The pattern and magnitude of T cell subsets reconstitution during ten years of ART with viral suppression in HIV-infected patients

Background: The extent of immune reconstitution in human immunodeficiency virus (HIV) infected persons receiving long-term antiretroviral therapy (ART) with controlled viral load has been controversial. We studied the extent and speed of T cell subsets retrieval after long-term antiretroviral treatment. Methods: 662 HIV-infected patients followed at least 2 years whose plasma HIV-1 RNA load <50 copies/mL were evaluated for longitudinal and functional phenotypic indices of immune restoration. Determinants of change in magnitude and importance of recovery have been evaluated using mixed linear regression models. Results: Almost all robust immune restorations achieved occurred after 2–3 years of ART. The median CD4 lymphocyte count increased 449 cells/μl (IQR 303–604) from 226 cells/μl (IQR 83–336) at baseline during the third year (P < 0.001); CD4+T lymphocyte rises during the sixth and tenth years were not significant. Naive and memory CD4+T cells‘reconstitution occurred in the sixth and eighth years of ART but no significant change thereafter. The change of CD45RA+Naïve and CD45RA-memory CD4+T cell reconstitution is different in baseline CD4+T cell counts <100 cells/μl group and in baseline CD4+T cell counts >100 cells/μl group. Activation antigen expression (CD38 or HLA-DR) on CD8 lymphocytes declined mostly during the first till second year, and after 4 years, activation antigen expression on patient lymphocytes showed no significant change. The proportion of CD4 cells expressing CD28 climbed during the first years and reached normal levels in the second year. Conclusions: Immune restoration was dependent on the capacity of immune system during the first 2–3 year of ART. But the significant change of CD4 and compartments of CD4+T cells could persist until 6–8 years. The pattern of CD38+CD8+, HLA-DR+CD8+, CD28+CD4+ T cells could quickly return to normal level and no significant change after sufficient time of ART. In general, the immune response compared to the baseline status may be the overall effect from the age and time of antiretroviral treatment.


INTRODUCTION
Infection with the human immunodeficiency virus (HIV) will lead to loss of CD4+T lymphocytes, increasing susceptibility to severe immunodeficiency, opportunistic infection and even death. The number of people infected with HIV is continually increasing worldwide and patients receiving antiretroviral therapy (ART) is climbing to 27.5 million worldwide, according to WHO reports up to 2020 [1]. The immune system of HIV-infected patients recovered after ART, which is generally evaluated by the increased number of CD4+T lymphocytes after undetectable viral load [2][3][4][5].
It has been acknowledged that CD4+T cells will increase over a period in HIV-infected patients with viral suppressed. However, the data were ambiguous with respect to the increasing period and degree of CD4+T cells. It is reported that CD4+ T cells climbed quickly within the 1-2 years of treatment, but slowly increased to 4-8years, regardless of baseline CD4+ T cell value [6][7][8][9], or weather CD4 count stabilizes. Most analyses on CD4 counts have set limit of times when plasma HIV-1 RNA (pRNA) was suppressed, e.g., to <400 copies/ml or <1000 copies/ml [7,8], while the lowest detection limit of HIV detection has become less than 20 copies/ml. Nowadays, the characteristics of immune system response in HIV patients that meet lower limits of what is considered viral suppression are likely different from many years ago, leading to possible selection biases. As a result, we still require up-to-date data for the CD4+T cell count increment after restricted viral suppression.
On the other hand, the comprehensive assessment of the immune response should cover up the increased number of CD4+ T cells and concentrate on the other T cell phenotype like T cell activation related to virus fluctuate during ART treatment. However, only a small number of investigations with a limited sample size have shown the kinetics of T cell subsets such as CD4+CD28+T cells and CD4+CD45RA+T cells after short-term follow-up [10][11][12]. Some researchers have focused on the subpopulation of T lymphocytes in children [13]. The characteristics of this research included a viral load limit detected below 400 copies/ml, patients enrolled having viral load higher than 400 copies/ml and a sample size of less than 50. In conclusion, these studies have not provided a complete explanation of the change in the T cell subpopulation after viral suppression according to current guidelines [14].
Overall, we aim to show the immune system response after long-term (10-year) ART with undetectable viral load in our patients. The assessment of immune system will include CD4+T cell counts, CD8 cell counts, CD4/CD8 ratio, and the number of naive CD45RA+CD4+T cells and memory CD45RA-CD4+T cells, the CD38+CD8+T cells, CD8+HLA-DR+T cells and CD28 expression on CD4+ and CD8+T cells in the peripheral blood. In this study, we analyze the data by fitting the kinetics of selected parameters and related risk factors with mixed linear model in patients who received ART at least two years and maintained viral load <50 copies/ml.

Clinical and immunology characteristics at baseline
In 662 HIV-infected patients, 88.2% (584) of them were male ( Table 1). The mean age was 36.4 ± 11.2 years. The sexual transmission was the most frequent in transmission routes, especially in patients having sex between men (63.0%). 65.1% of patients has one or more signs or symptoms of HIV infection. 212 participants (32.0%) had an opportunistic infection and 43 (6.5%) of enrolled patients were HBsAg positive or 24 (3.6%) were HCV-Ab positive. Mean log10 (viral load) of these patients was 4.74 ± 0.77. 36.3% of patients' baseline viral load is higher than 10 5 cps/ml, which represents a relatively high level of viral load. 225 patients (34.0%) did not achieve viral suppression at the first six months, most patients (74.8%) initiated ART with two Nucleoside Reverse Transcriptase Inhibitor (NRTIs) with non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). The median followup time was 5.67 (3.17, 8.33) years.

Dynamics of absolute counts of CD4+ T cell, naive CD4+ T cells, and memory CD4+T cells
The median number of CD4+T cell counts was 226 cells/µl (IQR 83-336) before treatment and increased to 449 cells/µl (IQR 303-604) after 3-year of ART (Table 1). Through fitting the increasing curve of CD4+T cells, an initial surge in CD4+T cell counts within 3 months was evident, but relatively slower accumulation in CD4+T cell count until the fourth year after treatment ( Table 2 and Figure 1A). After the fourth year of steady increment, the significant change period of CD4+T cell counts prolonged to 2-year until the seventh year while there was no significant difference of CD4+T cells between the next three year ( Figure 1A and Supplementary  Table 1). Unlike CD45RA+ naïve CD4+T cells, there was a significant change in CD45RA-CD4+T cells within 3 months of treatment initiation, but there was no evident change between first 3-month and 6-month. The significant increasing span went from one year during 0-3 years after ART and two years in 3-6 years ( Figure  1C), and there was no significant increase thereafter through multiple comparisons (Seen in Table 2 and  Supplementary Table 2).
In the mixed linear model, no significance of naïve and memory CD4+T cell counts between gender was found. Age was independently inversely linked to the CD45RA-Memory and CD45RA+CD4 T cell counts. CD4+CD45RA-T cells increased with age, while CD45RA+Naïve CD4+T cell counts decreased with age group. ART regimens (2NRTIs+PI and 2NRTIs+INSTI) have marked changes of naïve CD4+T cell and Memory CD4+T cells, which are consistent with results from CD4+T cell counts (Table 2). Since CD4+T cell counts were composed of CD4+naïve and CD4+memory T cells, it is unreasonable to put this indicator in the mixed model, so we calculated the trends of naïve and memory T cell ratio stratified by baseline CD4+T cell counts. It is shown that the kinetics of naïve/memory CD4+T cells decreased at first three months in patients whose CD4+T cell counts <100 cells/µl. However, in patients with CD4+T cell counts > = 100 cells/µl, the naïve/memory CD4+T cells would not transiently decrease at first three months ( Figure 2).

Dynamics of counts of CD8+ T cells and CD4/CD8 ratio
A significant decreased effect occurred on the absolute counts of CD8+ T cells during the first years. CD8+T cells decreased from 789 cells/µl (IQR, 532-1045) to 699 cells/µl (IQR, 527-925) after 3 years of treatment (Table 1). Hereafter, the CD8+T cell counts decreased  Figure 1D). In the mixed linear model, baseline CD8+T counts significantly influenced the CD8+T cells after ART. Higher CD4+T cell counts, delayed viral suppression may interfere with CD8+T cells descent. Patients who used ART in PI-based and INSTI-based achieved higher CD8+T cell counts than NNRTI-based patients after ART treatment (Table 3).
In our mixed linear model, baseline CD8+T cells, baseline CD4+T cell counts were independent with CD4/CD8 ratio normalization, but the ART regimen did not (Table 3).

DISCUSSION
Our results have illustrated the tragedies of naive and memory subpopulation of CD4+T cells change for 10-year of follow-up with ART. We fitted the increasing trends of CD45RA+CD4+ and CD45RA-CD4+T cells at different time-point with real-world data. In addition, ten years of ART with undetectable viremia enabled the complete reconstitution of CD45RA+CD4+T cells and CD45RA-CD4+T cells, but     The ability to continuously reconstitute CD4+T cells count in HIV-infected patients during suppressive ART for longer periods is quite variable in literature data.
Several longitudinal studies suggested that amount of CD4+T cells reconstitution for exceeding one or two years was restricted at the early era of the ART regimen [15,16]. Slowly, the results of longer follow-up studies showed that CD4+T cell count in HIV-infected patients continuously ascended after virologically depressed even after 4-6 years of treatment, although there was a part of patients that could not continuously climb to a normal level if their CD4+T cell counts were lower pretreatment [6,7,9,17,18]. Until now, the longitudinal study followed more than 10 years conducted in Canada, found that 16% of patients achieved comprehensive T cell subsets reconstitution through a 10-year of sustained successful ART [19].
Although Dawit Wolday's study suggested that 69.6% of patients achieved their amount of CD4+T cell normalization within 6.5 (IQR: 3.0-10.5) years. The CD4+T cell depicted in their study continuously climbed up till 5 years and plateaued until 14 years [20]. Compared with our research, the CD4+T cells would continuously increase until the seventh year of ART when we consider the baseline CD4+T cell counts, age, ART regimen, and virological suppression, which was consistent with the results of Dawit Wolday's study [20] and other similar researches [6,8,21]. Interestingly, Studies in French [22] and ALLRT team [23] suggested that CD4+Tcell counts ascended in most patients for at least seven years if HIV-patients remained on ART, which is slightly different from our results since our results showed no significant change thereafter.
The kinetics of CD45RA-memory CD4+T cells and CD45RA+naïve CD4+T cells increased was mainly explained by a triphasic progress: an early surge with sharp slope followed by a gradual climbing, and subsequently string along with indolent growth [3]. The first two phases of CD4+T cells were mainly composed of CD4+memory T cells migration to the peripheral blood and followed by naive T cells refreshed from thymic. Therefore, with higher CD4+T cells at baseline, the number of memory T cells redistributing would be higher in HIV-infected patients. Thus, we also observed the significant change in memory CD4+T cell at first 3month. CD45RA+Naïve CD4+T cells are often thought as a renewable and truly increased T cell from lymphoid organ. In the study [10] followed for four years, significant increase was observed in naive CD4+T cells at the second year after ART initiation. But there was little change of naïve CD4+T cells thereafter. In our results, CD45RA+ naïve CD4+T cells and CD45RAmemory CD4+T cells would be influenced by age, their change persisted until 4-6 years which was similar with CD4+T cell counts. The naïve/memory T cell ratio can be regarded as the competition of newly regeneration of T cells, which showed different model of recovery among groups of CD4<100 cells/µl and CD4>100/ cells/µl at baseline. This phenomenon can be partly explained by a severe degree of thymus damage in patients with CD4<100 cells/µl but homing CD4+T cell counts in peripheral lymphocyte organ than in patients with CD4>100 cells/µl. Besides, Longer ART treatment would lead to a more pronounced higher memory T cell and lower naïve T cell in HIV-infected patients than in healthy controls. Frederikke F Rönsholt [11] found lower percentage of Naive CD4 cells in 71 HIVinfected patients than in controls even after 12-year on ART, which was consistent with our study.
After persistent virus-related stimulation and activation and even inhibitor signal effect during HIV infection, CD28 expression on the CD8+T cells would downregulate. This phenomenon has been described previously and can be partly recovered through ART treatment in HIV-infected patients [10,24]. Clinical literature reported that the complete reconstitution of CD4 and CD8 cells expressing CD28 required for 4-year from baseline but lower than healthy controls [24]. Similarly, a total of 1291 patients in chronic phrase followed 96-week found that removing covariance of age, sex, and race/ethnicity, the percentage of CD28 expression on both CD4+ and CD8+ T cell subsets was lower and abnormal than those of controls [25]. However, the percentage of CD28 expression on CD4+T cell in our research showed a rapid increase in the first year within the normal range at 2-year, while CD28 expression on CD8+T cell kept increased until the fifth year. CD8+CD28+T cells showed a trend of decrease with aging after ART in our results, which is in line with research showing that CD8+CD28−T cells are often regarded as symbols of short telomere length, promoting pro-inflammatory molecules production, and reducing proliferation signal [26,27].
CD38 on CD8+ T lymphocytes as immune hyperactivation would make CD4+T cells more susceptible to HIV infection and contribute to HIV replication, which was proposed in the studies [12,28]. Therefore, evaluating the expression of activating immune signatures, for example, CD38 (on CD8+T cells) is essential for assessment of comprehensive immune reconstitution in HIV-infected patients. Valdez et al. [24] have shown that abnormal CD8 + T cell activation in HIV-infected patients with CD4>100 cells/µl did not completely recover after 3-year of ART. Comparing with our study, higher percentage of CD38 on CD8+T cells have normalized after 4 years of ART in our patients. The major reduction in CD38+ overexpression occurred within the first 3 months of treatment. This result may be mainly explained by the significant suppression of plasma virus replication at the first 3 months [29]. Another activating immune signature marker HLA-DR expression on CD8+T cells was related to AIDS progression [30], which perform immune regulation in normal situation but exaggerated abnormal immune response in HIV-1 infection status [31]. In our data, a significant descend was seen in the HLA-DR+CD8+/CD8+% T cells after 1-year of ART. Whereas, no evident difference between time intervals after 1-year was observed in HIV-infected subjects regardless of their CD4+T cell counts pretreatment.
In general, our study showed longitudinal research of the kinetics of the T cell subsets reconstitution after 10-year ART. To our knowledge, this study provided largest sample size and longest time of T cell subpopulation change, which depicted the aging and the ART effect on the HIV-patients. Besides, we depicted the model of T cell subpopulation change over 10-years of ART with mixed linear model, considering the covariate effect including ART regimen and viral suppression as well as status at baseline, and comprehensively explained the data measured repeatedly.
Comparing with cohort in European and other developed countries, the limitation of our study covered up the following: firstly, it is the relatively small sample size after long-term visit. The predictive role of pattern and kinetics of CD4+T cell count and other T cell subpopulations will be clearer with more subjects. Another limitation was that first-line recommended ART regimens have changed during the followed time. Therefore, CD4+T cell trajectory and T cell subsets change might be influenced by starting a different ART regimen. As current ART can be well tolerated and effective in viral control because of reduced side effects or tablets leading to improved adherence. Last but not least, we confirmed that among these indicators, age has a significant effect on the T cell subpopulation, especially CD28, CD45RA expression, but this effect on the healthy people did not have longitudinal data as HIV-infected patients, which may partly explain the difference between our study with others.
Overall, our data showed longitudinal research of the kinetics of the reconstitution of CD4+T cells, CD8+T cells and CD4/CD8 ratio, naive CD45RA+CD4+T cells and memory CD45RA-CD4+T cells, CD28+ expression on CD4+T and CD8+T cells, and activation markers (CD38+ and HLA-DR expression on CD8+T cells) induced by ART. In our study, we found that most part of immune restoration was achieved within the 2-3 years after ART whatever the CD4+T or the other lymphocyte subpopulations, but slower change for next 3-4 years. The gap between the baseline status and the immune reconstitution at different time points was different, but almost all T cell subpopulation did not change significantly after 7-8 years when there counted nearly 100-200 patients on. Conclusively, Immune restoration after ART robustly within first 2-3 years already reflect the immune system response to whether achieve fully reconstitution. Main laboratory analysis (including T cell subsets and HIV viral load) were conducted at baseline, followed by 3 months, 6 months, 12 months, and every 6 months thereafter. Plasma samples for laboratory tests were taken during routine diagnosis.

Laboratory test
In order to assess the T cell immune response in HIV infected patients, we compared lymphocyte sub-populations in subjects before and during treatment to reference range, which examined 56 healthy blood donors (36 male, mean age was 36 ± 10 years old) in PUMCH and established reference value range since 2002 [32,33]. Immune profiles were analyzed by three-color flow cytometry (Epics XL flow cytometry; Beckman Coulter, USA) as previously described [34]. Freshly EDTAanticoagulated whole blood was incubated, lysed, and tested. The combination of monoclonal antibodies panels is as following: CD3/CD8/CD4, CD3/CD16CD56/ CD19, HLA-DR/CD38/CD8, CD28/CD8/CD4, CD45RA/CD4 and isotype controls (Immunotech, France). By measuring white blood cell counts and lymphocyte differentials from routine blood tests, cell counts of lymphocyte subsets were calculated of the same sample. HIV-RNA plasma viral load was examined by COBAS Ampliprep/TaqMan 48 RT-PCR real-time test (Roche, CA, USA).

Statistical analysis
Kolmogorov-Smirnov test was used to examine data distribution. The mean standard deviation with mean were used for parametric data and 25% and 75% for non-parametric data. two groups and parametric data comparison need student-test, non-parametric data demands for Mann-Whit test, χ 2 test for categorical variables.
Analysis of lymphocyte subsets was performed in individuals with an initial CD4+T count and available CD4+T cell records for at least twice visits. The linear mixed model (MIXED) with repeated measurements intended for identifying the possible covariates and time effect on repeated measurements of the immune profile. The mixed linear model was used to represent T cell subpopulation trajectories. Let yit (i = 1, 2, … , n; t = 1, 2, … , ni) be the immune indicators of HIV infected patient i at follow-up visit time t. The longitudinal immune indicators dynamics yit depicted the following model: represents time-varying covariates with fixed effects β and random effects bi, respectively. Random effects represent the effects of each subject that cannot be explained by the observed covariates. The errors in our assumption are independent and follow a normal distribution, εi(t) ~N(0, σ), and that bi and εi(t) are independent. The unstructured covariance of fixed effect was assumed for the covariance structure. Model fit statistics (e.g., AIC, BIC, etc) were used to choose the best covariance structure. To check the effect of time on CD4, we used the Scheffe-Test as post-hoc comparison. P-value < 0.05 (two-side) was regarded as significant.
Statistical analysis was performed with SAS software version 9.4 and SPSS software (SPSS ® for Windows ™ version 22.0, SPSS Inc., Chicago, IL, USA). Plots were drawn by GraphPad Prism 8.

AUTHOR CONTRIBUTIONS
LLF acquired, analyzed all the data, interpreted the data and drafted the manuscript; LXD and LXS analyzed the data and revised the paper; QZF and HY performed laboratory experiments; XJS, YLL and XXL collected patient samples and organized clinical data; CW, LW, TSL designed the study, obtained funding and evaluated data,; ZHD designed the study and instructor for statistical analysis. CW, LW, TSL made contributions to the manuscript revision. All authors participated in the manuscript review.

ACKNOWLEDGMENTS
The authors would like to thank all participants for their contributions to this study, especially the patients and the staff in PUMCH. In particular, the authors gratefully acknowledged the support of all laboratory researchers and data collectors during the visit time.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this study.

ETHICAL STATEMENT AND CONSENT
The Ethic Committee of Peking Union Medical College Hospital approved this study and all the participant provided written informed consent.