Failure of T-cell homeostasis preceding AIDS in HIV-1 infection. The Multicenter AIDS Cohort Study.

We and others have postulated that a constant number of T lymphocytes is normally maintained without regard to CD4+ or CD8+ phenotype ('blind' T-cell homeostasis). Here we confirm essentially constant T-cell levels (despite marked decline in CD4+ T cells and increase in CD8+ T cells) in homosexual men with incident human immunodeficiency virus, type 1 (HIV-1), infection who remained free of acquired immunodeficiency syndrome (AIDS) for up to eight years after seroconversion. In contrast, seroconverters who developed AIDS exhibited rapidly declining T cells (both CD4+ and CD8+) for approximately two years before AIDS, independent of the time between seroconversion and AIDS, suggesting that homeostasis failure is an important landmark in HIV disease progression. Given the high rate of T-cell turnover in HIV-1 infection, blind T-cell homeostasis may contribute to HIV pathogenesis through a CD8+ T lymphocytosis that interferes with regeneration of lost CD4+ T cells.

Human immunodeficiency virus-1 (HIV-1) gp120 superantigen-binding serum antibodies. A host factor in homosexual HIV-1 transmission.

HIV-1 gp120 is an immunoglobulin superantigen which can bind to preimmune serum Ig. We hypothesize that levels of such preimmune antibodies vary in the population and might affect host resistance or susceptibility to viral transmission. This study tests two predictions: (a) levels of preimmune anti-gpl20 Igs are a polymorphic trait; and, (b) these levels are correlated with resistance or susceptibility to HIV-1 transmission. The first prediction was confirmed in a longitudinal study of a low-risk seronegative population. In this group, levels of both endogenous anti-gpl20 IgM and IgG varied widely, but were characteristic and stable for each individual. The second prediction was addressed in a study of participants of the Multicenter AIDS Cohort Study, in which men "susceptible" and "resistant" to HIV infection were identified based on numbers of sexual partners and eventual seroconversion. Specimens consisted of archival sera obtained > 2 yr before seroconversion. Men in the susceptible population (low-risk seroconverters) were distinguished by low levels of anti-gpl20 IgG. We conclude that the level of preimmune anti-gpl20 IgG is a polymorphic population trait, and low levels are a potentially specific and significant factor in homosexual transmission of HIV infection.

Applications of a computer simulation model of the natural history of CD4 T-cell number in HIV-infected individuals.

Data from the Multicenter AIDS Cohort Study (MACS) of 1637 gay men, recruited in 1984 and 1985 in Los Angeles and followed at 6-monthly intervals, are used to develop a computer simulation model of the typical pattern of CD4 T-cell number changes in HIV-infected AIDS-free subjects. The empirical model incorporates the following features: (1) within-person and between-person variability in CD4 measurements; (2) variation in the rates of decline of CD4 values; (3) variation in the level of CD4 at which clinical AIDS is diagnosed, and (4) greater absolute variation in CD4 values in men with high CD4 levels compared with men with low CD4 values. Three applications of the model to assist in the design and interpretation of clinical trials are given. Further applications to clinical trials and to estimate the current and future spectrum of HIV-mediated immunological disease in the USA, as measured by the CD4 values, are discussed.

Calculation and use of an HIV-1 disease progression score.

OBJECTIVE: The pathogenesis of human disease is often multifactorial. For fatal diseases, it is common to combine survival information in relation to different aspects of pathogenesis. Here we explored a scoring system for HIV disease markers that combines measures of immunodeficiency (CD4 cell count), plasma viral burden, and immune activation (CD38 expression on CD8 T cells) DESIGN: Observational data from 252 HIV-infected individuals from the Multicenter AIDS Cohort Study (MACS) was used for model development. METHODS: A statistical model was used to develop a system that related marker values to the outcomes of clinical AIDS or death. RESULTS: Mathematical formulae were derived to calculate AIDS and death progression scores. These scores give the number of days at which there is a 50% probability that a person may develop AIDS or die. Graphs were constructed that can be used to determine the probability that a person will be AIDS-free or alive for times between 6 months and 4 years. The model was valid when tested on data from 240 additional people from the MACS cohort. CONCLUSIONS: These formulae and graphs are a convenient way for individuals and their physicians to apply existing MACS cohort data to HIV disease markers in order to estimate probabilities of progression to AIDS or death. Further investigation is needed to determine whether modifications of the formulae are required to predict outcome accurately in those patients on highly active antiretroviral therapy or in other demographic groups.

Immunologic effects of combined protease inhibitor and reverse transcriptase inhibitor therapy in previously treated chronic HIV-1 infection.

OBJECTIVE: To evaluate the efficacy of combination protease and reverse transcriptase inhibitor therapy in correcting HIV-1-induced lymphocyte subset abnormalities in previously treated adults. DESIGN: A 48-week observational study of lymphocyte subsets in 12 participants in the Multicenter AIDS Cohort Study who were already taking at least one reverse transcriptase inhibitor and added a protease inhibitor to their treatment regimen. Comparison groups were HIV-seronegative homosexual men, HIV-seronegative heterosexual men, and homosexual HIV-1-infected men who were long-term non-progressors. METHODS: Three-color immunofluorescence and monoclonal antibodies were used to assess HIV-1-induced lymphocyte subset alterations related to immune deficiency and immune activation. Plasma HIV-1 RNA levels were monitored to assess suppression of viral replication. RESULTS: CD4+ cell counts significantly increased and lymphocyte activation measured as CD38 and HLA-DR expression on CD8+ T cells significantly decreased by 48 weeks. CD4+ cell values remained abnormal even in those who were fully suppressed. Some T-cell activation markers decreased to levels observed in long-term non-progressors. The increase in CD4+ T-cell numbers reached a plateau by week 24, but the increase in resting HLA-DR- CD38-T cells was sustained through week 48. Proportions of CD45RA+ CD62L-selectin+ and CD28+ CD4+ T-cell subsets and Fas expression were not abnormal at baseline compared with seronegative homosexual controls. CONCLUSIONS: The most significant impact of suppression of viral replication was reversal of T-cell activation. However, normalization of lymphocyte subset perturbations associated with chronic HIV-1 infection was not achieved after 1 year of treatment with current combination antiretroviral regimens. More profound viral suppression, therapy for longer than 1 year, or immunologic augmentation may be needed to fully reverse the abnormalities.

Enhanced levels of functional HIV-1 co-receptors on human mucosal T cells demonstrated using intestinal biopsy tissue.

OBJECTIVE: To examine compartmental differences in co-receptor expression on CD4 lymphocytes between blood and gut using endoscopic biopsies. DESIGN: Mucosal and peripheral CD4 T cells from healthy controls were compared for co-receptor expression and vulnerability to infection by HIV-1. METHODS: Expression of CCR5 and CXCR4 was quantified by flow cytometry on isolated mucosal CD4 lymphocytes obtained from endoscopic biopsies and blood from healthy controls. Vulnerability to in vitro infection by both R5 and X4 strains was assessed by measuring p24. RESULTS: Biopsies yielded sufficient lymphocytes for flow cytometric characterization and infectivity studies. The percentage of mucosal CD4 T lymphocytes that expressed CCR5 and the per cell expression of CCR5 were both significantly increased compared with that in peripheral blood CD4 T lymphocytes. CXCR4 was expressed on the majority of CD4 lymphocytes in both compartments. In vitro infection of mucosal mononuclear cells supported greater viral replication of both R5 and X4 strains than peripheral blood mononuclear cells. CONCLUSIONS: Enhanced expression of CXCR4 and CCR5 on CD4 lymphocytes in normal intestinal mucosa predicts increased vulnerability to infection by both R5 and X4 HIV-1. Endoscopic biopsies provide a useful mucosal tissue sampling technique to identify compartmental immunologic differences that may be exploited by HIV-1 in establishing initial mucosal infection.

Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis.

OBJECTIVE: To test the hypothesis that the expanded population of non-proliferative CD28-CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. DESIGN: CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28-CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28-CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. METHODS: CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. RESULTS: The TRF lengths of sorted CD28-CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28-CD8+ cells in uninfected subjects (P = 0.003). The TRF length in CD28-CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. CONCLUSIONS: The shortened telomeres in the CD28-CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

Prognostic significance of plasma markers of immune activation, HIV viral load and CD4 T-cell measurements.

OBJECTIVE: To evaluate the prognostic significance for AIDS occurrence of plasma levels of immune activation markers in comparison with and in conjunction with HIV viral load and CD4 T-cell measurements. DESIGN: A retrospective analysis was conducted of three plasma activation markers, the soluble tumor necrosis factor (TNF) receptor II (TNF-RII), neopterin and soluble interleukin-2 receptor levels, and of CD4 T-cell levels and plasma HIV viral load. SUBJECTS: The participants were 659 men taking part in the University of California Los Angeles Multicenter AIDS Cohort Study who were HIV-seropositive but AIDS-free in 1985. MAIN OUTCOME MEASURE: Clinically defined AIDS within 3 years. Failure time statistical regression models for the time to development of AIDS were used to assess prognostic capacity of the parameters alone and in combination. RESULTS: All the markers had prognostic capability. The levels of the three plasma activation markers correlated well with each other (median r = 0.61). They related less well with HIV RNA plasma levels (median r = 0.50) and least well with CD4 cell levels (median r = 0.36). Furthermore, plasma marker levels were shown to be able to stratify patients for prognosis within all the major categories of CD4 T-cell and HIV RNA levels. CONCLUSIONS: Plasma levels of soluble TNF-RII and other soluble markers of immune activation have prognostic capabilities which are different from HIV and CD4 T-cell levels. Combination of a single plasma activation marker measurement (such as soluble TNF-RII) with CD4 T-cell levels improved the prognostic capability of each. A new graphic technique for presenting prognostic capability indicated that plasma soluble TNF-RII and CD4 cell levels are better prognostic factors than HIV plasma level with CD4 cells < 200 x 10(6)/l. Inexpensive tests for one of the plasma activation markers, such as soluble TNF-RII or neopterin, can be useful for evaluations of HIV disease course, especially when expensive equipment, technical expertise and funding required for flow cytometry and for HIV load measurements are not readily available.

Prognostic value of plasma HIV RNA in the natural history of Pneumocystis carinii pneumonia, cytomegalovirus and Mycobacterium avium complex. Multicenter AIDS Cohort Study.

OBJECTIVES: To use follow-up on untreated HIV-positive men to assess the prognostic information provided by baseline data on plasma HIV RNA, CD4 cell count, age, and HIV-related symptom status, separately for three specific AIDS-defining illnesses: Pneumocystis carinii pneumonia (PCP), cytomegalovirus (CMV), and Mycobacterium avium complex (MAC). METHODS: The study population were 734 HIV-positive homosexual men enrolled in the Multicenter AIDS Cohort Study, with follow-up (1984-1985 through mid-1988) restricted to the antiretroviral treatment-free and prophylaxis-free era. Baseline marker values were categorized and assessed as predictor variables in separate time-to-event analyses for each of the three specific outcomes. RESULTS: A total of 138 cases of PCP, 25 cases of CMV, and 25 cases of MAC were observed. For PCP and CMV, higher categories of HIV RNA and lower categories of CD4 cell count were associated with increased risk relative to the respective reference groups. For MAC, oral candidiasis or fever and elevated HIV RNA at baseline were the primary risk factors. Further analysis highlighted the importance of monitoring HIV RNA levels in addition to CD4 cell counts when evaluating patients' risk of developing PCP. CONCLUSIONS: In the absence of treatment, plasma HIV RNA levels provide prognostic information about the risk of these three specific AIDS-defining illnesses, independently of the CD4 cell count. These data provide a useful reference as researchers investigate changing patterns in the incidence and predictors of opportunistic infections in the era of increasingly active antiretroviral therapies.

CD4 percentage, CD4 number, and CD4:CD8 ratio in HIV infection: which to choose and how to use.

Data from a 3 year longitudinal study of CD4 levels in 813 untreated HIV-seropositive men are presented. Baseline CD4 levels were used to stratify the men for the severity of HIV disease at the outset of the study, and each man's CD4 levels and progression to AIDS were evaluated. These data can be used to advise patients who are HIV seropositive on the likelihood that they will deteriorate immunologically or progress to AIDS based on their own CD4 cell level obtained from laboratory evaluation. Graphs are presented here for 1, 2, and 3 years of follow-up. As expected, each individual's absolute CD4 lymphocyte number, CD4 lymphocyte percent, and CD4:CD8 ratio were strongly correlated, and were similar in their ability to predict the development of AIDS. In this sense, all three are useful markers for monitoring individuals with HIV infection, and for many clinical situations from a practical point of view there is little to choose between the markers. However, the CD4 percent has slightly greater prognostic significance and shows slightly less variability on repeated measurements. Because of this, the CD4 percent may be preferable to the CD4 absolute number for use in stratifying individuals who are candidates for antiviral or immunotherapy for HIV infection, and for monitoring patients in clinical trials.

Elevated levels of CD38+ CD8+ T cells in HIV infection add to the prognostic value of low CD4+ T cell levels: results of 6 years of follow-up. The Los Angeles Center, Multicenter AIDS Cohort Study.

A cohort of 98 HIV-infected initially AIDS-free homosexual men from the Multicenter AIDS Cohort Study (MACS) was followed for 6 years to investigate whether CD8+ cell subsets have prognostic value for progression to AIDS. In the present study, four subsets of CD8+ T cells that previously have been shown to be selectively elevated in HIV-infected asymptomatic persons, specifically the CD8+ T cell subsets that were CD38+, HLA-DR+, CD57+ and L-selectin negative (Leu8-), were measured. Forty-nine of the 98 developed AIDS. Prognostic value of these CD8+ cell subsets was evaluated using the proportional hazards model. Levels of both CD38+ CD8+ and Leu8- CD8+ cells individually had prognostic value for progression to AIDS. In contrast, CD57+ CD8+ and HLA-DR+ CD8+ cell subsets levels did not have prognostic value. After adjustment for level of CD4+ T cells, however, only the elevation in the CD38+ CD8+ cell subset had additional prognostic value. These results suggest that the level of CD38+ CD8+ cells could be used together with the CD4+ T cell level to more accurately predict progression to AIDS among HIV-infected men. These results provide further support for the observation that dramatic and progressive activation of CD8+ T cells in HIV infection occurs. The power of elevated levels of the CD38+ CD8+ subset to predict poor prognosis in this cohort suggests these CD8+ T cells reflect an immune stimulation that is ultimately unable to control disease progression.

Changes in T and non-T lymphocyte subsets following seroconversion to HIV-1: stable CD3+ and declining CD3- populations suggest regulatory responses linked to loss of CD4 lymphocytes. The Multicenter AIDS Cohort Study.

We investigated changes in lymphocyte subsets (total, CD4+ and CD8+ T cells, as well as non-T cells) associated with human immunodeficiency virus type I (HIV-1) seroconversion in 321 homosexual or bisexual men in the Multicenter AIDS Cohort Study (MACS). These subjects had serial lymphocyte characterizations for up to 4 years before and 5 years after seroconversion. CD4+ lymphocyte levels declined rapidly in the first 18 months following seroconversion and less rapidly thereafter, while CD8 lymphocytes increased with similar kinetics. In contrast, total T (CD3+) lymphocytes declined only slightly in the first 18 months following seroconversion, and then remained stable. These results support the hypothesis of physiologic regulation of the total number of circulating T cells, such that lost CD4+ lymphocytes are replaced by newly generated CD4+ and CD8+ lymphocytes; over time, continued loss of CD4+ lymphocytes due to HIV-1 infection would result in net replacement of lost CD4+ lymphocytes with CD8+ lymphocytes. Non-T (CD3-) lymphocytes also declined after seroconversion, and this decline paralleled that of CD4+ lymphocytes. Thus, changes in both T and non-T lymphocytes after HIV-1 seroconversion may reflect the operation of homeostatic or regulatory mechanisms. Whether these mechanisms contribute to the development of immune deficiency requires further study.

The relationship between T-cell levels and CMV infection in asymptomatic HIV-1 antibody-positive homosexual men.

To investigate the relationship between cytomegalovirus (CMV) infection and progression of HIV-1 disease, a group of 234 asymptomatic, HIV-1 antibody-positive homosexual men were examined for CMV isolation and levels of CMV IgM antibodies, CMV IgG antibodies, and CD4+ and CD8+ T-lymphocytes. CMV IgG antibodies were present in 100% and CMV IgM antibodies in 22% of the men. CMV was isolated from the semen of 45% of the men. No relationship was observed between CMV IgM antibodies and CMV in semen or CD4+ levels. CD4+ cell levels were significantly lower in those from whose semen CMV was isolated. In addition, an inverse relationship was observed between the concentration of CMV in semen and CD4+ levels. We postulate that the seminal tract may be a reservoir for systemic CMV infection in HIV-infected homosexual men. Reinfection from this or other sources may result in recurrent stimulation of HIV-1 replication and lead to a further decline in CD4+ cells. Clarification of whether persistent CMV infection is secondary to HIV-1-induced immunodeficiency or, conversely, promotes a more rapid decline in immunocompetency will require follow-up studies.

Activation and differentiation antigens on T cells of healthy, at-risk, and HIV-infected children.

We examined the T-lymphocyte phenotypes of 67 human immunodeficiency virus (HIV)-infected children (P-1 or P-2) and 65 age-matched, healthy, control children stratified into four groups from < 1 to > or = 5 years of age to determine expression of antigens associated with cell activation/differentiation. Immunophenotyping was performed by laser flow cytometry using two-color immunofluorescent labeling. Although the control children showed a decline in total CD4 cell percent with age, the HIV-infected children in all age groups showed significantly decreased CD4 cell numbers compared with the age-matched controls. However, the slope of the CD4 cell decline with age was not significantly different in HIV-infected and control children. The CD4 cell decrease in infected children was reflected in both the CD45RA+ (naive) and CD45RA- (memory) CD4 cell subsets, although the CD45RA+ cells were decreased in greater proportion. Results assessing CD4 cells for expression of the L-selectin (Leu8) molecule were similar to those for CD45RA. The overall CD8 cell percentage was significantly increased in HIV-infected children compared with controls in all age groups. This was due primarily to increases in CD8 cells that were CD38+, CD57+, HLA-DR+, or CD45RA-. In a retrospective analysis of data from 23 P-0 children, we compared phenotype results from 5 children who were HIV+ with those 18 who were HIV-. Although the phenotypic changes seen in the 5 HIV+ children paralleled those described above for P-1 and P-2 subjects, there was no significant difference in the values for HIV+ compared with HIV P-0 children. Although the phenotypic alterations described did not appear to be diagnostic markers in P-0 children, they may serve as useful adjuncts for the evaluation of HIV-infected children.

Phenotypically defined memory CD4+ cells are not selectively decreased in chronic HIV disease.

Simultaneous measurements of phenotypically defined memory CD4+ cells and in vitro proliferation to three recall antigens (Ags; tetanus toxoid, influenza, and Candida albicans) were performed in 53 HIV-seropositive subjects and 39 HIV-seronegative controls. The results indicate that the low proliferative responses to recall Ags of those who were HIV infected could be partly, but not fully, explained by a decrease of phenotypically defined memory CD4+ cells. This is, to our knowledge, the first report of experiments that simultaneously measured memory CD4+ cell numbers and function and then examined whether the low responses observed in seropositive subjects could be explained by low numbers of phenotypically defined memory CD4+ cells. A central finding of the study, which argues against prevailing dogma, was that within the CD4+ lymphocyte population, the proportion of cells displaying the memory phenotype was not selectively decreased in HIV-seropositive subjects as compared with the proportion of these cells in seronegative homosexual controls. An entirely new finding of the study was that AIDS patients, many of whom were unresponsive to all three recall Ags tested, actually had a significant increase in the proportion of CD4+ cells with the memory phenotype, and this fraction approached 100% in subjects with CD4+ cell numbers that were near zero. A final observation of the study, possible because some patients were on zidovudine (ZDV), was that there was no evidence that ZDV treatment led to an increased proliferative response to recall Ags in vivo. An in vitro study also found no effect of ZDV, dideoxycytidine (ddC), or azido-dideoxyuridine (AZU) on proliferative responses to recall Ags.

Influence of HIV-1 infection and cigarette smoking on leukocyte profiles in homosexual men. The Multicenter AIDS Cohort Study.

The interaction between the effects of HIV-1 infection and cigarette smoking on leukocyte profiles was studied in 307 HIV-1 seroconverters in the Multicenter AIDS Cohort Study (MACS). Longitudinal data for white blood cell (WBC) counts, WBC differentials, T cell subsets, and smoking behavior were collected semiannually for up to 7 years. Prior to seroconversion, total WBC count increased in direct proportion to daily cigarette consumption, but this effect disappeared within 3 years of seroconversion. Analyses of WBC subsets (lymphocytes, monocytes, and granulocytes) and lymphocyte subsets (CD4+, CD8+ and non-T[CD3-]) showed that smoking had only minor effects on the proportions of these cells. In contrast, HIV-1 seroconversion was associated with a dramatic decrease in CD4+ lymphocyte percentage, a large increase in CD8+ lymphocyte percentage, a small increase in total lymphocyte percentage, and small decreases in the non-T lymphocyte and granulocyte percentages. These findings indicate that the effect of smoking on CD4 cell counts is (a) nonspecific, (b) maximal in seronegative individuals; and (c) lost by 3 years after seroconversion. Although the mechanism of the loss of smoking-induced leukocytosis after seroconversion remains to be determined, our results suggest that the effects of smoking are not likely to be important in the clinical use of leukocyte measurements, including measurements of CD4 lymphocytes, in individuals who have been HIV-1 seropositive for more than 3 years.

Patterns of CD4+ cell changes after HIV-1 infection indicate the existence of a codeterminant of AIDS.

Successive interval slopes of CD4+ cells each constructed from levels at three consecutive 6 month visits were compared over 3 years of follow-up among 565 persistently HIV-1 antibody-positive, 326 persistently antibody-negative, and 51 seroconverting homosexual men who had at least 500 CD4+ cells/mm3 at baseline and completed the first three 6 month visits. "Change" was defined as a difference between two successive interval slopes. Sixty-two percent of seroconverters meeting these criteria experienced a shift in one or more of their successive CD4+ interval slopes, the majority (56%) from a level slope to a negative slope (decreasing numbers of CD4+ cells), a significantly greater proportion than that observed among seronegatives (30%, p less than 0.0001). Fifty-eight percent of the seropositives maintained level interval slopes over the 3 years of follow-up. The majority (59%) of those men experiencing a shift went from a level to a negative interval slope, a significantly greater proportion than observed among seronegatives (30%, p less than 0.0001). The observed patterns of change in interval slopes are consistent with the laboratory observation that CD4+ cells must be activated to replicate HIV-1. The use of the interval slope strategy provides a method to identify a temporal focal point at which to examine possible codeterminants that trigger the production of HIV-1 and the subsequent decline in CD4+ cells.

A reproducible method to detect CD8 T cell mediated inhibition of HIV production from naturally infected CD4 cells.

CD8 T lymphocytes are an important component of the host immune response to human immunodeficiency virus (HIV). To characterize CD8 cell function, we have studied the in vitro phenomenon of CD8 cell-mediated inhibition of HIV replication from autologous, naturally infected CD4 cells. We describe here a reproducible assay of CD8 T cell-mediated inhibition in HIV-infected individuals. The method involves the use of a commercially available cell separation system and anti-CD3 monoclonal antibody to stimulate CD4 cells to produce HIV. Using this technique, we were able to detect HIV production from the CD4 cells of 25 of 27 HIV-infected individuals who had not progressed to AIDS. Further, in vitro CD8 cell-mediated inhibition of HIV production was noted in all of the 25 subjects whose CD4 cells produced viral p24 antigen. This assay may be useful as an in vitro correlate of protective immunity to HIV, with potential application for assessing disease progression, therapeutic efficacy, and immune mechanisms in HIV disease.

A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry.

A sensitive method for quantification of cells undergoing apoptosis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) cell surface staining. After staining, the samples can be treated with paraformaldehyde (PF) solution to eliminate the risk for exposure of laboratory personnel to biohazardous agents and to preserve the cells through fixation for later analysis on the flow cytometer. The value of the method is shown on the measurement of apoptosis in human thymocytes and in human peripheral blood mononuclear cells (PBMC) exposed to various inducers of active cell death. The method is validated by fluorescent activated cell sorting in combination with morphologic examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid multiparameter analysis of apoptosis in combination with cell surface phenotype on biohazardous samples with single laser instrumentation.

Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry.

We developed a method for simultaneous flow cytometric analysis of three-color immunofluorescence and DNA content. We show here that staining with 7-amino-actinomycin D (7-AAD) at 10 microg/ml using a phosphate-citrate buffer at low pH containing saponin for cell membrane permeabilization yields good resolution DNA histograms with low coefficients of variation. Furthermore, light scatter properties of cells are preserved after permeabilization; this permits gating on cell populations that differ in scatter signals on the flow cytometer. Because of the low pH of the phosphate-citrate staining buffer, Alexa488, a pH-independent green-fluorescent fluorochrome is used instead of fluorescein-isothiocyanate (FITC) for cell surface staining in combination with phycoerythrin (PE) and with allophycocyanin (APC) which are both pH insensitive. Removal of 7-AAD after staining and replacing it with non-fluorescent actinomycin D (AD) retains DNA staining and allows detection of Alexa488, PE and APC cell surface immunofluorescence without interference from fluorescent 7-AAD in solution for clear identification of cell subpopulations even after prolonged stimulation in culture. Thus, using a four-color benchtop flow cytometer, measurement of Alexa488, PE and APC three-color immunofluorescence can be combined with 7-AAD DNA content analysis. Furthermore, we demonstrate that sample storage overnight without fixation for later analysis on the flow cytometer is possible without compromising results. Application of the method to the assessment of the differential proliferative responses of lymphocyte subsets of human peripheral blood mononuclear cells (PBMC) that were costimulated with CD3 and with CD28.2 is presented.