analyzed with a BD LSRFortessa flow cytometer, and dead cells were excluded by staining with LIVE/DEAD Fixable Viability Stain 510. The cytometer was set up and tracking calibration particles were used to ensure that fluorescence intensity measurements were consistent across all experiments. Flow cytometry Comp-Beads kits (BD Biosciences) were used for compensation. Gating by forward scatter and side scatter light was established to exclude cell debris from the analysis; forward height and forward area were used to exclude doublet cells; and dead cells were excluded by staining with LIVE/DEAD Fixable Viability Stain 510 (BD Biosciences). PBMCs were analyzed with the BD LSRFortessa flow cytometer, as previously described,[20,21] and the data were analyzed with FlowJo Software version 10.0 (TreeStar, Ashland, OR, USA).
Data are expressed as the median with range or interquartile range. Statistical analysis was performed with GraphPad Prism software version 5.03 (GraphPad Software, San Diego, CA, USA). The significance of differences among multiple variables (parameters in 1st, 3rd, and 12th months of AHI and in CHI) was analyzed by one-way analysis of variance or Kruskal–Wallis test, and then a difference between two variables was determined by Student's t-tests or nonparametric Mann–Whitney U test. The difference of variables between HD and HIV individuals was analyzed by Mann–Whitney U test. The Wilcoxon matched pairs test was performed to analyze paired variables. Spearman's rank correlation analysis was performed to assess the relationship between two variables. Differences were considered statistically significant when the P value <0.05 in two-tailed tests.
Clinical parameters of the enrolled participants
In this study, differences in the levels of HIV-1 viral load, CD8 T-cell counts, and the CD4 T-cell/CD8 T-cell ratio in HIV-1 individuals at different stages of infection were insignificant [Table 1]. CD4 T-cell counts were observed to be significantly changed as HIV-1 infection was prolonged (one-way ANOVA, P = 0.023, Table 1), and CD4 T-cell counts in individuals in the third month of AHI were higher than those in individuals in CHI (P < 0.05) [Table 1].
Table 1 -
Demographics and characteristics of HIV-1-infected individuals.
|Number of cases
|Viral load (log10)
|CD4 counts (cells/μL)
|CD8 counts (cells/μL)
Data are presented as n or median (interquartile range). Among 19 patients in the AHI cohort, 17 men visited the hospital in months 1, 3, and 12, while 2 of these patients visited the hospital twice, at months 3 and 12.AHI: Acute HIV-1 infection; CHI: Chronic HIV-1 Infection; HIV: Human immunodeficiency virus; NS: Not significant.AHI cohort: Individuals with acute HIV-1 infection; CHI cohort: Individuals with chronic HIV-1 infection; 1, 3, 12 mon: In the 1st, 3rd, and 12th months after HIV-1 infection; NS: P value was not significant by Kruskal–Wallis test for multiple variables.
∗One-way ANOVA for variables in the 1st, 3rd, and 12th months of AHI and in chronic HIV-1 infection >2 years.
†CD4 counts in the third month of AHI was compared with those in chronic HIV-1 infection >2 years, and P value was <0.05.
Profound increase in CD3+CD8low T-cell number following HIV-1 infection were gradually reduced with HIV-1 infection progression
The flow charts in Figure 1A show that CD3+CD8low T cells expanded at different stages of HIV-1 infection. Compared with those in healthy donors, CD3+CD8low T cells were significantly increased in the 1st, 3rd, and 12th months of AHI and in CHI (all P values <0.001) [Figure 1B]. However, the number of CD3+CD8high T cells was significantly diminished at the first month of AHI (P = 0.033) [Figure 1B] and significantly elevated in CHI (P = 0.005) [Figure 1B]. Interestingly, the proportion of CD3+CD8low T cells was significantly larger than that of CD3+CD8high T cells at the first month (P = 0.012) [Figure 1B] and third months (P = 0.038) [Figure 1B] of AHI, and these levels were comparable to those of CD3+CD8high T cells at the 12th month of AHI, but significantly lower than those of CD3+CD8high T cells in CHI (P = 0.007) [Figure 1B]. By contrast, in healthy donors, the proportion of CD3+CD8low T cells was significantly lower than that of CD3+CD8high T cells (P < 0.0001) [Figure 1B].
Furthermore, the frequency of CD3+CD8low T cells was largely decreased (P = 0.034) [Figure 1B] as HIV-1 infection was prolonged to the stage of CHI, while the frequency of CD3+CD8high T cells was elevated (P = 0.002) [Figure 1B]. In CHI, the frequency of CD3+CD8low T cells was significantly lower than that at the first and third months of AHI (all P values <0.05) [Figure 1B], but that of the CD3+CD8high T cells was significantly higher (all P values <0.05) [Figure 1B].
In addition, an inverse correlation between the frequency of CD3+CD8low T cells and the CD4 T-cell/CD8 T-cell ratio was observed in the first month (r = −0.615, P = 0.009) [Figure 1C] and third month (r = −0.619, P = 0.005) [Figure 1C] of AHI, accounting for the expansion of CD3+CD8low T cells after HIV-1 infection. These outcomes demonstrate that the number of CD3+CD8low T cells had markedly expanded due to HIV-1 infection, and this expansion lasted for a short time before decline until the chronic stage of infection was reached. Moreover, the number of CD3+CD8high T cells diminished rapidly after HIV-1 infection and then increased gradually, and this meaningful increase was initiated when CHI was evident >2 years.
Interestingly, in the first and third months of AHI, significantly higher numbers of CD3+CD8low T cells than CD3+CD8high T cells were observed among HIV-1-infected individuals who presented with higher viremia (≥4.0 log10) (all P values <0.05, Supplementary Figure 1, https://links.lww.com/CM9/B379), but comparable numbers of CD3+CD8low T cells to CD3+CD8high T cells was observed in HIV-1-infected individuals with lower viremia (<4.0 log10). At the 12th month of AHI and in CHI, significantly higher numbers of CD3+CD8high T cells than CD3+CD8low T cells were identified in HIV-1-infected individuals with low viremia (<4.0 log10) (all P values <0.05, Supplementary Figure 1, https://links.lww.com/CM9/B379) but not in HIV-1-infected individuals with high viremia (≥4.0 log10). On the other hand, a significantly positive association between the numbers of CD3+CD8low T cells and viral load was identified at the 12th month of AHI (r = 0.55, P = 0.016) [Figure 1D], while an inverse correlation of the number of CD3+CD8high T cells to viral load was identified in CHI (r = −0.55, P = 0.012) [Figure 1E]. Thus, at the 1st and 3rd months of AHI, HIV-1-infected individuals presenting with high viremia produced more CD3+CD8low T cells than CD3+CD8high T cells, and the minor effect of CD3+CD8low T cells for reducing HIV-1 viremia was delayed and it was not evident until the 12th month of AHI. Later, with the decrease in CD3+CD8low T-cell number and the increase in CD3+CD8high T-cell number, the efficient impact of CD3+CD8high T cells on HIV-1 replication was observed in CHI.
High classical activation of CD3+CD8low T cells after the onset of HIV-1 infection was gradually reduced with the prolonged infection
After HIV-1 infects individuals, CD8 T cells are activated and rapidly expand to combat the virus. There are three phenotypes of activated CD8 T cells as indicated by the expression of CD38 and HLA-DR markers, namely, classical CD38+HLA-DR+ T cells and two nonclassical types, CD38+HLA-DR− and CD38−HLA-DR+ T cells. Many more CD3+CD8low T cells than CD3+CD8high T cells were classically activated in both the HIV-1 individuals and healthy donors, as shown in the flow charts presented in Figure 2A. The classical activation capacity of all CD8 T cells was the highest at the first month of AHI and then decreased as HIV-1 infection was prolonged and entered the chronic stage (P < 0.001) [Figure 2B]. Similarly, the classical activation kinetics of CD3+CD8low T cells were consistent with those of all CD8 T cells (P < 0.001) [Figure 2B]. Concurrently, the classical activation of CD3+CD8high T cells was low as compared with that of CD3+CD8low T cells, but did not further decrease with disease progression, remaining at a relatively stable level (P > 0.05) [Figure 2B].
Notably, a highly positive correlation between the number of CD3+CD8low T cells and the classical activation capacity of total CD8 T cells was observed at the 1st, 3rd, and 12th months of AHI (1 month: r = 0.919, P < 0.001; 3 months: r = 0.838, P < 0.001; 12 months: r = 0.674, P = 0.002) [Figure 2C] but not in CHI (r = 0.389, P = 0.090) [Figure 2C]. In addition, there was an inverse correlation or no correlation between the number of CD3+CD8high T cells and the activation capacity of all CD8 T cells [Figure 2D]. These outcomes indicate that CD8 T-cell classical activation capacity at the 1st, 3rd, and 12th months of AHI was largely dependent on the number of CD3+CD8low T cells not that of CD3+CD8high T cells, and the classical activation capacity of CD3+CD8low T cells was indeed much higher than that of CD3+CD8high T cells.
Furthermore, stronger CD3+CD8low T-cell classical activation at the third month of AHI correlated with a higher viral load (r = 0.518, P = 0.023) [Figure 2E], which was consistent with the correlation between total CD8 T-cell activation and viral load (r = 0.469, P = 0.043) [Figure 2E]. However, in CHI, the activation of CD3+CD8high T cells (r = 0.463, P = 0.040) [Figure 2F] but not CD3+CD8low T cells was related to a higher viral load, coincident with the relationship of total CD8 T-cell activation to viral load (r = 0.486, P = 0.030) [Figure 2F]. In addition, an inverse correlation of classical activation of total CD8 T cells, including CD3+CD8low and CD3+CD8high T cells, to the CD4 T-cell/CD8 T-cell ratio [Supplementary Figure 2, https://links.lww.com/CM9/B379] was observed at the 1st, 3rd, and 12th months of AHI. These outcomes suggest that the direct impact of HIV-1 viral load on the classical activation of CD3+CD8low T cells and CD3+CD8high T cells was delayed to the third month of AHI and CHI, respectively, although cell activation was elevated in the first month of AHI.
The number of CD38−HLA-DR+CD8+ T cells, particularly CD38−HLA-DR+CD8low T cells, which did not increase after the first month of AHI, was related to reduced HIV-1 viremia
The number of CD38+HLA-DR− activated cells among total CD8, CD3+CD8high and CD3+CD8low T cells at the 1st, 3rd, and 12th months of AHI and even in CHI increased compared with that in healthy donors (all P values <0.001) [Figure 3A], which did not reduce with HIV-1 disease progression (all P values >0.05) [Figure 3A]. By contrast, cells undergoing nonclassical activation, CD38−HLA-DR+-activated cells among total CD8, CD3+CD8high, CD3+CD8low T cells gradually and significantly increased as HIV-1 infection persisted from the first month of AHI to the stage of CHI (all P values <0.001) [Figure 3B], although the numbers of these activated cells did not increase at the first month of AHI (all P values <0.001) [Figure 3B].
At the first month of infection, higher numbers of CD38+HLA-DR−CD8+ T cells, especially CD38+HLA-DR−CD8high T cells, were positively associated with higher HIV-1 viremia (total CD8 T cells: r = 0.485, P = 0.048; CD8high T cells: r = 0.510, P = 0.037) [Figure 3C] but inversely associated with CD4 T-cell count (total CD8 T cells: r = −0.750, P < 0.001; CD8high T cells: r = −0.765, P < 0.001) [Figure 3D]. By contrast, the number of CD38−HLA-DR+CD8+ T cells, especially CD38−HLA-DR+CD8low T cells, which did not increase at the first month of AHI, was inversely associated with viral load (total CD8 T cells: r = −0.644, P = 0.005; CD8low T cells: r = −0.664, P = 0.004) [Figure 3E] but positively associated with CD4 T-cell count (total CD8 T cells: r = 0.619, P = 0.008; CD8low T cells: r = 0.586, P = 0.014) [Figure 3F]. These opposite relationships exhibited in the first month of AHI indicate the mutually antagonistic roles of these two phenotypes of activated CD8 T cells in early acute HIV-1 infection.
The reduction in the CD4 T-cell count related to increased PD-1 abundance on CD3+CD8low T cells was postponed to the chronic stage of infection
Inhibitory molecules such as TIGIT (T-cell immunoglobulin and ITIM domain) and PD-1 (programmed cell death-1) are upregulated upon T-cell activation due to HIV-1 infection, and they downregulated immune responses to prevent hyperimmune system activation. PD-1 expression on total CD8 T cells was significantly upregulated after the first month of AHI (all P values <0.001) [Figure 4A] but did not continue to increase as HIV-1 infection persisted [Figure 4A]. By contrast, TIGIT protein abundance on total CD8 T cells was not significantly increased at the 1st and 3rd months of AHI but was increased at the 12th month of AHI and in CHI (all P values <0.05) [Figure 4A]. Moreover, TIGIT protein abundance on total CD8 T cells in CHI was higher than that at the first and third months of AHI (all P values <0.05) [Figure 4A].
Intriguingly, the expression patterns of PD-1 and TIGIT differed between CD3+CD8low and CD3+CD8high T cells in HIV-1 individuals. Specifically, PD-1 abundance increase on CD3+CD8low and CD3+CD8high T cells was exhibited after the first month of AHI (all P values <0.001) [Figure 3B] and was maintained at high levels as HIV-1 infection was prolonged [Figure 4B]. Likewise, increased TIGIT abundance on CD3+CD8high T cells was first identified at the first month of AHI and remained at high levels with HIV-1 infection progression. By contrast, TIGIT abundance on CD3+CD8low T cells was significantly increased after the 12th month of infection (all P values <0.05) [Figure 4B].
Furthermore, in CHI, PD-1 upregulation on CD3+CD8+ T cells, especially CD3+CD8low T cells, was negatively correlated with CD4 T-cell count (total CD8 T cells: r = −0.395, P = 0.085; CD8low T cells: r = −0.456, P = 0.043) [Figure 4C]. Similarly, TIGIT abundance increase on CD3+CD8+ T cells, including CD3+CD8high and CD3+CD8low T cells, was inversely correlated with CD4 T-cell count (total CD8 T cells: r = −0.575, P = 0.008; CD8high T cells: r = −0.503, P = 0.024; CD8low T cells: r = −0.488, P = 0.029) [Figure 4D]. Together, the adverse effects of PD-1 expression on CD3+CD8 T-cell, including CD3+CD8high and CD3+CD8low T cells, and CD4 T-cell count were delayed to the early chronic stage of infection.
PD-1 abundance on classically activated CD3+CD8low T cells was associated with decreased HIV-1 replication in the first month of AHI
CD8 T cells were activated when exposed to HIV-1, and then, the levels of some inhibitory receptors were increased to resist CD8 T-cell activation and maintain CD8 T-cell homeostasis; therefore, the abundance of TIGIT and PD-1 on activated CD8 T cells was assessed. Increased PD-1 abundance on classically activated CD3+CD8+ T cells [Figure 4E], particularly CD3+CD8low T cells [Figure 4F], was initiated in the third month of AHI, while PD-1 abundance on classically activated CD3+CD8high T cells did not significantly increase during HIV-1 infection [Figure 4F]. By contrast, TIGIT level increases on classically activated CD3+CD8+ T cells [Figure 4E], particularly CD3+CD8low T cells [Figure 4F], started at the 12th month of AHI, which was later than the TIGIT level increase on CD3+CD8high T cells, which started from the 3rd month of AHI (all P values <0.05) [Figure 4F].
In addition, with the persistence of HIV-1 infection, PD-1 and TIGIT abundance on classically activated CD3+CD8+ T cells, including CD3+CD8high and CD3+CD8low T cells, was increased (all P values <0.05) [Figure 4E,F], except for PD-1 abundance on CD3+CD8high T cells (P = 0.061) [Figure 3D]. A correlation analysis showed that the levels of PD-1 abundance on CD38+HLA-DR+CD8+ T cells (r = −0.593, P = 0.012) [Figure 4G], including CD38+HLA-DR+CD8high (r = −0.498, P = 0.042) [Figure 4G] and CD38+HLA-DR+CD8low T cells (r = −0.578, P = 0.015) [Figure 4G], were inversely associated with viral load at the first month of AHI. These data suggest that the helpful effect of PD-1 expression on classically activated CD8 T cells, including CD3+CD8high and CD3+CD8low T cells, on inhibiting HIV-1 replication was evident in the first month of AHI; however, at that time, PD-1 expression was not upregulated.
Few CD3+CD8low T cells but comparable to the number of CD3+CD8high T cells were induced to produce HIV-1-specific CD8 T-cell responses in the first month of AHI
After HIV-1 infection of individuals, immune cells in the adaptive immune system, such as CD8 T cells, are not immediately induced to respond to HIV-1 infection. In this study, a small number of CD3+CD8low T cells, comparable to the number of CD3+CD8high T cells, was induced to produce CD107a, IFN-γ, and TNF-α in response to the Gag peptide pool in individuals at the first month of AHI. However, a significantly lower number of CD3+CD8low T cells than CD3+CD8high T cells was triggered to produce an HIV-1-specific immune response at the 3rd and 12th months of AHI and in CHI (all P values <0.05) [Figure 5A–C].
In addition, HIV-1-specific CD3+CD8high T-cell responses leading to CD107a, IFN-γ, and TNF-α production gradually increased as HIV-1 infection was prolonged (all P values <0.05) [Figure 5A–C], and the responses induced in CHI were significantly more consequential than those at the first month of AHI (all P values <0.05) [Figure 5A–C]. Additionally, as HIV-1 infection was prolonged, HIV-1-specific CD3+CD8low T-cell responses leading to CD107a and IFN-γ production were not significantly enhanced (all P values >0.05) [Figure 5A,B], but TNF-α-production in CD3+CD8low T cells was increased (P < 0.05) [Figure 5C].
Early immunological events are characterized by a marked increase in the CD8 T-cell counts during primary HIV-1 infection, and CD8 T-cell counts were much higher in Fiebig stages II/III. In this study, a significant increase in CD3+CD8low T cells was exhibited in the first and third months of AHI, and the number of CD3+CD8low T cells was higher than that of CD3+CD8high T cells. Subsequently, the number of CD3+CD8low T cells was comparable to that of CD3+CD8high T cells at the 12th month of infection but lower than that of CD3+CD8high T cells in CHI. The dynamic change in the number of CD3+CD8low and CD3+CD8high T cells in this study revealed the rapid expansion and subsequent gradual decline in the number of CD3+CD8low T cells following HIV-1 infection, while the dynamic change in the number of CD3+CD8high T cells showed the opposite trend. In addition, in AHI, the increase in CD3+CD8low T cells was related to higher HIV-1 viremia because more CD3+CD8low T cells than CD3+CD8high T cells were found in HIV-1-infected individuals who presented with a high viral load (log10 ≥ 4.0). Later, at the 12th month of infection, the decreased number of CD3+CD8low T cells was associated with a high HIV-1 viral load. By contrast, the increase in CD3+CD8high T cells in CHI was related to low HIV-1 viremia because HIV-1-infected individuals with a lower viral load harbored more CD3+CD8high T cells than CD3+CD8low T cells in the chronic stages of infection, particularly in CHI. These outcomes showed that a higher HIV-1 viral load was related to a higher number of CD3+CD8low T cells at the acute stage of HIV-1 infection. However, lower HIV-1 viremia was associated with a higher proportion of CD3+CD8high T cells at the chronic stage of HIV-1 infection. Nonetheless, the causes and effects of peripheral CD3+CD8low T-cell increases after HIV-1 infection need to be further probed.
In contrast to peripheral CD3+CD8low T cells in humans, CD3+CD8low thymocytes are generated in the HIV-1-infected thymus, which has been confirmed in a SCID-hu Thy/Liv mouse model. It has also been proven that HIV-1 can infect the human thymus,[24,25] which mediates the repopulation of peripheral T-cell pools. The HIV-1-initiated selection process in the thymus may contribute to the generation of dysfunctional CD8low T cells in HIV-1-infected patients. Hence, mature single-positive CD3+CD8low thymocytes in the thymus infected with HIV-1 possibly move into peripheral T-cell pools. In mice, CD8 expression can be downregulated by certain cytokines, including Interleukin 2 (IL-2), IL-4, IL-15,[26-28] and type I interferon, in the presence of Listeria monocytogenes antigen. Therefore, it is possible that CD8 downregulation may be regulated following HIV-1 infection due to “cytokine storms,” as reported in the Beijing PRIMO cohort in our previous study. In the previous study, we showed that the IL-4 level peaked 4 to 5 weeks after infection and then declined in parallel with a decrease in viral load. In addition, the IL-15 level was found to increase rapidly and was sustained >2 months after infection, and the IFN-α2 level significantly increased during both the acute and chronic stages of infection. Therefore, the increase in peripheral CD3+CD8low T cells after HIV-1 infection is elusive and complicated.
Antigen-driven stimulation through the TCR usually leads to cell activation. The onset of HIV viremia induces vigorous CD8 T-cell activation and proliferation. In the present study, CD8 T-cell activation reached its peak approximately 2 weeks after HIV-1 viremia reached the maximal level and then was reduced to a quasi-steady state approximately 80 days after the viremia was first detected (interquartile range [IQR] 73–107). The rapidity in which CD8 T-cell activation peaks and the magnitude of CD8 T-cell activation at this peak time have been previously associated with a low viremia threshold, which can be used to predict subsequent disease progression. Indeed, high levels of CD8 T-cell immune activation have been associated with poor outcomes for HIV-1 individuals, as confirmed in several studies.[33,34] In the present study, the classical activation of CD3+CD8+ T cells, particularly CD3+CD8low T cells, was the greatest in the first month of infection and then gradually decreased, whereas the classical activation of CD3+CD8high T cells remained relatively stable as HIV-1 infection progressed. As classical activation of CD3+CD8+ T cells, especially CD3+CD8low T cells, declined to some extent in the third month of infection, HIV-1 viral load was found to be positively related to the magnitude of classical activation. This relationship indicated that the direct effect of HIV-1 viremia on classical activation of CD3+CD8+ T cells, particularly CD3+CD8low T cells, was clearly evident in the third month of infection; at that time, the probability of HIV-1 replication reaching a relatively stable level was 76.3%. In contrast, the direct impact of HIV-1 viremia on classical activation of CD3+CD8high T cells was exhibited in CHI, until that time when CD3+CD8high T cells increased significantly and were proportionally larger than CD3+CD8low T cells.
Notably, HIV-1 viral load in the first month of AHI was inversely associated with a stable number of CD38−HLA-DR+CD8+ T cells, especially CD38−HLA-DR+CD8low T cells [Figure 3], suggesting that CD38−HLA-DR+CD8low T cells were probably among the contributors to a reduced viral load. Indeed, the unusual CD8 T-cell activation phenotype, CD38−HLA-DR+, has been reported to be associated with a greater capacity of multiple mechanisms that suppress viral replication, leading to better viral control and delayed progression to AIDS.[35-38]
During HIV-1 infection, CD8 T cells exhibit features of exhaustion and dysfunction linked to the increased expression of TIGIT, PD-1, CD160, 2B4, T-cell immunoglobulin and mucin domain-3 (TIM-3), and lymphocyte activation gene-3 (LAG-3),[39-41] and inhibitory molecule coexpression aggravates the poor functionality of CD8 T cells. It has been reported that, compared with that in HIV-uninfected adults, TIGIT abundance on CD8 T cells was increased in subjects with CHI who had not received continual treatment but not in individuals with acute infection. Antigen-specific TCR stimulation is the primary determinant of PD-1 abundance on CD8 T cells, and it is not limited to HIV-specific CD8 T cells but is observed in total CD8 T-cell populations in HIV-1-infected individuals. Chemnitz et al reported that PD-1 abundance was increased on the surface of T cells within 24 h of TCR stimulation, and the effects of PD-1 ligation were seen within a few hours. In this study, TIGIT abundance on total CD8 T cells was not significantly increased until the 12th month of infection, which was after a significant increase in PD-1 abundance identified in the 1st month of infection. Moreover, CD3+CD8low T cells exhibited the same TIGIT and PD-1 expression dynamics as total CD8 T cells [Figure 4]. For CD3+CD8high T cells, TIGIT and PD-1 upregulation was evident in the first month of AHI but did not continually increase as HIV-1 infection progressed. Up to CHI, PD-1 abundance on CD8 T cells was inversely associated with CD4 T-cell count, indicating the delayed impact of PD-1 expression on clinical HIV-1 progression, although increased PD-1 abundance on CD8 T cells appeared at an earlier stage of acute HIV-1 infection. Similarly, the adverse impact of TIGIT expression on CD4 T-cell count was delayed to the stage of CHI.
Importantly, in the first month of AHI, PD-1 expressing on classically activated CD3+CD8+ T cells, including CD3+CD8high and CD3+CD8low T cells, was considered to contribute to inhibited HIV-1 replication because an inverse association was observed between these two factors [Figure 4]. Our findings provide new insights into the possible role played by PD-1 abundance on classically activated CD3+CD8low T cells in inhibiting HIV-1 at the early stages of acute HIV-1 infection.
CD3+CD8low T cells were abundant in acute HIV-1 infection and substantially activated at different stages of HIV-1 infection; hence, TIGIT and PD-1 abundance was increased and was associated with negative activation feedback. However, CD3+CD8low T cells showed weak HIV-1-specific responses than CD3+CD8high T cells at all stages of infection, except at the first month of AHI [Figure 5]. Nonetheless, CD3+CD8low T cells appeared to be associated with poor clinical parameters, which was evident at the 3rd and 12th months of infection but not at the 1st month of infection. At the first month of infection, the HIV-1 viral load was inversely associated not only with the unreinforced CD38−HLA-DR+ activation capacity of CD3+CD8low T cells but also with PD-1 expression on classically activated CD3+CD8low T cells. Moreover, at the first month of infection, weak HIV-1-specific CD3+CD8low T-cell responses, namely, low IFN-γ, TNF-α, and CD107a secretion levels, were comparable to those elicited in CD3+CD8high T cells. Notably, the weak initial HIV-1-specific CD8+ T-cell responses contributed to the control of acute viremia in HIV-1 infection.[47,48] Hence, it is reasonable to think that CD3+CD8low T cells are effective in combating HIV-1 at the early stage of acute HIV-1 infection even though CD3+CD8low T-cell responses are inferior to CD3+CD8high T-cell responses at the late stage of acute HIV-1 infection and at the stage of CHI.
In total, the dynamics of the quantity, activation, and expression of TIGIT and PD-1 and the HIV-1-specific responses elicited in CD3+CD8low T cells at different stages of HIV-1 infection revealed the complexity of the roles played by CD3+CD8low T cells in combating the HIV-1. On the one hand, CD3+CD8low T cells were abundant at the first month of acute HIV-1 infection and played only an anti-HIV role. On the other hand, the number of CD3+CD8low T cells decreased gradually as the infection persisted, and their anti-HIV functions were inferior to those of CD3+CD8high T cells. Nonetheless, many issues, such as cell origin, proliferation rate, differentiation pattern, senescence and apoptosis rate, need to be investigated to remove the veil that covers CD3+CD8low T-cell function in HIV-1 infection.
This work was supported by grants from the National Natural Science Foundation of China (NSFC, 81974303), the High-Level Public Health Specialized Talents Project of Beijing Municipal Health Commission (2022-2-018), the Ministry of Science and Technology of China (CPL-1233), the “Climbing the peak (Dengfeng)” Talent Training Program of Beijing Hospitals Authority (DFL20191701 and DFL20181701), the Beijing Health Technologies Promotion Program (BHTPP2020), Beijing Key Laboratory for HIV/AIDS Research (BZ0089 and BZ0373), Beijing Natural Science Foundation (7191004), Beijing Municipal Science and Technology Project (Z211100002521024), the Natural Science Foundation of Capital Medical University (PYZ21126), and the Scientific Research Project of Beijing Youan Hospital (CCMU-2020-BJYAYY-2020YC-01 and CCMU-2021-YNKTXF2021001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflicts of interest
Data availability statement
The data for this study are available by contacting the corresponding authors upon reasonable request.
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