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.

Major expansions of select CD8+ subsets in acute Epstein-Barr virus infection: comparison with chronic human immunodeficiency virus disease.

CD8+ lymphocyte phenotypes were characterized during acute Epstein-Barr virus (EBV) infection, and a comparison was made to previous studies of human immunodeficiency virus (HIV). This was of interest because CD8+ cells contribute to immunologic control of both infections, but the usual outcome of EBV infection is benign, whereas untreated HIV infection is fatal. During acute EBV infection, CD8+ cells expressed elevated levels of the activation antigens CD38 and HLA-DR, similar to that during chronic HIV infection. Within 16 weeks, when EBV latency is established, CD8+ cell activation had resolved. In contrast, activation persists in HIV infection. Expression of CD38 and HLA-DR on CD8+ cells could be a marker for ongoing viral replication in both infections. Other CD8+ cell alterations observed in this study of acute EBV infection included increases in both CD62L- and CD62L+ CD8+ cells and unique kinetics in the expansion of the CD57+CD8+ cell subset.

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.

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.

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.