352
AIDS COMMENTARY
CD4+ Lymphocyte Cell Enumeration for Prediction of Clinical Course of Human Immunodeficiency Virus Disease: A Review Daniel S. Stein, Joyce A. Korvick, and Sten H. Vermund
Medical and Epidemiology Branches. Clinical Research Program. Division ofA IDS. National Institutes o.fAllergy and Infectious Diseases, National Institutes of Health. Bethesda. Maryland
Human immunodeficiency virus type 1 (HIV -1), a retrovirus, is the etiologic cause of the overwhelming majority of cases of AIDS [1, 2]. The HIV-l envelope glycoprotein, gp 120, specifically binds to the CD4 receptor that is expressed in greatest concentration in a subset of T lymphocytes and in lower amounts on monocytes and macrophages [3]. HIV can apparently enter both resting and stimulated CD4+ T lymphocytes, but replication only proceeds to completion in stimulated CD4+ T lymphocytes [4]. T lymphocyte subsets are defined by both functional activity and phenotypic assays using monoclonal antibodies. Cells expressing CD4 receptors are termed the "helper/ inducer" subset, reflecting their role as both helper cells for B cell responses for antigens expressed on cells bearing human leukocyte antigen (HLA) class II receptors and inducer cells that cause T cells to suppress immune responses. Activated CD4+ cells are major sources ofinterleukins 2, 3, and 5 and interferon-)', which are important in the growth, function, and differentiation of the cellular immune system [5). Less commonly, CD4+ cells function as cytotoxic cells. The selective loss ofCD4+ cells results in numerous immune defects
Received 16 July 1991; revised I I October 1991. Informed consent was obtained from all subjects whose data appear in the figure; guidelines of the US Department of Health and Human Services and of the participating institutions of the Multicenter AIDS Cohort Study were followed. Grant support for data in figure I: National Institutes of Health for Multicenter AIDS Cohort Study (AI-32535. -72632. -72634, -72631, -72676). Reprints or correspondence: Dr. D. S. Stein. MB/CRP/DAIDS/NIAID/ NIH. 6003 Executive Blvd., Bethesda. MD 20892. The Journal of Infectious Diseases This article is in the public domain.
1992;165:352-63
associated with susceptibility to the opportunistic infections that are the hallmark of AIDS [6). We discuss how the CD4+ cell depletion caused by HIV-l infection increases the risk of developing the clinical syndrome of AIDS in adults. In addition, the association of CD4+ cell depletion with other immune and nonimmune markers will be considered. Finally, we will discuss the effects of therapy on CD4+ cell counts and the problem of correlating changes in these counts with subsequent course of disease. A caveat for this review is that nearly all of the studies referenced here have cohorts of male participants secondary to the very high male preponderance of HIV infection in the developed world where these studies were done [7). Prospective CD4+ cell data for women with HIV infection are needed, particularly correlating women-specific clinical outcomes, such as genital tract infections and neoplasms.
Measurement and Variability Measurement oflymphocyte subsets is dependent on phenotypic identification by fluorescent-labeled monoclonal antibodies. Enumeration of cells is usually done in a flow cytometer that uses a laser beam to cause fluorescence of the bound antibody. The cells are separated by both light scatter and color of the attached fluorescent compound. They are counted as very short pulses of electric current that are generated in response to laser stimulation as detected by photomultiplier tubes. Variability occurs as a consequence of the techniques used in cell labeling, type of antibody, type of machine, expertise of the operators, standardization of ma-
Downloaded from http://jid.oxfordjournals.org/ at New York University on May 31, 2015
Over the last 10 years the appreciation of the full breadth of the spectrum of human immunodeficiency virus (HIV) infection has gradually increased; it is now known that AIDS represents merely the end stage of this progressive infectious process. The surrogate marker that most closely correlates with the stage of HIV infection is the CD4+, or T helper, cell count. Because this count is relied on in making important therapeutic decisions, it is of paramount importance that clinicians be cognizant of and fully understand the multiple factors that can influence this parameter: the variability of the count from day to day and from morning to night, the influence of intercurrent viral infections, the influence of drugs. In this AIDS Commentary, Sten H. Vermund and his colleagues at the National Institutes of Health discuss these issues and put the CD4 + cell count into a lucid and practical clinical perspective. Merle A. Sande
lID 1992; 165 (February)
AIDS Commentary
Seroconversion Studies Acute HIV infection is associated with a nonspecific clinical syndrome that commonly includes fever, pharyngitis, lymphadenopathy, rash, and myalgias-arthralgias. The HIV core antigen (p24) and high titers of cytopathic virus, similar
to those seen in late-stage disease [12, 13], can be detected before the appearance of HIV antibodies. After the appearance of HIV antibodies by the screening ELISA test, p24 antigenemia generally becomes undetectable, though it can occasionally persist and often will recur later in the disease [14, 15]. Recent investigations using acid pretreatment of patient sera (pH 2.5-3.0) have shown that most p24 antigen is in the form of circulating immune complexes [16, 17]. Data from a group of 29 seroconverters followed for 1 year indicate that during this undetectable phase for p24 antigen (using standard detection methods), >50% of patients have persistent p24 antigen immune complexes [16]. HIV-I titers found in plasma and peripheral blood mononuclear cell cultures also fall rapidly as specific antibodies are detectable, suggesting at least a transiently effective host immune response [12, 13]. Markers of immune stimulation include {j2microglobulin, a component of class I HLA cell receptors [18], and neopterin, a metabolite of guanosine triphosphate produced by macrophages after stimulation by interferon-')' [19]. Both have been shown to increase at the time of seroconversion and to continue to increase after seroconversion [20, 21]. Laboratory findings associated with the acute HIV infection syndrome include lymphopenia with lowered CD4+ cell numbers and thrombocytopenia [12]. CD3+CD4-CD8lymphocytes have cytolytic activity and increase in numbers at the time ofseroconversion and reach levels at 3-9 months after infection resembling the levels found in patients chronically infected with HIV [22]. Hofmann et al. [20] in their UCLA MACS series showed that 73% of patients (33/45) had clinically and statistically significant CD4+ cell declines at seroconversion, and 83% experienced significant declines within I year. CD8+ cell counts were noted to rise at time of seroconversion. Detels et al. [23], in another UCLA MACS study of 51 homosexual men who were followed through seroconversion, demonstrated a mean CD4+ cell decline from 854 cellslJ.tl to 651 cells/,ul 6 months after seroconversion. However, it was rare for seroconverters to have a consistent decline in their percentage ofCD4+ cells over the next 3 years. In the interval between visits, stable or declining levels of CD4+ cell percentages were found in 38% of subjects, with 12% experiencing declines followed by a leveling in their rates ofloss ofCD4+ cells. Overall, 62% experienced declines in their CD4+ cell percentage over 3 years of follow-up. In another study of predominantly homosexual men (the San Francisco Men's Health Study), the mean CD4+ cell count fell from 1078 to 716 cells/,ul by 12 months after seroconversion (n = 37) and to 636 ± 69 cells/,ul by 18 months. The latter value was similar to that noted among seropositives at the time of their recruitment in 1984 [24]. The CD8+ cell count rose significantly at seroconversion from 794 to 1198 ± 117 cells/,ul, then declined slightly to a level still above baseline (1073 ± 87 cells/,ul) 6 months after serocon-
Downloaded from http://jid.oxfordjournals.org/ at New York University on May 31, 2015
chine alignment, number of cells counted, and normal variations in cell populations. The quality control program of the Multicenter AIDS Cohort Study (MACS) was established in an attempt to decrease the variability in CD4+ cell enumeration [8]. The MACS is a study of -- 5000 homosexual and bisexual men in Baltimore, Chicago, Los Angeles, and Pittsburgh [9]. Identical flow cytometers, monoclonal antibodies, analytic procedures, and sample preparation protocols were used in the four laboratories. Still, significant variations in CD3+, CD4+, and CD8+ lymphocyte subset percentages were found. Variation over time occurred within center, between centers, and within patients. The within-person standard deviations ranged from 4.5% to 7.9% for the percentages of CD3+, CD4+, and CD8+ lymphocytes and was 303-517, 202-334, and 156-231 cellslJ.L1 for absolute numbers, respectively. Coefficients of variation for lymphocyte subset measurements at each laboratory ranged from 6% to 24% and for the absolute counts from 19% to 40%. Absolute counts demonstrate a larger coefficient of variation than the subset measurements because the absolute measures also depend on the variability of total white blood cell counts and differential cell count. Intercenter coefficients of variation ranged from 7.6% to 15.5% depending on sample and subset being compared across laboratories. The investigators' recommendations to maintain maximal consistency over time included the ongoing evaluation of CD4+ cell counts in a healthy control group and comparison of common specimens between laboratories to minimize intra- and interlaboratory variation. No evidence was found for seasonal or age-related variation. While technical improvements in flow cytometry can be expected to reduce some sources of variation, the MACS data confirm the need for strict quality assurance programs to minimize variability for lymphocyte subsets within and between laboratories. Biologic factors that cause T cell variations include other illnesses, pharmacologic agents, and circadian variation. Heterogeneity of CD4 epitopes to some monoclonal antibodies among different ethnic and racial groups was reported by Parker et al. [10] to cause large differences in reported CD4+ cell counts. Both Parker et al. and a study by the UCLA MACS group [11] suggested use ofCD4+ cell percentage in monitoring of individual patients rather than absolute cell number, though the two measures are highly correlated. The UCLA MACS group reported that the prognostic value over time was quite similar for CD4+ cell percentage, CD4+ cell absolute number, and CD4+:CD8+ cell ratio.
353
AIDS Commentary
354
Table 1.
Host and virus mediators of human immunodeficiency virus (HIV) infection. Potential surrogate markers or associated factors
Events of cofactors Events/potential surrogate markers T4 cell activation, HIV replication, T4 cell lysis T8 cell immunologic dysregulation Macrophage/monocyte activation B cell activation Host-related cofactors/associated factors Immune responsiveness Increased lymphocyte activation Other infections NOTE.
lID 1992; 165 (February)
CD4+ cell loss; decline in percentage of CD4+ and CD4+:CD8+; P2-microglobulin; p24 antigenemia; cytokines (e.g., IL-6, soluble IL-2 receptors) Increase in activated CD8+ cells; cytokines (e.g., soluble CD8+ receptors, IFN--y) Neopterin production; cytokines (e.g., IL-2, IL-6, TNF) Increased immunoglobulins (e.g., 19A, IgM, IgG), specific autoantibodies, functional unresponsiveness (e.g., to pokeweed mitogen) Age, specific antigen responses Smoking History of other sexually transmitted diseases or ongoing high-risk behavior
Items in bold have been epidemiologically linked to risk of progression to AIDS. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.
Correlation of Changes in CD4+ Cell Count with Progression to AIDS In patients followed from the time of seroconversion, CD4+ cell decline has been correlated with progression to AIDS [28-32]. In a cohort of 288 seropositive homosexual men followed at the San Francisco General Hospital, progres-
sion rates of 87%, 46%, and 16% were observed after 3 years of follow-up for patients with CD4+ cell counts of 400, respectively [28]. Serum levels of f32microglobulin and detection of p24 antigen in blood were also both independently correlated with rates of progression. Combined with CD4+ cell counts, use of f32-microglobulin and p24 antigen increased prognostic accuracy for progression to AIDS compared with CD4+ cell count alone. The patient's age was a significant cofactor for progression as men aged ~35 years had a relative hazard of2.1 (P = .015) compared with men
AIDS COMMENTARY
CD4+ Lymphocyte Cell Enumeration for Prediction of Clinical Course of Human Immunodeficiency Virus Disease: A Review Daniel S. Stein, Joyce A. Korvick, and Sten H. Vermund
Medical and Epidemiology Branches. Clinical Research Program. Division ofA IDS. National Institutes o.fAllergy and Infectious Diseases, National Institutes of Health. Bethesda. Maryland
Human immunodeficiency virus type 1 (HIV -1), a retrovirus, is the etiologic cause of the overwhelming majority of cases of AIDS [1, 2]. The HIV-l envelope glycoprotein, gp 120, specifically binds to the CD4 receptor that is expressed in greatest concentration in a subset of T lymphocytes and in lower amounts on monocytes and macrophages [3]. HIV can apparently enter both resting and stimulated CD4+ T lymphocytes, but replication only proceeds to completion in stimulated CD4+ T lymphocytes [4]. T lymphocyte subsets are defined by both functional activity and phenotypic assays using monoclonal antibodies. Cells expressing CD4 receptors are termed the "helper/ inducer" subset, reflecting their role as both helper cells for B cell responses for antigens expressed on cells bearing human leukocyte antigen (HLA) class II receptors and inducer cells that cause T cells to suppress immune responses. Activated CD4+ cells are major sources ofinterleukins 2, 3, and 5 and interferon-)', which are important in the growth, function, and differentiation of the cellular immune system [5). Less commonly, CD4+ cells function as cytotoxic cells. The selective loss ofCD4+ cells results in numerous immune defects
Received 16 July 1991; revised I I October 1991. Informed consent was obtained from all subjects whose data appear in the figure; guidelines of the US Department of Health and Human Services and of the participating institutions of the Multicenter AIDS Cohort Study were followed. Grant support for data in figure I: National Institutes of Health for Multicenter AIDS Cohort Study (AI-32535. -72632. -72634, -72631, -72676). Reprints or correspondence: Dr. D. S. Stein. MB/CRP/DAIDS/NIAID/ NIH. 6003 Executive Blvd., Bethesda. MD 20892. The Journal of Infectious Diseases This article is in the public domain.
1992;165:352-63
associated with susceptibility to the opportunistic infections that are the hallmark of AIDS [6). We discuss how the CD4+ cell depletion caused by HIV-l infection increases the risk of developing the clinical syndrome of AIDS in adults. In addition, the association of CD4+ cell depletion with other immune and nonimmune markers will be considered. Finally, we will discuss the effects of therapy on CD4+ cell counts and the problem of correlating changes in these counts with subsequent course of disease. A caveat for this review is that nearly all of the studies referenced here have cohorts of male participants secondary to the very high male preponderance of HIV infection in the developed world where these studies were done [7). Prospective CD4+ cell data for women with HIV infection are needed, particularly correlating women-specific clinical outcomes, such as genital tract infections and neoplasms.
Measurement and Variability Measurement oflymphocyte subsets is dependent on phenotypic identification by fluorescent-labeled monoclonal antibodies. Enumeration of cells is usually done in a flow cytometer that uses a laser beam to cause fluorescence of the bound antibody. The cells are separated by both light scatter and color of the attached fluorescent compound. They are counted as very short pulses of electric current that are generated in response to laser stimulation as detected by photomultiplier tubes. Variability occurs as a consequence of the techniques used in cell labeling, type of antibody, type of machine, expertise of the operators, standardization of ma-
Downloaded from http://jid.oxfordjournals.org/ at New York University on May 31, 2015
Over the last 10 years the appreciation of the full breadth of the spectrum of human immunodeficiency virus (HIV) infection has gradually increased; it is now known that AIDS represents merely the end stage of this progressive infectious process. The surrogate marker that most closely correlates with the stage of HIV infection is the CD4+, or T helper, cell count. Because this count is relied on in making important therapeutic decisions, it is of paramount importance that clinicians be cognizant of and fully understand the multiple factors that can influence this parameter: the variability of the count from day to day and from morning to night, the influence of intercurrent viral infections, the influence of drugs. In this AIDS Commentary, Sten H. Vermund and his colleagues at the National Institutes of Health discuss these issues and put the CD4 + cell count into a lucid and practical clinical perspective. Merle A. Sande
lID 1992; 165 (February)
AIDS Commentary
Seroconversion Studies Acute HIV infection is associated with a nonspecific clinical syndrome that commonly includes fever, pharyngitis, lymphadenopathy, rash, and myalgias-arthralgias. The HIV core antigen (p24) and high titers of cytopathic virus, similar
to those seen in late-stage disease [12, 13], can be detected before the appearance of HIV antibodies. After the appearance of HIV antibodies by the screening ELISA test, p24 antigenemia generally becomes undetectable, though it can occasionally persist and often will recur later in the disease [14, 15]. Recent investigations using acid pretreatment of patient sera (pH 2.5-3.0) have shown that most p24 antigen is in the form of circulating immune complexes [16, 17]. Data from a group of 29 seroconverters followed for 1 year indicate that during this undetectable phase for p24 antigen (using standard detection methods), >50% of patients have persistent p24 antigen immune complexes [16]. HIV-I titers found in plasma and peripheral blood mononuclear cell cultures also fall rapidly as specific antibodies are detectable, suggesting at least a transiently effective host immune response [12, 13]. Markers of immune stimulation include {j2microglobulin, a component of class I HLA cell receptors [18], and neopterin, a metabolite of guanosine triphosphate produced by macrophages after stimulation by interferon-')' [19]. Both have been shown to increase at the time of seroconversion and to continue to increase after seroconversion [20, 21]. Laboratory findings associated with the acute HIV infection syndrome include lymphopenia with lowered CD4+ cell numbers and thrombocytopenia [12]. CD3+CD4-CD8lymphocytes have cytolytic activity and increase in numbers at the time ofseroconversion and reach levels at 3-9 months after infection resembling the levels found in patients chronically infected with HIV [22]. Hofmann et al. [20] in their UCLA MACS series showed that 73% of patients (33/45) had clinically and statistically significant CD4+ cell declines at seroconversion, and 83% experienced significant declines within I year. CD8+ cell counts were noted to rise at time of seroconversion. Detels et al. [23], in another UCLA MACS study of 51 homosexual men who were followed through seroconversion, demonstrated a mean CD4+ cell decline from 854 cellslJ.tl to 651 cells/,ul 6 months after seroconversion. However, it was rare for seroconverters to have a consistent decline in their percentage ofCD4+ cells over the next 3 years. In the interval between visits, stable or declining levels of CD4+ cell percentages were found in 38% of subjects, with 12% experiencing declines followed by a leveling in their rates ofloss ofCD4+ cells. Overall, 62% experienced declines in their CD4+ cell percentage over 3 years of follow-up. In another study of predominantly homosexual men (the San Francisco Men's Health Study), the mean CD4+ cell count fell from 1078 to 716 cells/,ul by 12 months after seroconversion (n = 37) and to 636 ± 69 cells/,ul by 18 months. The latter value was similar to that noted among seropositives at the time of their recruitment in 1984 [24]. The CD8+ cell count rose significantly at seroconversion from 794 to 1198 ± 117 cells/,ul, then declined slightly to a level still above baseline (1073 ± 87 cells/,ul) 6 months after serocon-
Downloaded from http://jid.oxfordjournals.org/ at New York University on May 31, 2015
chine alignment, number of cells counted, and normal variations in cell populations. The quality control program of the Multicenter AIDS Cohort Study (MACS) was established in an attempt to decrease the variability in CD4+ cell enumeration [8]. The MACS is a study of -- 5000 homosexual and bisexual men in Baltimore, Chicago, Los Angeles, and Pittsburgh [9]. Identical flow cytometers, monoclonal antibodies, analytic procedures, and sample preparation protocols were used in the four laboratories. Still, significant variations in CD3+, CD4+, and CD8+ lymphocyte subset percentages were found. Variation over time occurred within center, between centers, and within patients. The within-person standard deviations ranged from 4.5% to 7.9% for the percentages of CD3+, CD4+, and CD8+ lymphocytes and was 303-517, 202-334, and 156-231 cellslJ.L1 for absolute numbers, respectively. Coefficients of variation for lymphocyte subset measurements at each laboratory ranged from 6% to 24% and for the absolute counts from 19% to 40%. Absolute counts demonstrate a larger coefficient of variation than the subset measurements because the absolute measures also depend on the variability of total white blood cell counts and differential cell count. Intercenter coefficients of variation ranged from 7.6% to 15.5% depending on sample and subset being compared across laboratories. The investigators' recommendations to maintain maximal consistency over time included the ongoing evaluation of CD4+ cell counts in a healthy control group and comparison of common specimens between laboratories to minimize intra- and interlaboratory variation. No evidence was found for seasonal or age-related variation. While technical improvements in flow cytometry can be expected to reduce some sources of variation, the MACS data confirm the need for strict quality assurance programs to minimize variability for lymphocyte subsets within and between laboratories. Biologic factors that cause T cell variations include other illnesses, pharmacologic agents, and circadian variation. Heterogeneity of CD4 epitopes to some monoclonal antibodies among different ethnic and racial groups was reported by Parker et al. [10] to cause large differences in reported CD4+ cell counts. Both Parker et al. and a study by the UCLA MACS group [11] suggested use ofCD4+ cell percentage in monitoring of individual patients rather than absolute cell number, though the two measures are highly correlated. The UCLA MACS group reported that the prognostic value over time was quite similar for CD4+ cell percentage, CD4+ cell absolute number, and CD4+:CD8+ cell ratio.
353
AIDS Commentary
354
Table 1.
Host and virus mediators of human immunodeficiency virus (HIV) infection. Potential surrogate markers or associated factors
Events of cofactors Events/potential surrogate markers T4 cell activation, HIV replication, T4 cell lysis T8 cell immunologic dysregulation Macrophage/monocyte activation B cell activation Host-related cofactors/associated factors Immune responsiveness Increased lymphocyte activation Other infections NOTE.
lID 1992; 165 (February)
CD4+ cell loss; decline in percentage of CD4+ and CD4+:CD8+; P2-microglobulin; p24 antigenemia; cytokines (e.g., IL-6, soluble IL-2 receptors) Increase in activated CD8+ cells; cytokines (e.g., soluble CD8+ receptors, IFN--y) Neopterin production; cytokines (e.g., IL-2, IL-6, TNF) Increased immunoglobulins (e.g., 19A, IgM, IgG), specific autoantibodies, functional unresponsiveness (e.g., to pokeweed mitogen) Age, specific antigen responses Smoking History of other sexually transmitted diseases or ongoing high-risk behavior
Items in bold have been epidemiologically linked to risk of progression to AIDS. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.
Correlation of Changes in CD4+ Cell Count with Progression to AIDS In patients followed from the time of seroconversion, CD4+ cell decline has been correlated with progression to AIDS [28-32]. In a cohort of 288 seropositive homosexual men followed at the San Francisco General Hospital, progres-
sion rates of 87%, 46%, and 16% were observed after 3 years of follow-up for patients with CD4+ cell counts of 400, respectively [28]. Serum levels of f32microglobulin and detection of p24 antigen in blood were also both independently correlated with rates of progression. Combined with CD4+ cell counts, use of f32-microglobulin and p24 antigen increased prognostic accuracy for progression to AIDS compared with CD4+ cell count alone. The patient's age was a significant cofactor for progression as men aged ~35 years had a relative hazard of2.1 (P = .015) compared with men
