Homeostatic regulation occurs in all living organisms and is critical for preserving a variety of biologic parameters within a normal physiologic range . The maintenance of T-cell homeostasis (TCH) is essential for the functional integrity of the immune system. TCH is achieved through multiple complex and tightly regulated processes such as thymic output, access to cytokines, interaction with self-peptide-major histocompatibility complexes (sp-MHC), and antigen-independent peripheral T-cell proliferation [2,3].
TCH is characterized by the maintenance of T-cell (CD3+) percentages within a normal physiologic range (NPR) representing 65–85% of peripheral blood lymphocytes . The inability to maintain T cells within that range denotes an impairment of TCH regulation. TCH imbalance, in certain situations, can be used as a diagnostic tool. For instance, T-cell lymphocytosis (abnormally high levels of T cells) is associated with viral and bacterial infections, autoimmune diseases, lymphocytic leukemias, and other lymphoproliferative disorders [5–7]. Similarly, T-cell lymphopenia (abnormally low level of T cells) can be seen during acute infections, in hematologic disorders, and in both primary and secondary immune deficiency [8,9].
In a study by Margolick et al., a group of 372 HIV seroconverters enrolled in the Multicenter AIDS Cohort Study were followed for 8 years post-seroconversion. It was found that individuals who did not progress to AIDS maintained an intact TCH, whereas those who progressed experienced a failure of their TCH approximately 18 months prior to the onset of AIDS. This TCH failure was characterized by a substantial decline in the total level of T cells. Therefore, although progressive CD4+ T-cell depletion is the hallmark of HIV disease, impaired TCH can also occur during the course of the infection and is associated with progression to AIDS [11,12].
Impaired TCH has also been linked to deleterious clinical outcomes in non-HIV settings such as systemic lupus erythematosus, burn injury with subsequent decreased resistance to infections, and active pulmonary tuberculosis [13–16].
Despite findings that impaired TCH is associated with negative clinical outcomes in both HIV and non-HIV settings, few, if any, studies have assessed the effect of long-term successful combination antiretroviral therapy (cART) on TCH maintenance. The dynamics of CD3+ T-cell normalization in treated HIV-positive patients are, therefore, unknown. We, therefore, investigated the probability and predictors of transitioning in or out of the NPR during the course of therapy. Furthermore, we also assessed the clinical impact of impaired TCH on AIDS-defining illness (ADI) or death.
The Canadian Observational Cohort (CANOC) collaboration is a study of antiretroviral-naive HIV-positive patients initiating cART on or after 1 January 2000 . CANOC participants represent nearly a quarter of Canadians currently on cART. This collaboration is open to all Canadian HIV treatment cohorts with more than 100 eligible patients and currently includes eight participating cohorts across Canada. Eligibility criteria for inclusion into CANOC include documented HIV infection, residence in Canada, age 18 years and older, initiation of a first antiretroviral regimen consisting of at least three individual agents, and at least one measurement of HIV-1 RNA viral load and CD4+ T-cell count within 6 months of initiating cART. Patient selection and data extraction are performed locally at the data centers of the participating cohort studies. Nonnominal data from each cohort on a predefined set of demographic, laboratory, and clinical variables are then pooled at the Project Data Centre in Vancouver, British Columbia. The last date of follow-up in the cohort for the current analysis was 22 August 2010. All participating cohorts have received approval from their institutional ethics boards to contribute nonnominal patient-specific data.
Participants included in this study were first treated with at least three individual antiretroviral agents on or after 1 January 2000. Participants were eligible for the analysis if they came from sites able to collect and provide electronic data on CD3+ T-cell percentages, had at least one CD3+ T-cell measurement within 2 years prior to starting cART, and had at least two follow-up CD3+ T-cell measurements greater than 30 days apart following treatment initiation. Additionally, individuals from sites providing electronic ADI data without an ADI diagnosis prior to cART initiation were eligible for inclusion in the clinical impact analysis.
TCH was defined as the maintenance of CD3+ T-cell percentages within an NPR, representing 65–85% of circulating blood lymphocytes. These values are based on the historical phenotyping of 124 healthy HIV seronegative controls recruited through the Montreal General Hospital. All controls received a thorough physical examination and were screened for immune deficiency and autoimmune diseases. Altered TCH was defined as having T-cell percentages that were lower or higher than the NPR. We followed patients with available baseline and follow-up CD3+ T-cell measurements. Markov states were defined according to the CD3+ T-cell percentage intervals at each visit as follows: very low (<50%), low (50–64%), normal (65–85%), and high (>85%). Patients required at least two consecutive values outside their CD3+ T-cell percentage state to be considered as having transitioned.
Primary outcomes of interest were as follows:
- Achievement of TCH – defined as remaining or transitioning into the NPR for CD3+ T-cell percentages (65–85%).
- Time from cART initiation to ADI or death – defined as developing an ADI or dying during the course of the follow-up.
Demographic and clinical characteristics
Baseline characteristics were summarized using medians and interquartile ranges (IQRs) for continuous variables, and frequencies for categorical variables. Baseline values were defined as the closest values within 2 years of initiating cART. Duration of the follow-up period was measured from the time of cART initiation.
Longitudinal dynamics of CD3+ T-cell percentages
A multistate Markov model was used to model CD3+ T-cell dynamics in treated HIV-positive patients. Markov models depict the evolution of a disease as a gradual progression of mutually exclusive health states. These models are characterized by transition intensities, which represent the probability of transitioning from one state to another. Transition intensities are computed using the number of observed transitions between states and the time at which these transitions took place [18,19].
In this article, we explored a continuous-time Markov process with four states defined by CD3+ T-cell percentage levels. We used a state structure that assumes an individual must pass through all intermediate states in order to move from the lowest state to the highest and vice versa (Fig. 1). We estimated the mean amount of time that patients spent in a specific state prior to transitioning to a new state. We then performed univariate and multivariable analyses to assess the effect of covariates of interest on the transition probabilities between individual states. Based on previous studies analyzing the effect of HIV infection on the immune system, sociodemographic and clinical covariates potentially associated with immune dysregulation were included in the multivariable analysis [20,21]. These variables included age, sex, region, baseline treatment regimen, time-dependent detectable HIV viral load, and hepatitis C virus (HCV) coinfection.
Clinical impact of CD3+ T-cell percentages on AIDS-defining illness or death
The primary covariate of interest was time-updated CD3+ T-cell state. Patients were stratified according to their CD3+ T-cell values, as detailed above, and time from first cART initiation to ADI or death was calculated. Individuals were censored at their last CD3+ T-cell measurement if they did not experience an event (ADI or death). The association between the patient's current CD3+ T-cell state and his progression to ADI or death was determined by Cox proportional hazards models .
All analyses were performed using SAS software version 9.3 (SAS Institute, Cary, North Carolina, USA). P values<0.05 were considered significant.
Demographic and clinical characteristics
Among the 6673 initially ART-naive HIV-positive individuals followed within CANOC, 2210 were excluded from the analysis. Of these, 1071 came from two sites that did not have available electronic records of CD3+ T-cell percentages, 480 had insufficient follow-up data due to recent initiation of ART, and 659 had missing baseline and/or insufficient follow-up data. A total of 4463 patients met the inclusion criteria for the multistate analysis. Of these, 605 were diagnosed with ADI prior to cART initiation and were, therefore, excluded from the clinical impact analysis. The median follow-up times were 3.15 (IQR = 1.48–5.47) years and 3.06 (3.63–6.54) years for the multistate and the survival analyses, respectively. The median year of cART initiation was 2005 (IQR = 2002–2007). Most individuals were on a nonnucleoside reverse transcriptase inhibitor (NNRTI)-based or boosted protease inhibitor-based regimens at the time of treatment initiation.
At baseline, for the multistate analysis, median age was 40 (IQR = 34–47) years, median CD4+ T-cell count was 190 (IQR = 106–280) cells/μl, and median log10 viral load was 5.0 (IQR = 4.0–5.0) copies/ml. Sixty-eight percent had CD3+ T-cell percentages within the NPR (65–85% of circulating lymphocytes), and 29% were coinfected with HCV. These values were similar in the survival analysis. The demographic and clinical baseline characteristics of the study population for both analyses are summarized in Table 1.
Multistate modeling for transition between CD3+ T-cell percentage states
Transition intensities between states were estimated and summarized in Fig. 1. Individuals in the high CD3+ T-cell percentage state were 1.7 times more likely to transition to the NPR than those in the low state (transition intensities: 0.055 and 0.032, respectively). The mean time spent in each specific state was also estimated. Patients in the normal CD3+ T-cell percentage state remained stable for the longest time, with a mean period of 87.30 months (CI: 82.90–91.93 months) prior to transitioning. Patients in the very low, low, and high CD3+ T-cell percentage states transitioned after 15.34 months (95% confidence interval, CI: 13.52–17.39), 25.76 months (95% CI: 24.25–27.36), and 18.08 months (95% CI: 16.88–19.37), respectively.
Demographics and clinical characteristics by transition history are depicted in Table 2. Individuals were classified based on never transitioning from their baseline state or transitioning to the adjacent state. A total of 2508 (56%) patients never transitioned from their baseline CD3+ T-cell percentage state; 85% of these had normal TCH at baseline. After adjusting for treatment regimen, multivariable analysis showed that individuals with time-updated low CD4+ T-cell count and detectable HIV RNA were less likely to maintain TCH, and more likely to transition to lower CD3+ T-cell percentage states. Older age (>50 years old) and HCV coinfection were also associated with increased likelihood of transitioning out of the NPR (Fig. 2).
Survival analysis for time to AIDS-defining illness or death
Among the 3656 patients included in the survival analysis, we observed 438 events; of these, 217 were ADI diagnoses, and 221 were deaths. Survival analysis for time to ADI or death revealed that those in the very low CD3+ T-cell state (CD3+ T-cell percentages <50%) had the fastest progression to ADI or death compared with the other groups. A Kaplan–Meier curve of time to ADI or death by CD3+ T-cell percentage states is shown in Fig. 3.
In the multivariable proportional hazards model, after adjusting for region and sex, both very low and high CD3+ T-cell percentages were associated with increased risk of ADI or death (adjusted hazard ratio = 1.91, P <0.01 and hazard ratio = 1.49, P <0.01, respectively). Older age, HCV seropositivity, time-updated CD4+ T-cell count and updated detectable viral load were also associated with poor clinical outcomes (Table 3).
Previous reports of untreated individuals revealed that failure of TCH is an important landmark in HIV disease progression [11,12,23]. However, to our knowledge, no studies monitoring changes in circulating CD3+ T cells exist in treated HIV-positive patients. We studied TCH in a well described, large cohort of treatment-naive HIV-seropositive patients initiating cART. We constructed a Markov multistate model with reversible CD3+ T-cell percentage states, in order to evaluate covariates that influence the restoration and maintenance of TCH in these treated individuals. At baseline, two thirds of the study population had a normal TCH. Most of these patients never transitioned from that state. Those who transitioned, maintained their TCH for approximately 7 years prior to progressing to other states. This indicates that the TCH process is tightly regulated by an immune system that strives to maintain constant T-cell levels in the face of external perturbations from viral infections. Patients with baseline CD3+ T-cell percentage states outside the NPR spent less time in their respective state prior to transitioning. When they did transition, the most likely trajectory was toward the NPR from both high and low states. Thus, following TCH disruption, the immune system seems to have a natural tendency to return to its equilibrium. Interestingly, the majority of the patients who never transitioned from the very low CD3+ T-cell percentage state had CD4+ T-cell counts less than 200 cells/μl. This is in line with previous findings from Gange et al. showing that TCH is generally lost during the last phase of the disease. Previous studies measured TCH in terms of absolute CD3+ T-cell counts. However, in this study, TCH was defined in terms of CD3+ T-cell percentages, as we consider this parameter to be a more reliable marker of the proportional stability of the circulating T-cell pool. Indeed, T-cell percentages vary less than absolute counts as they are not affected by fluctuations in total lymphocyte counts [25–27].
Our study revealed that sustained virological suppression and higher CD4+ T-cell counts are important factors for the achievement of TCH. On the contrary, older age and HCV coinfection appear to be detrimental to TCH. This is of particular interest, given the increasing prevalence and incidence of HIV among individuals who are above 50 years of age . This group is expected to represent 50% of the US HIV population by 2015 . Furthermore, HCV coinfection occurs in approximately 20% of HIV-infected Canadians, with a prevalence of up to 90% among seropositive intravenous drug users [30–32].
Altered TCH has been associated with poor prognosis in non-HIV immune and infectious settings [13–16]. In untreated HIV patients, it has been associated with progression to AIDS . However, the relationship between CD3+ T-cell percentages and clinical outcomes in treated HIV patients has not been investigated until now. Our survival analysis showed that individuals with very low or high CD3+ T-cell percentages were more likely to develop an ADI or to die. It is possible that these two states reflect different immune disorders: immune deficiency vs. chronic inflammation. Indeed, the persistence of very low CD3+ T-cell percentages might connote a permanent damage of regenerative processes. Patients with severe T-cell lymphopenia have a compromised immune system that renders them susceptible to recurrent infections [33,34]. Conversely, high CD3+ T-cell percentages might reflect an underlying chronic antigenic stimulation leading to abnormal T-cell expansion. This is a phenomenon known as memory inflation. It occurs in the presence of persistent reactivating viruses such as cytomegalovirus (CMV), where a large fraction of effector T cells avoid apoptosis and become terminally differentiated memory T cells [35,36]. These cells occupy the immunological ‘space’ that would normally be allocated to naive T cells. The ensuing shrinkage of the T-cell repertoire diversity, thus, predisposes the host to increased risk of infections and poor response to vaccination .
In this study, we could not assess the association between CMV coinfection and CD3+ T-cell expansion, because CMV serology is not available throughout all CANOC centers. Therefore, the role of CMV in CD3+ TCH disruption remains to be elucidated.
The introduction of cART has resulted in a significant reduction in morbidity and mortality among HIV-positive patients. However, with HIV-positive patients living longer, a change in the mortality/morbidity profile of these individuals has been observed. Proportion of deaths attributable to non-AIDS-defining illnesses (NADIs) rose from 13.1% in 1996 to 42.5% in 2004 . Although CD4+ T-cell recovery might be adequate to control opportunistic infections and viral-mediated neoplasms, residual immune dysregulation such as altered CD3+ TCH might impact on age-related comorbidities that are linked to chronic inflammation. The specific immune pathways through which altered TCH may lead to death and/or comorbidities are still unclear. Therefore, there is a need for future studies to identify the key immune and functional disorders characterizing this phenotype.
One of the challenges faced in the evaluation of true immune recovery with cART stems from the limited number of clinically relevant and available surrogate markers. Monitoring CD3+ T-cell levels, as a supplement to CD4+ T-cell counts, may provide further insight into immune reserve and restoration. To our knowledge, this is the first study linking altered TCH and morbidity/mortality in a cohort of cART-treated HIV-positive patients.
The CANOC Collaboration includes the following: Community Investigators: Sean Hosein and Shari Margolese; Investigators: Gloria Aykroyd (Ontario HIV Treatment Network, OHTN), Louise Balfour (University of Ottawa, OHTN Cohort Study, OCS Co-Investigator), Ahmed Bayoumi (University of Toronto, OCS Co-Investigator), Ann Burchell (Ontario HIV Treatment Network), John Cairney (University of Toronto, OCS Co-Investigator), Liviana Calzavara (University of Toronto, OCS Co-Investigator), Angela Cescon (British Columbia Centre for Excellence in HIV/AIDS), Curtis Cooper (University of Ottawa, OCS Co-Investigator), Kevin Gough (University of Toronto, OCS Co-Investigator), Silvia Guillemi (British Columbia Centre for Excellence in HIV/AIDS, University of British Columbia), P. Richard Harrigan (British Columbia Centre for Excellence in HIV/AIDS, University of British Columbia), Marianne Harris (British Columbia Centre for Excellence in HIV/AIDS), George Hatzakis (University of Southern California), Robert Hogg (British Columbia Centre for Excellence in HIV/AIDS, Simon Fraser University), Don Kilby (University of Ottawa, Ontario HIV Treatment Network), Marina Klein (Montreal Chest Institute Immunodeficiency Service Cohort, McGill University), Richard Lalonde (The Montreal Chest Institute Immunodeficiency Service Cohort and McGill University), Viviane Lima (British Columbia Centre for Excellence in HIV/AIDS, University of British Columbia), Mona Loutfy (University of Toronto, Maple Leaf Medical Clinic, OCS Co-Investigator), Nima Machouf (Clinique Medicale l’Actuel, Université de Montréal), Ed Mills (British Columbia Centre for Excellence in HIV/AIDS, University of Ottawa), Peggy Millson (University of Toronto, OCS Co-Investigator), Julio Montaner (British Columbia Centre for Excellence in HIV/AIDS, University of British Columbia), David Moore (British Columbia Centre for Excellence in HIV/AIDS, University of British Columbia), Alexis Palmer (British Columbia Centre for Excellence in HIV/AIDS), Janet Raboud (University of Toronto, University Health Network, OCS Co-investigator), Anita Rachlis (University of Toronto, OCS Co-Investigator), Stanley Read (University of Toronto, OCS Co-Investigator), Sean Rourke (Ontario HIV Treatment Network, University of Toronto), Marek Smieja (McMaster University, OCS Co-Investigator), Irving Salit (University of Toronto, OCS Co-Investigator), Darien Taylor (Canadian AIDS Treatment Information Exchange, OCS Co-Investigator), Benoit Trottier (Clinique Medicale l’Actuel, Université de Montréal), Chris Tsoukas (McGill University), Sharon Walmsley (University of Toronto, OCS Co-Investigator), and Wendy Wobeser (Queen's University, OCS Co-Investigator)
Analysts and Staff: Guillaume Colley (British Columbia Centre for Excellence in HIV/AIDS), Svetlana Draskovic (British Columbia Centre for Excellence in HIV/AIDS), Mark Fisher (OHTN), Sandra Gardner (University of Toronto), Nada Gataric (British Columbia Centre for Excellence in HIV/AIDS), Suzanne Humphreys (British Columbia Centre for Excellence in HIV/AIDS), David Milan (British Columbia Centre for Excellence in HIV/AIDS), Sergio Rueda (OHTN), and Benita Yip (British Columbia Centre for Excellence in HIV/AIDS).
CANOC is funded by an Emerging Team Grant from the Canadian Institutes of Health Research (CIHR) and is supported by the CIHR Canadian HIV Trials Network (CTN242). P.N. is supported through a CANOC Scholarship Award, a collaborative program of CANOC, CTN, and REACH. J.M.R. and C.C. have Career Scientist Awards from the Ontario HIV Treatment Network. M.R.L. receives salary support from the Canadian Institutes of Health Research. J.S.G.M. is supported by an Avant-Garde Award from the National Institute on Drug Abuse, National Institutes of Health.
Conflicts of interest
There were no conflicts of interest for this study.
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