Increased levels of activated subsets of CD4 T cells add to the prognostic value of low CD4 T cell counts in a cohort of HIV-infected drug users
Carbone, Javiera; Gil, Juanaa; Benito, José M.a; Navarro, Joaquína; Muñóz-Fernández, Angelesa; Bartolomé, Joaquína; Zabay, José M.a; López, Franciscob; Fernández-Cruz, Eduardoa
From the aDepartment of Immunology, University General Hospital Gregorio Marañón, Complutense University, Madrid, Spain; and bNufarm-21 Madrid, Spain.
Correspondence and reprint requests to: Dr Javier Carbone, Department of Immunology, University General Hospital Gregorio Marañón, Dr Esquerdo, 46, 28007 Madrid, Spain. Tel: +34 91 5868423; fax: +34 91 5866698; e-mail: firstname.lastname@example.org
Received: 23 August 1999;
revised: 22 August 2000; accepted: 26 September 2000.
Sponsorship: This study was supported by grants from the ‘Comunidad Autónoma de Madrid’ and the ‘Fondo de Investigación Sanitaria’ to E.F.-C.
Objective: To identify subsets of CD4 T lymphocytes that can predict the development of AIDS and to assess whether increased levels of these cellular markers could provide additional independent prognostic information to the CD4 T cell count and plasma HIV-1-RNA levels.
Design and methods: In a prospective study, a cohort of 85 HIV-positive intravenous drug users [clinical categories of the CDC classification A (n = 48) and B (n = 37)] were followed for a period of 37 ± 13 months. Memory and activated CD4 and CD8 T cells were quantitated by three-colour flow cytometry at baseline and expressed as a percentage of total CD4 and CD8 lymphocytes. Clinical evaluations were performed at 6 month intervals. The relationships between these lymphocyte subsets and progression to AIDS were studied using Kaplan–Meier plots and proportional hazards regression models.
Results: After adjustment for the level of CD4 T cells and plasma HIV-1-RNA levels, the elevation in the subset CD4+CD38+DR+ was the marker within the functionally distinct subsets of CD4 T lymphocytes with additional prognostic value in bivariate Cox regression models. In multivariate models, increased percentages of CD4+CD38+DR+ T cells provided the strongest independent prognostic information for progression to AIDS (relative hazard, 1.07;P < 0.0001).
Conclusion: Our results suggest that high levels of CD4+CD38+HLA-DR+ T cells reflect the increasing degree of CD4 T cell activation during the progression of HIV infection, and could be used together with the CD4 T cell and HIV-RNA levels to evaluate more accurately the progressive cellular immune impairment associated with the risk of progression to AIDS.
Profound changes of immune cellular activation occur shortly after infection with HIV and persist throughout the disease course. The identification of cellular immune activation markers is important to aid in the monitoring of disease progression [1–12] and the response to therapy [13,14]. The decline in the number of CD4 T lymphocytes is considered one of the most reliable cellular laboratory markers used as predictor of progression to AIDS [1,2,5,6,15,16]. Previous studies have focused on changes in the representation of phenotypically defined functionally distinct subsets of CD4 and CD8 T cells in different disease stages of HIV infection [17–33]. However, few studies have addressed the prognostic value of the memory and activated subsets of CD4 and CD8 T lymphocytes in long-term follow-up studies [7,12,34–39]. Using prospective follow-up studies it has been shown that an increase in the percentages of CD8 T lymphocytes expressing CD38 predict the rate of CD4 decline  and the development of AIDS independently of the CD4 cell count [35,37,38]. To date, very few studies have assessed whether the subsets of CD4 T cells could add prognostic value to the information provided by the level of CD4 T cells . We performed a prospective follow-up lasting 54 months in a cohort of 85 HIV-positive injection drug users (IDU) to assess which of the lymphocyte activation antigen changes in CD4 T cells can provide additional prognostic value to the CD4 cell number and plasma HIV-1-RNA levels for the development of clinical AIDS.
Materials and methods
We performed a prospective follow-up lasting from 8 to 54 months (mean 37 ± 13 months) of the evolution of HIV infection in a cohort of IDU. The patients included in the study were 85 HIV-infected heroin users admitted to our General Hospital of the Community of Madrid or attending the outpatient department for a variety of medical reasons, not necessarily related to HIV infection. Seropositivity for HIV-specific antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) (Abbot recombinant HIV-1/HIV-2; Abbott GmBh Diagnostika, North Chicago, IL, USA) and Western blot confirmation (LAVBlot; Diagnostic Pasteur, Marnes La Coquette, France). In our clinical study, HIV-positive IDU with asymptomatic infection and with different clinical manifestations of early stage infection voluntarily entered into the study at different times. As a result the length of follow-up period differs among patients. Each participant was scheduled to return at 6 month intervals for laboratory tests and clinical evaluation of disease progression. There were 67 men and 18 women (mean age 34 ± 5 years). At entry the clinical stages together with the CD4 cell categories of the patients were defined according to the definitions of the Centers of Disease Control and Prevention (CDC) . CDC category A included 48 asymptomatic patients (A1 21; A2 21; A3 6) and category B included 37 symptomatic patients without AIDS-defining diseases (B1 12; B2 12; B3 13). IDU HIV-infected patients who had a history of recent drug use, active infections or opportunistic infections or AIDS-defining diseases were not included in the study. Nineteen out of 85 (22%) HIV-positive IDU, asymptomatic or symptomatic non-AIDS at entry (initial visit), progressed to clinical AIDS. Of the 19 patients who progressed to AIDS, 14 (74%) died during follow-up. Specific AIDS conditions diagnosed after entry were Candida oesophagitis (n = 6), Pneumocystis carinii pneumonia (n = 3), toxoplasmosis of the brain (n = 3), recurrent pneumonia (n = 5), cytomegalovirus retinitis (n = 1), tuberculosis (n = 4), wasting syndrome caused by HIV (n = 2), Kaposi's sarcoma (n = 1), Salmonella septicaemia (n = 1), Mycobacterium avium disseminated (n = 1) and progressive multifocal leukoencephalopathy (n = 1). All the patients who died had a diagnosis of AIDS by the 1993 definition. Regarding the final status of the group of 66 IDU who did not progress to clinical AIDS, 49 showed, at the last follow-up visit, the same clinical status as at entry. In the remaining 17 IDU of this group there was a progressive course to more advanced stages of the disease (stage B from stage A, n = 17). In relation to antiviral treatments, a total of 35 (41%) HIV-infected individuals in this study were receiving antiretroviral therapy as either zidovudine (n = 29), dideoxyinosine (n = 4), dideoxycytidine (n = 1) or both zidovudine and dideoxycytidine (n = 1) at study entry. During the follow-up period, antiretroviral therapy was used by 69 (81%) patients for various time periods as either monotherapy (n = 5), double therapy (n = 33) or highly active antiretroviral therapy (HAART; one protease inhibitor and two nucleoside analogues) (n = 31). Prophylaxis of opportunistic infections was used by 26 out of 85 (31%) patients as either isoniazid (n = 10), trimethoprim/sulfamethoxazole (n = 9) or both (n = 7) at study entry. During the follow-up period, prophylaxis for opportunistic infections was employed by 44 out of 85 patients (51%) as either isoniazid (n = 15), trimethoprim/sulfamethoxazole (n = 17) or both (n = 12).
Lymphocyte phenotype analysis
Three-colour immunophenotyping of peripheral blood lymphocytes was performed by flow cytometry. The monoclonal antibodies used were directly conjugated with the fluorochrome fluorescein isothiocyanate (FITC; FL1), phycoerythrin (PE; FL2), or peridinin chlorophyll protein (PerCP; FL3). We enumerated T cell subsets using FITC/PE/PerCP combinations of CD38/HLA-DR/CD4 or CD8; CD38/CD45RO/CD4 or CD8; HLA-DR/CD45RO/CD4 or CD8; CD25/CD45RO/CD4 or CD8; CD45RA/CD45RO/CD4 or CD8; CD38/CD11b/CD8; HLA-DR/CD11b/CD8; CD3FITC/CD4PerCP or CD8; CD45/CD14 and isotope controls. Lymphocyte staining was carried out using a whole-blood lysis technique, according to the recommended methodology and quality control procedures . Stained samples were analysed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) with lysis II software (Becton Dickinson). Data were collected for 5000 events in a lymphocyte gate defined by both forward and side scatter characteristics and by phenotypic evaluation (CD45/CD14; Leucogate; Becton Dickinson). Background staining was assessed with the appropriate isotype- and fluorochrome-matched control monoclonal antibodies directed against an irrelevant target and was subtracted from all the results. Only CD4 and CD8 lymphocyte populations that express CD3 were considered in the analysis. Percentages of memory and activated CD4 and CD8 T cell subsets are expressed as a percentage of total CD4 and CD8 lymphocytes. To determine the percentage of CD4 or CD8 T cells co-expressing one or both of the additional markers under analysis, gates were set that included only PerCP-positive cells with the same intensity of fluorescence as the CD4 and CD8 cells that express CD3. In the present study we used a combination of anti-CD45RO and anti-CD45RA monoclonal antibodies for quantitative analysis of phenotypically defined functionally distinct subsets of CD4 and CD8 T cells. Transitional (RA+RO+) cells were included within the CD45RO+ T cells and all the lymphocytes defined as CD45RO− T cells express the CD45RA molecule .
Plasma HIV-1 RNA levels were measured by reverse-transcriptase polymerase chain reaction (Ultrasensitive Amplicor HIV Test, Roche Molecular Systems, Branchburg, NJ, USA) performed according to the instructions of the manufacturer, and the threshold was 50 HIV-1 RNA copies/ml (1.7 log10 copies/ml).
Each of the functionally distinct lymphocyte subsets of CD4 and CD8 T lymphocytes was split into two groups using the median value observed as the cut-off. The HIV-1-RNA serum concentration was analysed after log10 transformation. Categories of RNA level were defined in three groups [< 5000 copies/ ml (< 3.7 log10 copies/ml), 5000–30 000 (3.70–4.48 log10) and > 30 000 (> 4.48 log10) according to the commonly used categories for HIV-1-RNA levels . Time-to-event analyses were conducted using the Kaplan–Meier method . An event was considered when CDC-defined AIDS conditions were present. Breslow statistics  were performed for testing the equality of progression curves. For the Kaplan–Meier curve of the combined lymphocyte subset levels and CD4 T cell count, categories were merged into two groups. For the CD4 T cell count, the median value was used to select the cut-off in Kaplan–Meier analysis (400 cells/mm3). Association between variables was measured by means of the non-parametric Spearman's rank-correlation coefficient. The Cox proportional hazards model was used to evaluate the prognostic value of the functionally distinct lymphocyte subsets of CD4 and CD8 T lymphocytes for the development of AIDS. Initially, each variable was included in a univariate model. Bivariate Cox regressions, including each of the CD4 and CD8 T cell subsets as categorical variables and adjusting for CD4 T cell counts or plasma HIV-1-RNA levels as continuous variables, were then performed in order to identify independent markers of progression. The CD4 and CD8 T cell subsets with the higher independent predictive value that reached statistical significance at the 5% level along with CD4 T cell counts and HIV-RNA concentration as well as treatment were then included in a multivariate model. CD4 and CD8 T cell subsets (percentages) were included either as categorical or continuous variables. When lymphocyte subsets were included as continuous variables in the multivariate model, increases of 1% or 5% units were used. CD4 T cell counts (cells/mm3) and plasma HIV-1-RNA levels (log10 copies/ml) were included as continuous variables. A variable assuming a value of 1 if the patient was receiving antiretroviral treatment at entry and zero otherwise was included in the Cox model. We have also included in the Cox analysis three variables in relation to the number of drugs used in the antiretroviral treatment: time in monotherapy, double therapy and HAART. We also included a combined variable consisting of the sum of the time in each therapy multiplied by one, two or four, depending of the type of treatment received (monotherapy, double or triple drug combinations, respectively). A backward procedure was then used to eliminate variables that were not significant at the P = 0.05 level. The variability of the proportional hazards assumptions was tested for each variable, X, by including a time-dependent term (X/6 months or X/12 months) in the Cox analysis. We found no evidence of non-proportionality. Statistical computations were performed using the Statistical Package for the Social Sciences (SPSS).
The mean percentages (absolute numbers) of CD4 and CD8 lymphocyte counts for all HIV-positive patients at entry were 21 ± 10 (445 ± 308 cells/mm3) and 56 ± 12 (1128 ± 605), respectively. The mean percentages (absolute numbers) of CD4 T cells in the three groups, A1, A2 and A3, of HIV-positive asymptomatic patients were 29 ± 9 (731 ± 71 cells/mm3), 20 ± 8 (428 ± 51), 11 ± 9 (144 ± 59), respectively. The mean percentages (absolute numbers) of CD8 T cells were 53 ± 11 (1359 ± 97), 58 ± 13 (1361 ± 161), 56 ± 9 (662 ± 94), respectively. The mean percentages (absolute numbers) of CD4 T cells in the three groups, B1, B2, B3, of HIV-positive patients were 28 ± 7 (578 ± 59 cells/mm3), 21 ± 8 (367 ± 49), 10 ± 5 (116 ± 19), respectively. The mean percentages (absolute number) of CD8 T cells were 54 ± 9 (1160 ± 131), 51 ± 10 (990 ± 165), 63 ± 9 (693 ± 129), respectively.
Table 1 shows the 48 month actuarial progression rates to AIDS and the relative hazards (RH) for subjects stratified according to different levels of CD4 T cell number, plasma HIV-1-RNA level and activated and memory subsets of CD4 and CD8 T lymphocytes. The 95% confidence intervals (CI) and P values of the Cox analysis are also included in Table 1. Percentages of memory and activated CD4 and CD8 T cell subsets are expressed as a percentage of total CD4 and CD8 lymphocytes. Within the CD4 T cell subsets, increases in the percentages of circulating CD4+CD38+DR+ [Breslow statistic value (BS), 14.27;P = 0.0002], CD4+CD45RO+ (BS, 12.56;P = 0.0004), CD4+ CD45RO+DR+ (BS, 11.7;P = 0.0006) and CD4+ CD45RO+CD38+ T cells (BS, 8.14;P = 0.004) were highly predictive of AIDS. Among the different subsets of CD8 T lymphocytes, the increases in the percentages of circulating CD8+CD38+ (BS, 12.28;P = 0.0005), CD8+CD38+DR+ (BS, 5.95;P = 0.014), CD8+CD45RO+CD38+ (BS, 10.3, P = 0.0013), CD8+CD11b−CD38+ (BS, 11.7;P = 0.0006) and CD8+CD11b+CD38 T cells (BS, 6.67;P = 0.0098) showed the highest prognostic value for time to development of AIDS. Using Cox bivariate regression analysis adjusted by CD4 absolute numbers, we found that among the different subsets of CD4 T lymphocytes the increase in the percentage of CD4 T cells that co-express CD38 and DR antigens and of CD4+CD45RO+ T cells were the variables that showed additional predictive value (Table 1). Using this analysis we also found that among the different subsets of CD8 T lymphocytes, the increase in the percentage of total CD8+CD38+ T cells was the only variable with additional predictive value of progression to AIDS after adjustment by CD4 T cell count. After adjustment for plasma HIV-1-RNA levels, the RH of the CD4 T cell subsets CD4+CD38+DR+ and CD4+CD45RO+DR+ and the activated CD8 T cell subsets CD8+CD38+ and CD8+CD45RO+CD38+ retained significant prognostic value.
After adjustment for antiviral treatment, the RH for AIDS associated with CD4+CD38+DR+, CD4+ CD45RO+DR, CD8+CD38+ and CD8+CD45RO+ CD38+ levels were 5.95 (CI, 1.90–18.55), 5.24 (1.81–15.11), 10.96 (2.51–47.79) and 4.10 (1.33–12.66) respectively, similar to those of the unadjusted RH [6.35 (CI, 2.09–19.33), 5.62 (2.00–15.79), 10.53 (2.42–45.87) and 4.43 (1.45–13.47)] shown in Table 1.
The CD4 T cell count, plasma HIV-1-RNA levels and the most predictive activated CD4 (CD4+CD38+ DR+) and CD8 (CD8+CD38+) T cell subsets as well as treatment were included in a Cox multivariate analysis. The correlation between these variables included in the multivariate regression model is shown in Table 2. A Cox model with CD4 and CD8 T lymphocyte subsets modelled as categorical variables (Table 1) showed that the CD4+CD38+HLA-DR+ T cell subset and plasma HIV-1-RNA level were the variables that retained significant prognostic value for progression to AIDS (RH 4.36;P = 0.026; χ2 = 6.22 and RH 2.69;P = 0.0005; χ2 = 17.61, respectively). When CD4 and CD8 T cell subsets were included as continuous variables, we also found that the CD4+CD38+HLA-DR+ T cell subset and plasma HIV-1-RNA levels retained significant prognostic value (RH 1.07; CI 1.04–1.11;P < 0.0001; χ2 = 21.4321 and RH 3.7; CI 1.73–7.89;P = 0.0007; χ2 = 18.4940, respectively). The increased risk of AIDS associated with a 5% increase in the percentage of CD4+CD38+DR+ T lymphocyte was 42% (RH 1.42; CI 1.23–1.65;P < 0.0001). Our analysis demonstrated that the increase in the percentages of CD4+CD38+DR+ T cells provided independent prognostic information for progression to AIDS. None of the treatment variables were found to affect progression to AIDS when using the multivariate Cox models.
Fig. 1 shows the cumulative incidence of AIDS up to 48 months for participants stratified by both baseline CD4 cell count [< 400 and > 400/mm3 (median value 414)] and baseline CD4+CD38+HLA-DR+ T cell percentages [< 12 and > 12% (median value)]. Increased levels (> 12%) of CD4+CD38+HLA-DR+ T cells added significant prognostic information to the CD4 cell count among HIV-positive IDU with CD4 cell counts greater than 400/mm3 (BS, 4.35;P = 0.03) and with CD4 cell counts less than 400/mm3 (BS, 5.01;P = 0.02). Among the group of patients with CD4 cell counts greater than 400/mm3, the 12, 24, 36 and 48 month rates of progression to AIDS were 0, 0, 10 and 10%, respectively, for 35 cases with percentages of CD4+CD38+DR+ T cells below 12%, compared with 23, 23, 23 and 38% for nine patients with higher percentages of CD4+CD38+DR+ T cells (> 12%). Among HIV-positive IDU with CD4 cell count less than 400/mm3, the 12, 24, 36 and 48 month rates of progression to AIDS were 0, 8, 8 and 8%, respectively, for 16 cases with percentages of CD4+CD38+DR+ T cells below 12%, compared with 17, 26, 48 and 48% for nine patients with higher percentages of CD4+CD38+DR+ T cells (> 12%).
These results suggest that measurements of the percentage of CD4+CD38+HLA-DR+ T cells in combination with the CD4 T cell count and viral burden may be useful to evaluate more accurately the risk of progression to AIDS.
In a longitudinal follow-up study we have determined which functionally distinct subsets of CD4 T lymphocytes were associated with subsequent AIDS in a cohort of parenteral drug users who were seropositive at the time of enrolment. For the first time, using a prospective study, we have demonstrated that increased levels of CD4+CD38+HLA-DR+ T cells, after adjustment for the level of CD4T cells and plasma HIV-1-RNA levels, had additional prognostic value to the CD4T cell count and plasma HIV-1-RNA levels. Using the proportional hazards model, progression to AIDS was most strongly associated with increased percentages of CD4+CD38+DR+ T cells than with reduced absolute numbers of CD4 lymphocytes or other subsets of CD4 and CD8 T lymphocytes analysed in this study. The Kaplan–Meier curves demonstrated that at every CD4 cell strata, those individuals with high levels of CD4+CD38+DR+ cells had a higher incidence of progression to AIDS. We found in a previous cross-sectional study  that in asymptomatic HIV infection the increase in the fraction of CD4 T cells that co-expressed HLA-DR and CD38 antigens was, within the phenotypically defined functionally distinct subsets of CD4 and CD8 T cells, the variable most strongly associated with the decline in total CD4 T cell count. Other authors using cross-sectional studies [23,24] have reported an increase in this activated population in HIV-positive patients in the late stages of HIV infection. The findings in the current study suggest that a high level of CD4+CD38+HLA-DR+ T cells reflects the increasing state of immune activation during the progression of HIV infection and could be used together with the CD4 T cell count to evaluate more accurately the progressive cellular immune impairment associated with chronic HIV disease and to predict the risk of progression to AIDS.
In the current study, within the functionally distinct subsets of CD8 T cells, the increase in the percentages of CD8+CD38+ provided additional independent prognostic information on the development of AIDS. In other long-term follow-up studies performed mainly in homo/bisexual and haemophilic men [34,35,37,38], the CD8+CD38+ measurement has also been shown to have prognostic value for progression to AIDS, in addition to that of the absolute CD4 cell number and plasma HIV-1-RNA level . Other studies  have shown that increased numbers of activated CD8+CD38+CD45RO+ T cells predicted the decline of CD4 T cells in HIV-1-infected patients.
It has been suggested that cellular immune activation itself rather than merely an association with HIV-RNA levels is an additional contributing factor to subsequent disease progression . As previously demonstrated for patients with high levels of CD8+CD38+ T cells [34–38] among HIV-infected patients, the strong prognostic value of CD4+CD38+DR+ measurement in our analysis suggests that this marker, in association with plasma HIV-1-RNA levels, may help to identify a high risk of progression to clinical AIDS and might have utility in the clinical management of HIV-infected patients.
However, given the small size of our sample of IDU, future prospective studies with larger numbers of cases are necessary to rule out the potential independent roles of other markers as predictors and to ascertain the clinical utility as prognostic markers of the activated subsets of T cells.
The authors would like to thank Dr Salvador Resino and Mr José M. Bellón (Department of Immunology) for their contribution to the data analysis. They also acknowledge Dr Manuel Desco (Department of Experimental Medicine) for helpful discussions.
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CD4+CD38+DR+ lymphocyte subset; CD4 lymphocyte counts; CD8 T cell subsets; disease progression; HIV; HIV RNA; immune activation; intravenous drug users; longitudinal cohort study; prognosis
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