Cytokine flow cytometry: multiparametric approach to immune function analysis.

More precise quantitation of cellular immune responses has become possible with the advent of single-cell assays of immune function, such as cytokine flow cytometry, enzyme-linked immunospot (ELISPOT), and MHC-peptide multimers. Cytokine flow cytometry is an attractive technique because it allows the detection of responses to whole antigens without regard to MHC restriction, while also collecting additional information on responding cells via multiparameter flow cytometry. In this review, we compare cytokine flow cytometry with other assays of immune function, summarize some of that data that have been collected in various disease states using cytokine flow cytometry, and describe some methodological improvements designed to increase the robustness, throughput, and information content of this technique. We hypothesize that a new generation of automated cytokine flow cytometry assays will allow elucidation of the correlates of protection for diseases involving cellular immunity, through application of these assays in more and large clinical trials.

Decreased HIV-specific CD4 T cell proliferation in long-term HIV-infected individuals on antiretroviral therapy.

The effect of highly active antiretroviral therapy (HAART) on HIV-specific CD4 T cell proliferation in long-term HIV-infected individuals was studied. Subjects receiving treatment for over a year were compared with individuals not receiving therapy. The absolute number of HIV-specific memory CD4 T cells proliferating in response to HIV antigen was substantially higher in untreated subjects than in those on HAART. A decrease in HIV-specific memory CD4 T cells could explain the rebound in HIV replication after the termination of HAART.

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens.

CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.

Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens.

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65.

Antigen-specific CD8(+) T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-gamma) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8(+) T-cell IFN-gamma expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a (51)Cr release assay and frequencies of peptide-activated CD8(+) T cells expressing IFN-gamma at 6 h (r(2) = 0.72) or 7 days (r(2) = 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-gamma expression can be a functional surrogate for identification of CTL precursor cells.

Affinity isolation of neuron-reactive antibodies in MRL/lpr mice.

Autoantibodies from the MRL/lpr mice react with numerous proteins on neuronal cell surfaces. The purpose of this study was to isolate and characterize a population of autoantibodies reactive preferentially or exclusively with nervous system tissue. Using a purified plasma membrane preparation from brain cortex of balb/c mice coupled to diaminopropylamine agarose gel, we affinity-isolated antineuronal antibodies from pooled MRL/lpr immunoglobulins. The isolated immunoglobulins reacted with brain cortex plasma membranes and neuroblastoma cells (but not liver, kidney, or fibroblasts) by Western blot and indirect immunofluorescence with confocal microscopy. By Western blot, the epitopes in the brain cortex were proteins of apparent molecular weights 101, 63, 53, 43, 39, and 33, kd; the epitopes in the neuroblastoma cells were 63, 57, and 53 kd. Lectin column isolation revealed that the 101 and 63 kd epitopes were glycosylated. Indirect immunofluorescence revealed that the antibodies bound to the cell soma more intensely than to the cell processes of viable cultured neuroblastoma cells. The cell surface localization of this binding was confirmed by confocal microscopy. Within the central nervous system the antibodies bound more intensely to primary cultures of isolated neurons from fetal cortex than to hippocampal or neostriatal cells. With these antibodies we can begin studies of their potential pathogenic effects.

Characterization of & induction of immune response to anti-idiotypic antibodies for JE virus.

Anti-idiotypic antibodies (anti-Ids, Ab2s) were prepared by immunizing rabbits with two murine monoclonal antibodies (Ab1) having specificities for two independent haemagglutinin (HA) epitopes on JE virus [viz., Hs-1, monoclonal antibody (MAb) specific for Japanese encephalitis virus (JEV) and Hx-1, MAb common to flaviviruses]. Anti-Hs-1 (S-Ab2) and Anti-Hx-1 (X-Ab2) reacted specifically with the immunizing Ab1. In addition, they could react with other MAbs whose reactivity was similar to their immunizing homologous Ab1. The paratope inhibition assay indicated that both anti-idiotypes recognized paratope related idiotopes on their respective Ab1 and could therefore be designated as Ab2 beta. Experimental animals (Swiss mice, Balb/c mice and guineapigs) immunized with S-Ab2 or X-Ab2 produced anti-JE virus antibodies (Ab3) which could be detected by enzyme linked immunosorbent assay, immunofluorescence, haemagglutination inhibition and neutralization tests. The anti-idiotypes were also found to stimulate a cellular immune response in vitro as assessed by 3H thymidine incorporation by lymphocytes from JE vaccinated individuals and experimentally immunized Balb/c mice. The findings of the present study suggest that both the anti-Id antibodies are homobodies which may act as surrogate antigens to manipulate the immune response against JEV.