Preferential depletion of gut CD4-expressing iNKT cells contributes to systemic immune activation in HIV-1 infection.

Chronic inappropriate immune activation is the central defect-driving loss of CD4(+) T helper cells and progression to AIDS in persons with HIV-1 infection, but the mechanisms remain controversial. We examined key regulatory invariant receptor natural killer T (iNKT) cells in the gut, the largest reservoir of lymphocytes and a key arena of HIV-1 pathogenesis. In healthy control persons, the anti-inflammatory CD4(+) iNKT-cell subset predominated over the pro-inflammatory CD4(-) iNKT-cell subset in the gut, but not in the blood, compartment. HIV-1 infection resulted in a preferential loss of this anti-inflammatory CD4(+) iNKT-cell subset within the gut. The degree of loss of the CD4(+) iNKT-cell subset in the gut, but not in the blood, correlated to the systemic immune activation and exhaustion that have been linked to disease progression. These results suggest a potentially important contribution of gut iNKT-cell imbalance in determining the systemic immune activation that is the hallmark of HIV-1 pathogenesis.

Testing the efficiency of aerosol containment during cell sorting.

Production of droplets and microdroplets (aerosols) is part of the normal operation of a cell sorter. These aerosols may contain toxic, carcinogenic, or teratogenic fluorophores or known or unknown pathogens from viable biological specimens. Most newer models of commercially available instruments incorporate features designed to reduce the production of aerosols and prevent their release into the room. This unit presents two protocols for assessment of aerosol containment on jet-in-air flow sorters. In both procedures, lytic T4 bacteriophage is run through the instrument at high concentrations to tag aerosol droplets. The instrument is tested in normal operating mode and in simulated failure mode. Aerosols are detected by plaque formation on susceptible E. coli lawns. With the continuing increase in the sorting of viable human cells, it is vital for cytometrists to be aware of the potential dangers.

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.

Generation of functional thymocytes in the human adult.

Reconstituting the immune response will be critical for the survival of HIV-infected individuals once viral load is brought under control. While the adult thymus was previously thought to be relatively inactive, new data suggest it may play a role in T cell reconstitution. We examined thymopoiesis in adults up to 56 years of age and found active T cell receptor (TCR) rearrangement, generating a diverse TCR Vbeta repertoire. The resulting thymocytes are functional and are capable of responding to costimulatory signals. These data demonstrate that the adult thymus remains active late in life and contributes functional T cells to the peripheral lymphoid pool.

Detailed immunophenotype of CD8+ memory cytotoxic T-lymphocytes (CTL) against HIV-1 with respect to expression of CD45RA/RO, CD62L and CD28 antigens.

We have previously reported that circulating effector cytotoxic CD8+ T-lymphocytes (CTLs) against HIV-1 express CD38 and HLA-DR activation antigens. In this study, we performed two series of FACS sorts to phenotype and characterize precursors of CTL effectors. First we looked at memory CTL activity against HIV-1 stimulated by antigen as well as CTL activity stimulated by CD3 mAb with regard to whether the precursors expressed CD45RA and/or CD62L. We found that the precursor cells that could be stimulated with antigen to become effectors within 7 days predominated in the CD45RA CD62L subset. However. in donors with low levels of CD8+ T-cell activation as measured by CD38 antigen expression, memory cells could also be found in the CD45RA+ CD62L+ subset. Our data indicate that reversion of memory cells to the CD45RA+ CD62L+ phenotype can occur in humans, especially in donors with low levels of virus replication and minimal CD8 + T-cell activation. Next, we looked at CD28 expression with regard to antigen specific memory cells and again found that the level of virus replication and CD8+ T-cell activation influenced the subset that contained the memory cells. In donors with high levels of virus replication, our results indicated that CTL were being actively recruited from both CD28+ and CD28 subsets, while in donors with undetectable levels of viral replication, the memory cells were entirely in the CD28 compartment.

Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage.

To define predictors of survival time in late human immunodeficiency virus type 1 (HIV-1) disease, long- and short-duration survivors were studied after their CD4+ T cells fell to

Quantitation of CD38 activation antigen expression on CD8+ T cells in HIV-1 infection using CD4 expression on CD4+ T lymphocytes as a biological calibrator.

For some membrane-associated antigens, the number of molecules expressed per cell carries information about the cell's differentiation and activation state. Quantitating antigen expression by flow cytometry has immediate application in monitoring CD38 expression on CD8+ T cells in human immunodeficiency virus 1-associated disease, where elevated CD38 antigen expression is a marker of CD8+ T-cell activation and a poor prognostic indicator. Reproducible methods are needed in order to quantify such antigens. Here we describe a reproducible method for quantitative fluorescence cytometry (QFCM) that depends on the tightly regulated expression of CD4 antigen on human CD4+ T lymphocytes, which we estimated in a study of 57 normal donors to have an interperson coefficient of variation of 4.9%. Using phycoerythrin (PE)-conjugated CD4 monoclonal antibody (mAb) with a nominal fluorochrome to protein ratio of 1:1 and a nominal published value of approximately 50,000 CD4 antibody molecules bound per CD4+ T lymphocyte, we estimated the number of PE molecules detected per relative fluorescence intensity (RFI) unit on our flow cytometer to be 41 (19, 20). This value is called the "RFI multiplier." To estimate the number of CD38 antibodies bound per CD8+ T cell (CD38-ABC) on patient samples, we multiply the measured CD38 RFI value of CD38 staining using a nominal 1:1 conjugate of CD38-PE by the "RFI multiplier." The measurements for CD4 and CD38 were stable for 2 years despite the use of different mAb lots and the potential for drift in instrumentation. We used this approach in a study of nine flow cytometers in which the interinstrument interlaboratory coefficients of variation for CD3-ABC ranged from 3.3% to 5.8% and those for CD38-ABC ranged from 9.8% to 13.8%. These data indicate that CD4 expression can serve as a biological calibrator to standardize fluorescence intensity measurements in longitudinal and multicenter studies.

Quantitation of CD38 expression using QuantiBRITE beads.

The QuantiBRITE bead method was compared with the CD4 biological calibration method for quantitation of CD38 expression on CD8+ T-lymphocytes of Multicenter AIDS Cohort Study participants. Results were expressed as CD38 antibodies bound per cell (ABCs) and were the same with the two methods provided two conditions were met. These were the use of repurified (> 95% of the monoclonal antibodies [mAbs] have 1 phycoerythrin [PE] molecule per mAb) CD38-PE for both methods and use of repurified CD4-PE to calculate the relative fluorescence intensity multiplier for the CD4 biological calibration method. Our results indicate that the prognostic significance of CD38 values obtained using the QuantiBRITE method can be interpreted using previously published reports (Liu et al.: J Acquir Immune Defic Syndr Hum Retrovirol 16:83-92, 1997 and 18:332-340, 1998). Sample preparation using NH4Cl and FACS lysing solution gave similar results for CD38 relative fluorescence intensity. Dilution into either phosphate-buffered saline with 2% fetal calf serum and 0.1% sodium azide or fixation in 1% paraformaldehyde for 1 or 24 h also gave similar results. In experiments using Raji cells, which express high levels of CD38, the valence of binding of the intact Leu 17 antibody was approximately 68% bivalent and approximately 32% monovalent. This emphasizes the complexity of determining antigen density from ABCs. We conclude that repurified PE conjugates of CD38, which can be consistently made, together with QuantiBRITE PE beads, provide a convenient and reliable method for quantitation of CD38 expression as ABCs.

CD8+ T-lymphocyte activation in HIV-1 disease reflects an aspect of pathogenesis distinct from viral burden and immunodeficiency.

The CD8+ T-cell response is central to control and eventual elimination of persistent viral infections. Although it might be expected that CD8+ T-cell activation would be associated with a better clinical outcome during viral infections, in long-term HIV-1 infection, high levels of CD8+ T-cell activation are instead associated with faster disease progression. In this study, cell surface expression of CD38, a flow cytometric marker of T-cell activation of CD8+ T cells, had predictive value for HIV-1 disease progression that was in part independent of the predictive value of plasma viral burden and CD4+ T-cell number. Measurements of CD38 antigen expression on CD8+ T cells in HIV-1-infected patients may be of value for assessing prognosis and the impact of therapeutic interventions. The pathogenetic reason why CD8+ T-cell activation is associated with poor outcome in HIV-1 disease remains unknown. Possibly CD8+ T-cell activation contributes to immunologic exhaustion, hyporesponsiveness of T cells to their cognate antigens, or perturbations in the T-cell receptor repertoire.

Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression.

The prognostic value of several immunologic markers were compared in Los Angeles Multicenter AIDS Cohort Study (MACS) participants, most of whom had been infected with HIV for >8 years. Markers studied included CD4+ cell number, flow cytometric measurements of CD8+ cell expression of CD38 and HLA-DR antigens, and serum markers of immune activation including neopterin, beta2-microglobulin, soluble interleukin-2 receptor, soluble CD8, and soluble tumor necrosis factor receptor-alpha (TNF-alpha) type II. Cox proportional hazards models indicated that elevated CD38 on CD8, a flow cytometric measurement of CD8+ T-lymphocyte activation, was the most predictive marker of those studied for development of a clinical AIDS diagnosis and death. As compared with the reference group, who had CD38 on CD8 <2470 molecules per CD8+ cell and in whom 4 of 99 developed clinical AIDS within 3 years, participants with CD38 on CD8 between 2470 and 3899, 3900 and 7250, and >7250 had relative risks (and numbers developing AIDS within 3 years) of 5.0 (15 of 81), 12.3 (24 of 60), and 41.4 (36 of 49), respectively. The strong prognostic value of CD38 on CD8 measurements and the fundamental importance of chronic immune activation in the pathogenesis of HIV disease suggests that this marker might have utility in the clinical management of HIV-infected persons.

The immunopathogenesis of retroviral diseases: no immunophenotypic alterations in T, B, and NK cell subsets in SIVmac239-challenged rhesus macaques protected by SIV delta nef vaccination.

Immunophenotype analysis was used to characterize circulating lymphocyte subset levels in both rhesus monkeys that were chronically infected with SIVmac239 and in those that had resisted SIVmac239 infection as a result of prior vaccination with an attenuated SIV strain. Alterations in T, NK, and B cell subsets were compared with those previously identified in humans chronically infected with HIV [8-11, 14, 22]. The well-known decrease in CD4+ cell levels was observed in the SIVmac239-infected animals. However, these animals had relatively little activation of circulating CD8+ T cells as compared with uninfected monkeys. This contrasts with chronically HIV-infected humans who have substantial activation of circulating CD8+ cells as evidenced by elevated HLA-DR and CD38 antigen expression on CD8+ cells as well as substantially increased percentages and numbers of total CD8+ cells. NK cells of the SIVmac239-infected animals, on the other hand, demonstrated the same changes recently described in HIV-infected humans, i.e., a decrease in circulating percentages and a decreased amount of FcRIII (CD16). B cell percentages were markedly increased in the SIVmac239-infected animals, a finding also noted in some children with HIV infection but not in HIV-infected adults. SIV delta nef-vaccinated/SIVmac239-challenged animals showed none of the immune alterations found in the SIVmac239-infected monkeys, providing further confirmation of lack of SIV disease in these vaccinated animals.

Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ T cells is a marker of poor prognosis in HIV infection: results of 6 years of follow-up.

Relative fluorescence intensity measurements from a flow cytometer were used to evaluate expression of CD38 and HLA-DR antigens. These molecules are associated with cellular activation and are present at increased levels on the CD8+ lymphocytes of HIV-infected subjects. In the current study, the prognostic value of mean fluorescence intensity measurements of CD38 and HLA-DR on CD8+ cells was compared to results from our previous study in which we reported prognostic value for an elevated percentage of CD8+ cells that were positive for expression of the CD38 antigen (Giorgi et al.: JAIDS 6:904-912, 1993). Using the proportional hazards model, elevated mean fluorescence intensity of CD38 expression on CD8+ cells had prognostic value for development of AIDS that was almost identical to the prognostic value of the percentage of CD8+ cells that were positive for expression of CD38. This prognostic value was in addition to that provided by the patient's CD4+ cell measurement. To our knowledge, this is the first report that a measurement of fluorescence intensity can be used as a prognostic marker in an immunodeficiency disease. Efforts are needed to establish methods that will allow widespread application of this observation in the clinical management of HIV-infected subjects.

Natural killer cell immunodeficiency in HIV disease is manifest by profoundly decreased numbers of CD16+CD56+ cells and expansion of a population of CD16dimCD56- cells with low lytic activity.

Natural killer (NK) cells were enumerated by three-color immunofluorescence in 255 uninfected and 399 human immunodeficiency virus-infected adults. Several dramatic alterations were observed. First, the median number and percentage of CD16+CD56+ NK cells, the subset that comprises > 90% of the NK cells in healthy adults, were severely decreased (median, 175/mm3 in uninfected controls; 63/mm3 in HIV-infected non-AIDS subjects). Even subjects with > 800 CD4+ cells/mm3 had decreased CD16+CD56+ NK cell levels (97/mm3). Second, the number of CD16+CD56- cells, an NK population that is rare in healthy adults, was elevated (median, 20/mm3 in uninfected controls; 64/mm3 in HIV-seropositive non-AIDS subjects). Third, the expression of CD16 on the NK cells was markedly reduced; some CD56+ cells and virtually all CD56- cells were CD16dim. Fourth, fluorescence-activated cell-sorting studies revealed little NK- or antibody-dependent cellular cytotoxic activity in the CD16dimCD56- cell population. These results indicate that the pathogenesis of HIV disease includes numerical alterations in subpopulations of NK cells. A better understanding of how HIV infection causes this aspect of pathogenesis is needed.

Virus burden in long-term survivors of human immunodeficiency virus (HIV) infection is a determinant of anti-HIV CD8+ lymphocyte activity.

Persons infected with human immunodeficiency virus (HIV) for > 8 years were studied to delineate virologic and immunologic attributes of long-term survival. Whereas those with 300-700 CD4+ cells/microL often had circulating cytotoxic T lymphocytes (CTL) against HIV antigens, those with > 1000 CD4+ cells/microL did not. The subjects with > 1000 CD4+ cells/microL had low virus burden, low levels of Gag-specific CTL precursors, and minimal CD8+ cell activation. Overall, elevated levels of CD8+ cells, CD38 antigen expression on CD8+ cells, and anti-HIV functions were correlated with increased virus burden, provirus load, and HIV plasma RNA levels. A factor that suppressed HIV replication was spontaneously secreted from CD8+ cells of most subjects but not from those with high CD4+ cell counts. CD8+ cell activities, therefore, may reflect chronic viral stimulation of the immune system. Long-term survivors with high levels of CD4+ cells maintained control of viral replication but lacked the CD8+ cell activities.

CD8+ lymphocyte activation at human immunodeficiency virus type 1 seroconversion: development of HLA-DR+ CD38- CD8+ cells is associated with subsequent stable CD4+ cell levels. The Multicenter AIDS Cohort Study Group.

Subsets of activated CD8+ lymphocytes defined by membrane expression of the activation antigens HLA-DR and CD38 were counted by three-color flow cytometry in homosexual men who subsequently became seropositive for human immunodeficiency virus type 1 (HIV). Profound CD8+ cell activation was seen in all subjects at seroconversion and 6 and 12 months later. The HLA-DR+ CD38+ CD8+ cell population, which has potent direct HIV cytotoxic T cell activity, was markedly elevated at seroconversion in all subjects. In some men, these levels remained elevated throughout the first year of infection. During the next 5 years, these men had stable CD4+ cell levels, whereas the others did not. Long-term survivors (seropositive for 9 years, > 800 CD4+ cells/mm3) also had elevated levels of this subset, despite few other activated CD8+ cells. Thus, selective elevation of HLA-DR+ CD38- CD8+ cells was a marker of subsequent stable HIV disease.

B cell dysfunction after bone marrow transplantation is associated with decreased Ca2+ flux upon membrane Ig crosslinking.

Patients undergoing bone marrow transplantation have a long-lasting defect of B cell-mediated immunity. Both quantitative (decreased blood B cell counts) and qualitative (decreased Ig production) abnormalities of B cells have been described. To better understand the mechanism of the qualitative defect and its potential relation to B cell immaturity, we studied the in vitro responsiveness of B cells to polyclonal stimuli in patients at 2-12 months post-transplant and in normal neonates. Several key steps of the B cell program were deficient in the patients while they were relatively normal in the neonates. These included (i) early activation as assessed by Ca2+ flux; (ii) late activation as assessed by the increase in cell size and upregulation of the activation antigens CD25 and CD71; and (iii) proliferation as assessed by the number of cycling cells after stimulation. We conclude that the functional B cell defect during the early (< 1 year) post-transplant period extends back to the level of early activation and cannot be simply attributed to the relative immaturity of post-transplant B cells.

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.

Circulating HIV-specific CD8+ cytotoxic T cells express CD38 and HLA-DR antigens.

CD38, a molecule with multilineage distribution but unknown function, and the MHC class II molecule HLA-DR (DR) have markedly elevated levels of expression on CD8+ cells of HIV-infected people. This study investigated the expression of CD38 and DR Ag on circulating HIV-specific CD8+ CTL in HIV-seropositive subjects. Purified CD8+ lymphocytes from 22 participants in the University of California at Los Angeles Multicenter AIDS Cohort Study were screened for CTL activity against autologous EBV-immortalized lymphoblast targets infected with vaccinia vectors that carried HIVIIIB gag, pol, and env genes. Sixty-seven percent (14 of 21), 64% (14 of 22), and 9% (2 of 22), respectively, of the subjects had HIV-specific CD8+ CTL activity against gag, pol, and env proteins. CD8+ cells from 11 of the subjects who had high CTL activity were then FACS-separated using three-color immunofluorescence sorting. Circulating DR-CD38- CD8+ cells had little activity. Highly purified DR+CD38+ CD8+ cells had higher HIV-specific CTL activity than other CD8+ cells. DR+CD38- or DR-CD38+ CD8+ cells also mediated significant activity, but only about half as much on a per cell basis as DR+CD38+ CD8+ cells. This is the first report that the CD38 molecule is expressed in vivo on Ag-specific CD8+ CTL, and confirms previous reports that DR is expressed on these cells. Both asymptomatic HIV-seropositive subjects (144 +/- 132/mm3) and AIDS patients (253 +/- 178/mm3) had markedly elevated levels of DR+CD38+ CD8+ cells compared with the levels in HIV-seronegative controls (7 +/- 3/mm3). However, the level of anti-HIV CTL activity was not correlated with the level of DR+CD38+ CD8+ cells, indicating that enumeration of this lymphocyte population by flow cytometry most likely will not be a useful surrogate for measuring functional CTL activity. Low levels of HIV-specific CTL activity, especially against gag, were correlated with lower CD4+ cells numbers, suggesting that the loss of CD8+ T cell cytotoxic activity against HIV that has been reported to occur with advancing HIV disease progression may reflect in part the extent of CD4+ cell immunodeficiency in HIV-infected subjects.

Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program.

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16 + PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1 1/2 year period, 78 shared peripheral blood specimens were prepared and analyzed in each laboratory. The CD45bright CD14- percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were < 3% for total T cells; < 5% for CD4+ T cells and CD8+ T cells; < or = 17% for B and NK cells; and < 8% for CD4T/CD8T ratios. The 6-tube basic immunophenotyping panel has several notable features: a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and c) for within-sample quality assurance, it provides several quality control checks, including the lymphosum, i.e., the sum of an individual's corrected T+B+NK values, a sum that was generally 100 +/- 5% on the HIV-seronegative specimens analyzed in this study.

CD20 (pan-B cell) antigen is expressed at a low level on a subpopulation of human T lymphocytes.

Despite the previous description of the leukocyte differentiation antigen CD20 as B cell restricted, the findings reported here indicate that a small subset of human T cells expresses low levels of CD20 or a cross-reacting antigen. Three different CD20 monoclonal antibodies (mAb), Leu16, B1, and 1F5, reacted with the T cell subset. B cells that expressed CD20 were CD20bright and constituted an average of 9.2 +/- 3.3% of adult PBL. Meanwhile, T cells that expressed CD20 were CD20dim and represented 2.4 +/- 1.5% of the PBL. This population may have been overlooked in previous studies due to the low level of CD20 expression per T cell and the small size of the subset in most individuals. Blocking studies indicated that CD20 mAb binding to CD3+ cells was due to the antigen-reactive regions of the CD20 antibodies and was not a result of Fc receptor binding, or non-specific fluorochrome or protein binding. The T cell nature of the CD20dim CD3+ cells was confirmed by the rapid rise in the intracellular calcium concentration ([Ca2+]i) of CD20dim cells observed following treatment with CD3 mAb but not following treatment with anti-human immunoglobulin (Ig). Extensive three-color immunophenotypic analyses indicated that CD20dim T cells were phenotypically heterogeneous and displayed a leukocyte differentiation profile that was slightly different than that of CD20- T cells. Thus, the CD20dim T cells were more likely than CD20- T cells to be gamma/delta T cell antigen receptor positive (14% vs. 3.4%), CD8+ (57% vs. 33%), and CD45RO+ (82% vs. 51%); fewer were CD38+ (5% vs. 24%) or CD4+ (35% vs. 61%).(ABSTRACT TRUNCATED AT 250 WORDS)