Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

New analytic methods that permit absolute CD4 and CD8 T-cell determinations to be performed entirely on the flow cytometer have the potential for improving assay precision and accuracy. In a multisite trial, we compared two different single-platform assay methods with a predicate two-color assay in which the absolute lymphocyte count was derived by conventional hematology. A two-color method employing lymphocyte light scatter gating and Beckman Coulter Flow-Count fluorospheres for absolute counting produced within-laboratory precision equivalent to that of the two-color predicate method, as measured by coefficient of variation of replicate measurements. The fully automated Beckman Coulter tetraONE System four-color assay employing CD45 lymphocyte gating, automated analysis, and absolute counting by fluorospheres resulted in a small but significant improvement in the within-laboratory precision of CD4 and CD8 cell counts and percentages suggesting that the CD45 lymphocyte gating and automated analysis might have contributed to the improved performance. Both the two-color method employing Flow-Count fluorospheres and the four-color tetraONE System provided significant and substantial improvements in between-laboratory precision of absolute counts. In some laboratories, absolute counts obtained by the single-platform methods showed small but consistent differences relative to the predicate method. Comparison of each laboratory's absolute counts with the five-laboratory median value suggested that these differences resulted from a bias in the absolute lymphocyte count obtained from the hematology instrument in some laboratories. These results demonstrate the potential for single-platform assay methods to improve within-laboratory and between-laboratory precision of CD4 and CD8 T-cell determinations compared with conventional assay methods.     

Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults. Site Investigators and The NIAID DAIDS New Technologies Evaluation Group

A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was -8% and -3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of -1% for both CD4 and CD8 with 6-h samples and -2% and -3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter- and intralaboratory variability in determining absolute CD4 and CD8 counts.     

Inter- and intrainstitutional evaluation of automated volumetric capillary cytometry for the quantitation of CD4- and CD8-positive T lymphocytes in the peripheral blood of persons infected with human immunodeficiency virus. Site Investigators and the NIAID New CD4 Technologies Focus Group.

Volumetric capillary cytometry (VCC) is a new technology that involves the detection and enumeration of dually fluorochrome-labeled cells in a precise volume. We compared the accuracy and precision of VCC with the accuracy and precision of flow cytometry and hematology (F&H) for the measurement of the absolute numbers of CD4 and CD8 T cells in the whole blood of patients infected with human immunodeficiency virus. Five laboratories, each with a different F&H system and certified by the National institute of Allergy and Infectious Diseases flow cytometry proficiency testing program, were shipped aliquots of the same samples from a central site in addition to procuring samples locally. In general, the VCC technology generated CD4 and CD8 T-cell counts which were lower than those obtained with F&H. Intralaboratory variability of replicate CD4 T-cell determinations was similar for both technologies except in the local samples with CD4 counts less than 200/microliters, where the VCC variability was higher than the F&H variability. Interlaboratory variability on replicate CD4 T-cell counts made by VCC was significantly less than that when counts were made by F&H. The VCC instrument has automated CD4 and CD8 T-cell enumeration in whole blood and has consolidated the process to a single platform. Its performance in this evaluation indicates that it may represent a viable alternative to F&H for obtaining absolute T-cell subset counts.     

Distribution and cytokine production of CD4 and CD8 T-lymphocyte subsets in patients with acute asthma attacks.

BACKGROUND: The activation of T cells and the elevation of Th2-type cytokines have been observed in asthmatic patients, but the relative role of CD4 and CD8 T cell is still unclear. OBJECTIVE: To investigate the role of T cell subset in patients with acute asthma attacks, we analyzed the distribution, activation status, and cytokine production of CD4 and CD8 cells. METHODS: The percentages of the CD4 and CD8 cell in peripheral blood (PB) and bronchoalveolar lavage (BAL) fluid were analyzed by flow cytometry. The cytokines (IL-4, IL-5, and IFN-gamma) and soluble IL-2 receptor (sIL-2R) were measured by ELISA in culture supernatants of CD4 and CD8 cells purified from PB. RESULTS: The CD4/CD8 ratio in PB of asthmatic patients was significantly higher than that of controls, which was significantly reduced after treatment. In contrast, there was a tendency to high percentage of CD8 cells in asthmatic patients as compared with controls in BAL, which resulted in a decreased CD4/CD8 ratio. Comparing the T cell subsets in BAL with paired PB in asthma, the CD4 cells were higher in PB, but CD8 cells were higher in BAL. The IL-4, IL-5, and sIL-2R produced by CD4 cells were significantly higher than those produced by CD8 cells in asthmatic patients. CONCLUSIONS: Our results provide evidence that activated CD4 T cells increase and produce type 2 cytokines in PB, but CD8 T cell are more sequestrated than CD4 T cells in the airway during an acute asthma attack.     

HIV-1-specific cytolytic T-lymphocyte activity correlates with lower viral load, higher CD4 count, and CD8+CD38-DR- phenotype: comparison of statistical methods for measurement.

OBJECTIVES: The objective of this study was to use novel statistical methods to determine the correlation between HIV-1-specific cytolytic T-lymphocyte (CTL) activity and HIV-1 plasma viral load, in a blinded study of HIV-infected patients at various stages of clinical disease. METHODS: Peripheral blood mononuclear cells (PBMC) were collected and stored at enrollment and 2 weeks later, from 15 HIV-infected individuals who were receiving stable antiretroviral therapy for the previous 6 weeks and during the study period. HIV-1-specific CTL activity was measured using an antigen-specific PBMC in vitro stimulation method. Measurements of plasma viral load, as well as CD4+ and CD8+ T lymphocytes expressing T-cell activation markers (DR and CD38) were also performed at each time point. CTL activity was quantified using three separate statistical methods: area under the net HIV-specific lysis curve (AUC), lytic units (LU20), and linear regression (LR) of net HIV-specific lysis. RESULTS: HIV-1 nef-, pol- and gag-specific CTL activity (AUC method) was significantly higher in subjects with a plasma viral load < or = 30,000 RNA copies/ml, than in those with viral load >30,000 RNA copies/ml. When plasma viral load was analyzed as a continuous variable, there was a strong correlation between higher CTL activity and lower viral load for nef (r2 = .77; p < .001), pol (r2 = .63; p < .001) and gag (r2 = 0.75; p < .001) targets by the AUC, but not for the LU20 analysis. Using the LR analysis, which is less dependent on in vitro PBMC growth than the AUC analysis, an independent association was demonstrated between nef- and gag-specific CTL activity and lower viral load. Measurement of CTL activity was also significantly correlated with a higher percentage of circulating CD8+DR-CD38- T lymphocytes. CONCLUSIONS: In this blinded study using an in vitro stimulation of frozen PBMC, higher HIV-1 nef-, pol-, and gag-specific CTL activity correlated with lower plasma viral load, particularly in patients with a CD4 count <500 cells/mm3. Two new statistical methods for estimating CTL activity, AUC and LR analyses, were superior to the standard lytic unit (LU20) method for demonstrating this correlation. These data also demonstrated that higher circulating CD8+ T lymphocytes with a DR-CD38-phenotype, correlate with a lower plasma viral and load and higher HIV-specific CTL activity. This suggests that lymphocytes with this double-negative phenotype may include circulating HIV-specific CD8+ CTL.     

Patterns of membrane TcR alpha beta and TcR gamma delta chain expression by normal blood CD4+CD8-, CD4-CD8+, CD4-CD8dim+ and CD4-CD8- lymphocytes.

Enriched CD4+CD8-/CD4-CD8-, CD4-CD8+/CD4-CD8- and CD4-CD8- cell suspensions were prepared from normal peripheral blood by selective immunomagnetic depletion of monoclonal antibody-defined lymphocyte populations. Subsequent examination of these modified cell fractions by two-colour flow cytometry provided a means of determining the expression of membrane T-cell receptor (TcR)alpha beta and TcR gamma delta chains by both major (CD4+ and CD8+) and minor (CD3+CD4-CD8dim+ and CD3+CD4-CD8-) lymphocyte subpopulations. Normal CD4+CD8- lymphocytes were almost invariably (greater than 99%) TcR alpha beta+, whereas lymphocytes expressing membrane CD8, which could be further subdivided according to differences in fluorescent staining intensity into CD3+CD4-CD8+, CD3+CD4-CD8dim+ and CD3-CD4-CD8dim+ components, were characterized by distinct differences in patterns of TcR chain expression. In contrast to CD3+CD4-CD8+ cells, which were predominantly (99%) TcR alpha beta+, CD3+CD4-CD8dim+ lymphocytes showed a significant proportion (33%) of TcR gamma delta+ cells (natural killer-associated CD3-CD4-CD8dim+ cells were uniformly TcR-). The highest proportion (62%) of TcR gamma delta+ cells was associated with the CD3+CD4-CD8- fraction, but these studies also revealed that a significant minority of this population was TcR alpha beta+. Despite some evidence for normal inter-individual variation, further analysis of membrane CD8 fluorescent intensities confirmed clear differential relationships for TcR alpha beta and TcR gamma delta chain expression.     

Peripheral CD4+/CD8+ T-lymphocyte counts estimated by an immunocapture method in the normal healthy south Indian adults and HIV seropositive individuals.

BACKGROUND: Flow cytometry is the standard method for the estimation of CD4/CD8 counts, but the high initial investment for this instrument and costly reagents make it unaffordable to most of the centers in a developing country like India. OBJECTIVES: To evaluate the feasibility of an alternate system for the estimation of CD4 and CD8 counts in normal south Indian adults and validate the usefulness of this assay to monitor the counts in HIV seropositive individuals. STUDY DESIGN: Forty-six normal healthy adults and 68 HIV seropositive individuals both belonging to south Indian linguistic groups were enrolled in this cross-sectional study. The HIV seropositive individuals included 54 HIV-1, 9 HIV-2 and 5 HIV 1&2 infected individuals serologically confirmed by one of the commercial Immunoblot kits. The Capcellia CD4/CD8 whole blood assay, an immuno-capture ELISA based kit from Sanofi DIAGNOSTICS Pasteur, (France) was used with a few modifications in the procedure to measure the CD4 and CD8 counts. RESULTS: The mean CD4 cell counts were 1048 (central 95 centile only), 746 and 424 for the normal healthy adults, asymptomatic HIV seropositives and symptomatic HIV patients, respectively, and the mean CD8 counts were 595, 889 and 732, respectively. Statistically significant differences were observed in the CD4 cell counts between HIV seronegative healthy adults and asymptomatic (P < 0.001) as well as asymptomatic and symptomatic (P < 0.05) HIV infected individuals. The mean CD4 counts of asymptomatic HIV-2 infected individuals was significantly higher than the counts of asymptomatic HIV-1 infected individuals (P < 0.05). CONCLUSIONS: This is an user friendly test and can be an alternate to flow cytometry for the estimation of peripheral T-lymphocyte subsets in developing countries. The assay system has certain limitations inherent to ELISA techniques.     

A direct comparison of rejection by CD8 and CD4 T cells in a transgenic model of allotransplantation

INTRODUCTION: The relative contributions of CD4+ and CD8+ T cells to transplant rejection remain unknown. The authors integrated a previous model of CD4-mediated graft rejection with a complementary model of CD8-mediated rejection to directly compare the function of graft-reactive CD4+ and CD8+ lymphocytes in vivo in a model where rejection requires transgenic T cells. These studies allow direct comparison of CD4 and CD8 T cell responses to the same antigen without the confounding effects of T cell depletion or homeostatic proliferation. MATERIALS AND METHODS: Clone 4 and TS1 mice possess MHC class I- and II-restricted CD8+ and CD4+ T cells, respectively, which express transgenic T cell receptors that recognize the influenza hemagglutinin antigen (HA). We compared the in vivo response of CFSE-labeled, HA-specific transgenic CD8+ and CD4+ T cells after adoptive transfer into syngeneic BALB/c mice grafted with HA-expressing skin. RESULTS: As in the authors' CD4+ model, HA104 skin was consistently rejected by both Clone 4 mice (n=9, MST: 14.2) and by 5 x 10(5) Clone 4 lymphocytes transferred to naive BALB/c hosts that do not otherwise reject HA+ grafts. Rejection correlated with extensive proliferation of either graft-reactive T cell subset in the draining lymph nodes, and antigen-specific CD4+ and CD8+ cells acquired effector function and proliferated with similar kinetics. CONCLUSIONS: These data extend the authors' unique transgenic transplantation model to the investigation of CD8 T cell function. The initial results confirm fundamental functional similarity between the CD4 and CD8 T cell subsets and provide insight into the considerable redundancy underlying T cell mechanisms mediating allograft rejection.     

The role of CD4 and CD8 T cells in viral infections.

Presentation of viral antigens to T cells does not require uptake by 'professional' antigen-presenting cells. Viruses have specialized to enter the cells in which they replicate. Virus entry, uncoating and new viral protein synthesis can load both the cytosolic and the endosomal pathway of antigen processing, resulting in viral peptide presentation to CD8 and CD4 T cells by MHC class I and II molecules, respectively. Although a role of CD8 T cells in the control of viral infection has been well documented, current research interest centers on the contribution of the different CD4 T-cell subsets.     

Antibody to native human immunodeficiency virus type 1 envelope glycoproteins induced by IIIB and MN recombinant gp120 vaccines. The NIAID AIDS Vaccine Evaluation Group.

The ability of antibody induced by MN and IIIB recombinant gp120 (rgp120) human immunodeficiency virus type 1 (HIV-1) vaccines the bind to oligomeric native and monomeric recombinant HIV-1 envelope glycoproteins (rgp 120) was measured in 25 uninfected, healthy adult volunteers. A major focus was to evaluate the effect of simultaneous and sequential immunization with vaccines representing different strains of HIV-1 on the ability to broaden cross-reactivity of antibodies against these and other HIV-1 strains. A flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to envelope glycoprotein expressed by infected and rgp120-coated target cells was used, MN rgp120 HIV-1 vaccine given alone and coadministered with IIIB rgp120 HIV-1 vaccine elicited antibody which bound to cells infected with HIV-1MN, HIV-IIIB, HIV-1RF, and HIV-1-SF2. The presence of envelope glycoprotein-binding antibody detected by FIFA correlated to a moderate degree with functional antibody against HIV-1MN and HIV-IIIB. Priming immunization with IIIB rgp120 HIV-1 vaccine followed by booster injections of MN rgp120 HIV-1 vaccine resulted in increased cross-reactive antibody binding to these and heterologous clade B HIV-1 strains infecting cells. MN rgp120 HIV-1 vaccine given alone was better able to induce cross-reactive antibody to cells infected with heterologous HIV-1 laboratory strains than was IIIB rgp120 HIV-1 vaccine given alone. The vaccines induced binding antibody to rgp120 possessing the amino acid sequence of a clade E HIV-1 strain as measured by enzyme-linked immunosorbent assay. Levels of antibody binding to cells infected with clade B HIV-1 and cells coated with monomeric rgp120 were greater than that induced by HIV-1IIIB-based gp160 vaccines in previous studies.     

Determination of CD4 and CD8 lymphocyte subsets by a new alternative fluorescence immunoassay.

The purpose of this study was to evaluate a new alternative fluorescence immunoassay method (Zymmune CD4/CD8 Cell Monitoring Kit; Zynaxis, Inc., Malvern, Pa.) for determining the absolute CD4+ and CD8+ T-lymphocyte concentrations in whole blood. The investigation was performed as a two-site comparison of the reference whole blood flow cytometric method with the Zymmune method. In this investigation, a total of 166 patient samples were evaluated of which approximately 20% were from human immunodeficiency virus-positive individuals. The mean value for samples performed by the Zymmune CD4 assay was 1,094 (range, 74 to 2,586) cells per microliters, while the reference method yielded a mean of 890 (range, 35 to 2,033) cells per microliter. The correlation coefficient for regression analysis was 0.940. The mean value for samples performed by the Zymmune CD8 assay was 700 (range, 212 to 1,813) cells per microliter, while the reference method yielded a mean of 546 (range, 82 to 2,158) cells per microliter. The correlation coefficient for regression analysis was 0.921. No site-specific differences or trends in CD4 or CD8 values were seen when the data were analyzed by site of collection. The average precision of the CD4 assay varied from 6 to 14%, corresponding to the high and low concentration ranges. For CD8, the average precision varied from 8.3 to 16% over the respective high to low concentration ranges. We conclude that the Zymmune CD4/CD8 Cell Monitoring Kit method provides absolute CD4+ and CD8+ T-lymphocyte concentrations which are equivalent to those given by the reference flow cytometric method.     

Cryptosporidium muris in adult mice: adoptive transfer of immunity and protective roles of CD4 versus CD8 cells.

The aim of this study was to investigate the role of the CD4 and CD8 T cells in immunity to cryptosporidia by using Cryptosporidium muris and a mouse model of infection. Two approaches were used, each involving the use of rat anti-T-cell surface marker monoclonal antibodies (MAbs). In the first, the adoptive transfer of immunity was studied by using the CB.17 SCID mouse (which lacks T and B cells) as the host; in the second, the effect on susceptibility of BALB/c mice to infection was examined following depletion of T cells or subsets of T cells. In adoptive immunity experiments, the conditions which differentiated between resistance associated with reconstitution of SCID mice with naive BALB/c lymphocytes and the transfer of immunity with primed lymphocytes from infected animals were determined. Primed spleen or mesenteric lymph node cells conferred better protection to recipients than naive cells when obtained from donors which had developed resistance to infection. Adoptive immunity was abrogated when Thy.1 cells or CD4 cells were depleted from primed cells, while depletion of CD8 cells could reduce the level of protection. In the study of C. muris in BALB/c mice, treatment with either anti-Thy.1 plus anti-Lyt.1 or anti-CD4 MAbs increased susceptibility to a primary infection as determined by the size and duration of oocyst production, but an anti-CD8 MAb produced an increase only in oocyst shedding. Thus, both CD4 and, to a lesser extent, CD8 cells appeared to be involved in resistance to primary and secondary C. muris infection.     

Evaluation of serum levels of soluble CD4, CD8 and beta 2-microglobulin in visceral human leishmaniasis.

The levels of soluble CD4 (sCD4), sCD8 and beta 2-microglobulin (beta 2-M) were measured in sera from patients with visceral leishmaniasis during the course of infection. Levels of sCD4, sCD8 and beta 2-M were raised significantly above levels in normal sera and returned to the normal range after recovery. The decrease in the levels of sCD8 was related to a reduction of anaemia, leukopenia and thrombocytopenia. In contrast, sCD4 levels fluctuated during the period of infection. beta 2-M returned within normal range more rapidly than sCD8 secretion. Our results suggest that T cells are activated during infection, and that it is also possible that the raised levels of these soluble molecules play a role in the impairment of protective immunity.     

Reevaluation of the origin of CD44(high) "memory phenotype" CD8 T cells: comparison between memory CD8 T cells and thymus-independent CD8 T cells.

CD44(high)CD8 T cells in naive mice, which increase with age, and are often referred to as memory CD8 T cells. However, since thymus-independent CD8 T cells have also been shown to be CD44(high), the origin of the CD44(high)CD8 T cells in naive mice remains unclear. In this study, we compared the characteristics of memory CD8 T cells and thymus-independent CD8 T cells in TCR transgenic mice to clarify the origin of the CD44(high)CD8 T cells in naive normal mice. The memory and thymus-independent CD8 T cells showed differences in surface molecules, spontaneous cell death, cytokine production, and response to IL-2R binding of cytokines. Importantly, the "memory phenotype" CD8 T cells in naive normal mice showed similar characteristics to the thymus-independent CD8 T cells, but differed greatly from "true" memory CD8 T cells in the TCR transgenic mice. Therefore, we conclude that a significant part of the CD44(high) memory phenotype CD8 T cells in naive normal mice represents thymus-independent CD8 T cells, which may participate in age-related changes in immune responses.     

Analysis of blood CD4+ and CD8+ T-lymphocyte subsets with monoclonal antibodies. Comparison of the Genetic Systems CD4/CD antigen typing kit with conventional indirect immunofluorescence microscopy and the automated Technicon-H1 Enzyme Immunoassay (EIA)

The Genetic Systems Technique (GS: direct immunofluorescence microscopy with ethidium bromide counterstaining of nuclei) was tested for quantitative analysis of T-lymphocyte subsets in human peripheral blood. The monoclonal antibodies anti-CD4 and anti-CD8 were used for detection of T-helper and T-suppressor cells respectively and the results compared to those obtained by conventional indirect immunofluorescence microscopy and the Technicon Enzyme Immunoassay (EIA) with automated reading. The GS technique provided results correlating well with both indirect immunofluorescence and EIA techniques. Moreover, this method has two advantages: it is less time-consuming than the indirect immunofluorescence microscopy and necessitates less expensive and more commonly available equipment than the automated EIA technique.     

The relative contribution of IL-4 and IL-13 to human IgE synthesis induced by activated CD4 or CD8 T cells

The relative contribution of IL-4 and IL-13 to the regulation of IgE synthesis has remained relatively poorly characterized, partially because of lack of suitable animal models. We have studied the roles of IL-4 and IL-13 in human IgE synthesis induced by supernatants derived from activated CD4⁺⁺ or CD8⁺ T cell clones. Neutralizing anti–IL-4 and anti–IL-13 monoclonal antibodies (mAbs) inhibited IgE synthesis induced by anti-CD40 mAbs and supernatants from CD4⁺ T cells by an average 61% and 42%, respectively (n = 25). Recombinant IL-13 had additive effects on IL-4-induced IgE synthesis, but only when IL-4 was present at low concentrations. Accordingly, IL-4 was the dominant IgE synthesis–inducing cytokine derived from highly polarized T helper (TH)₂ cells. However, anti–IL-13 mAbs also significantly inhibited IgE synthesis induced by two of three supernatants derived from allergen-specific T_H2-like cell lines generated from the skin of patients with atopic dermatitis. Furthermore, anti–IL-13 mAbs almost completely inhibited IgE synthesis induced by supernatants from T_H1 cells or CD8⁺ T cell clones. Taken together, these data indicate that IL-13, in addition to IL-4, contributes to IgE synthesis induced by all T helper cell subsets, including allergen-specific T_H2 cells. Moreover, IL-13 appears to be the major IgE synthesis–inducing cytokine derived from T_H1 cells or CD8⁺ T cells. (J Allergy Clin Immunol 1997;100:792-801.)     

Clinical field site evaluation of the FACSCount for absolute CD3+, CD3+ CD4+, and CD3+ CD8+ cell count determinations in Thailand.

The FACSCount flow cytometer absolute CD3+, CD3+ CD4+, and CD3+ CD8+ cell counts measured at a field site hospital laboratory in Thailand were compared to FACScan absolute counts obtained at a nearby research laboratory. Correlation coefficients for 208 samples were > or = 0.95. The FACSCount was accurate, and it was easier and less expensive to operate than the FACScan. Additionally, the FACScan-generated lymphocyte percentage value was accurate for use with the FACScan SimulSET software.     

Microglial cells qualify as the stimulators of unprimed CD4+ and CD8+ T lymphocytes in the central nervous system.

The potential of central nervous system (CNS)-derived cells for initiating T cell responses is not known. Using the capacity of unprimed T cells to respond to allogeneic determinants on antigen-presenting cells (APC), we assessed the ability of microglial cells to act as stimulators of primary T cell responses in vitro. For this purpose, microglial cells were activated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or by phagocytosis of progenitor oligodendrocytes and subsequently tested for their ability to induce a proliferative response of naive, resting T cells. Activated microglial cells induced a significant proliferation of virgin, alloreactive CD4+ and CD8+ T lymphocytes, with a more substantial response of highly purified CD4+ than of CD8-expressing T cells. Phagocytosis activation was the most efficient stimulus to induce this APC competence on microglial cells. By contrast, IFN-gamma-pretreated, MHC-expressing astrocytes were unable to induce similar responses of alloreactive CD4+ or CD8+ T cells under the same experimental conditions. Collectively, our data suggest the role of activated microglia as the fully immunocompetent accessory cell population of the CNS.     

Inhibition of HIV replication by CD8+ T cells correlates with CD4 counts and clinical stage of disease.

We sought to evaluate the relationship of CD8+ T cell-mediated inhibition of autologous HIV replication in vitro to disease stage in HIV+ individuals. Depletion of CD8+ T cells from peripheral blood lymphocytes of 16 HIV+ subjects increased the percentage of virus-producing cultures from 56% to 81%. CD4+ T cells were purified from 52 HIV+ individuals and cultured alone or in the presence of autologous CD8+ T cells. In 13 (25%) subjects HIV replication was only detected in the absence of CD8+ T cells (inhibition positive); in 26 (50%) viral replication occurred both in the absence and presence of CD8+ cells (inhibition negative). In the remaining 13 (25%) subjects, CD8+ T cell-mediated inhibitory activity could not be evaluated because stimulation of their purified CD4+ T cells did not result in p24 production. In some virus culture-negative individuals, the inability to demonstrate HIV replication was due to the presence of low numbers of CD8+ T cells that co-purified with CD4+ T cells. Detection of inhibitory CD8+ T cells was associated with significantly higher CD4 counts and better clinical status compared with inhibition-negative subjects. These results demonstrate that CD8+ T cell-mediated inhibition of HIV replication correlates with disease stage, and thus may play a role in preventing disease progression. CD8+ T cells did not inhibit autologous HIV replication across a semipermeable membrane. Further, the ability of CD8+ T cells to prevent HIV replication did not correlate with lysis of autologous CD4+ T cells. Thus, CD8+ T cells inhibited autologous HIV replication in vitro through a contact-mediated non-lytic mechanism.