Decreased expression of IL-2 in central and effector CD4 memory cells during progression to AIDS in rhesus macaques.

OBJECTIVE: HIV-1 infection in humans has been reported to lead to a shift in the cytokine balance, with a relative decrease in T helper 1 type cytokines, especially IL-2. On the basis of the expression of CD45RA, in combination with homing markers CD62L or alpha4beta7, T helper cells can be sub-divided into naive, activated naive, central memory and effector memory cells as well as gut-homing subpopulations. In addition, each subset may have the potential to express distinct cytokines. At present it is unclear whether the changes in cytokine expression observed in HIV-1-infected individuals are secondary to changes within the composition of CD4 T cell subsets or are caused by changes in cytokine expression within each subset. MATERIALS AND METHODS: A new technique was developed to detect cytokine expression in phorbol 12-myristate 13-acetate/ionomycin-activated CD62L and alpha4beta7-expressing CD4 T cell subsets, using the protease inhibitor KD-IX-73-4. RESULTS: In SIV-infected macaques that develop AIDS a marked decrease in IL-2 expression was found within central, effector, or gut-homing memory cell subsets, whereas the expression of IL-2 in naive T cell subsets remained unaffected. This reduced IL-2 expression by memory cells and not a loss of the frequency of CD4 memory cells accounted for the reduced expression of IL-2 by CD4 T cells during SIV infection. CONCLUSION: As defined by the cell surface markers utilized, it appears that progression to AIDS is associated with functional impairment of memory cells, but not changes in lymphocyte circulation patterns.

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.

Clonotypic structure of the human CD4+ memory T cell response to cytomegalovirus.

High steady-state frequencies of CMV-specific CD4(+) memory T cells are maintained in CMV-exposed subjects, and these cells are thought to play a key role in the immunologic control of this permanent infection. However, the essential components of this response are poorly defined. Here, we report the use of a step-wise application of flow cytometric and molecular techniques to determine the number and size of the TCR Vbeta-defined clonotypes within freshly obtained CMV-specific CD4(+) memory T cell populations of four healthy, CMV-exposed human subjects. This analysis revealed a stable clonotypic hierarchy in which 1-3 dominant clonotypes are maintained in concert with more numerous subdominant and minor clonotypes. These dominant clonotypes accounted for 10-50% of the overall CMV response, and comprised from 0.3 to 4.0% of peripheral blood CD4(+) T cells. Two subjects displayed immunodominant responses to single epitopes within the CMV matrix phosphoprotein pp65; these single epitope responses were mediated by a single dominant clonotype in one subject, and by multiple subdominant and minor clonotypes in the other. Thus, the CMV-specific CD4(+) T cell memory repertoire in normal subjects is characterized by striking clonotypic dominance and the potential for epitope focusing, suggesting that primary responsibility for immunosurveillance against CMV reactivation rests with a handful of clones recognizing a limited array of CMV determinants. These data have important implications for the understanding of mechanisms by which a genetically stable chronic viral pathogen such as CMV is controlled, and offer possible insight into the failure of such control for a genetically flexible pathogen like HIV-1.

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

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.

Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.

The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.

Assessment of donor T-cell function in cellular blood components by the CD69 induction assay: effects of storage, gamma radiation, and photochemical treatment.

BACKGROUND: Functional donor T-lymphocytes in blood components may cause a variety of transfusion complications. A flow cytometric assay based on the measurement of induced CD69 expression may be an alternative to cell proliferation methods in determining the functional status of these cells in blood components. STUDY DESIGN AND METHODS: Seven units of whole blood, RBCs, and platelet concentrates (PCs) were stored under blood bank conditions. Half of 3 PCs each were gamma-radiated or treated with UVA+psoralen; the other half served as controls. Samples were analyzed for phorbolester-induced expression of CD69 as an indicator of cell responsiveness and for exclusion of propidium iodide as a measure of cell membrane integrity and viability. RESULTS: CD69 inducibility and propidium iodide exclusion decreased exponentially (half-life, 3. 3 and 8.1 days, respectively) during cold blood storage. Irradiation and UVA+psoralen treatment of PCs immediately reduced CD69 inducibility to 21 percent (controls, 82%; p = 0.004) and 12 percent (controls, 95%; p = 0.0008), respectively. The proportion of cells capable of propidium iodide exclusion was similar in treated samples and controls, but it declined faster in the treated samples during subsequent storage. CONCLUSION: Flow cytometric measurement of CD69 induction can be adapted to provide quantitative assessment of T-cell function in blood components. Results obtained by the CD69 assay are in general agreement with those previously reported by use of proliferation methods; the assay may be useful for special applications in transfusion medicine.

The CD4(+) T cell response to HIV-1.

Although virus-specific CD4(+) T cells have proven to be a critical component of the immunologic control of chronic viral infections in a number of models, the role and even the existence of HIV-1-specific CD4(+) T cells in human HIV-1 infection has been controversial. Recent advances in quantifying and functionally characterizing HIV-1-specific T cells may shed light on the participation of these cells in anti-HIV-1 host defense.

Enhancement of HIV type 1 antigen-specific CD4+ T cell memory in subjects with chronic HIV type 1 infection receiving an HIV type 1 immunogen.

We examined HIV-1 specific memory helper T immune responses in chronically HIV-infected subjects who received an immune-based therapy (HIV-1 immunogen, Remune). Subjects in this study exhibited significant increases (p < 0.05) in the frequency of helper T memory cells expressing interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in response to HIV-1 antigens in vitro. The frequencies of HIV-specific memory T cells increased after successive immunizations and exhibited a correlation with the standard tritiated thymidine incorporation lymphocyte proliferation assay (r = 0.72, p < 0.0008). These results support the notion that HIV-specific memory immune responses can be stimulated in subjects with chronic HIV infection. Further investigations are warranted to determine whether the induction of such responses is associated with virologic control.

Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy.

The detection of tumor-specific T cells in immunized cancer patients usually relies on lengthy and difficult CTL assays; we now report on flow cytometry to detect the intracellular cytokines interleukin 2 (IL-2), IL-4, IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) produced by CD4+CD69+ and CD8+CD69+ activated T cells after MUC1 antigen stimulation. Peripheral blood mononuclear cells were obtained from 12 patients with adenocarcinoma injected with mannan-MUC1; cells were exposed in vitro for 18 h to MUCI peptide in the presence of CD28 monoclonal antibody and Brefeldin; permeabilized cells were used for the expression of cytokines. After stimulation in vitro with MUC1-variable number of tandem repeats peptides, CD8+CD69+ T cells from all immunized patients generated 3-9 times higher levels of TNF-alpha(P < 0.038) and IFN-gamma (P <0.010) than did cells from 12 normal subjects; minor increases in IL-4 occurred. By contrast, CD4+CD69+ cells showed no overall alteration in TNF-alpha and IFN-gamma cytokine production, although in some patients, their measurement was informative; the measurement of IL-2 was not useful in either CD4+CD69+ or CD8+CD69+ cells. We conclude that in MUC1-immunized patients, the measurement of TNF-alpha and IFN-gamma in activated CD69+CD8+ T cells may be indicative of their immune status.

Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression.

Memory T cells specific for varicella-zoster virus (VZV), herpes simplex virus (HSV), and human cytomegalovirus (HCMV) were compared in immune adults by intracellular cytokine (ICC) detection. The mean percentages of CD4+ T cells were 0.11% for VZV and 0.22% for HSV by interferon (IFN)-gamma production; the frequency for HCMV was significantly higher at 1.21%. Percentages of VZV-, HSV-, and HCMV-specific CD4+ T cells were similar by use of tumor necrosis factor (TNF)-alpha. HCMV-stimulated CD8+ T cells produced IFN-gamma (1.11%) and TNF-alpha (1.71%); VZV- and HSV-specific CD8+ T cells were not detectable. VZV CD4+ T cell numbers were similar in young adults with natural or vaccine-induced immunity. VZV CD4+ T cells were significantly less frequent in older adults. Secondary varicella immunization did not increase VZV-specific CD4+ T cell frequencies by ICC assay. Numbers of memory T cells specific for herpesviruses may vary with sites of viral latency and with host age.

Immunofluorescence analysis of T-cell responses in health and disease.

The use of flow cytometry to study the functional responses of T cells by immunofluorescent staining for intracellular cytokines and other markers is a growing field of clinical interest. In this article, we describe methods for the rapid evaluation of T-cell responses to mitogens and specific antigens and explore how these assays might be valuable in various clinical settings.

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression.

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0-0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10-1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4-24 weeks, and 0.03% in subjects treated for 47-112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.

Normal human CD4+ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis.

CD4+ memory T cells coordinate immune responses against viruses and other pathogens via the Ag-induced secretion of potent effector cytokines. The efficacy of these responses depends on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. Here, we provide evidence that heterogeneity in Ag triggering thresholds constitutes an additional critical determinant of memory T cell function. Using a novel assay that allows single-cell detection of Ag-specific T cell cytokine production, we demonstrate that CMV-specific CD4+ memory cells from human peripheral blood display pronounced differences in their costimulatory requirements for Ag-induced triggering of IFN-gamma and IL-2 secretion, ranging from cells that trigger with little costimulation (e.g., resting APC alone) to cells requiring potent costimulation through multiple pathways (resting APC plus multiple costimulatory mAbs, or activated APC). These differences in costimulatory requirements are independent of clonal differences in TCR signaling intensity, consistent with an intrinsic activation-threshold heterogeneity that is "downstream" from the TCR. Thus, "effective" frequencies of Ag-specific CD4+ memory T cells appear to depend on the activation status of available APC, a dependence that would allow the immune system to rapidly adjust the number of functional Ag-specific memory T cells in a particular effector site according to local conditions.

Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression.

Methods for analysis of T cell function have traditionally relied upon measurements of proliferation or cytokine expression in bulk cultures of PBMC in long term incubations with polyclonal mitogens or putative antigen. These techniques suffer from the drawback that they do not enable analysis of single cell responses in the context of unselected cellular backgrounds. In addition these methods are not sensitive enough to rapidly assess rare event responses characteristic of cognate memory T cell responses. This review discusses recently developed flow cytometric methods designed to rapidly assess leukocyte subset cytokine responses to polyclonal activators and specific antigen in PBMC and whole blood samples. These procedures determine the percentages of activated cells and the identification of leucocyte subsets capable of expressing various cytokines and cell surface antigens. The ability to assess key intracellular functional markers by multiparameter flow cytometry offers some unique advantages in a number of clinical applications. The technical simplicity and rapidity of the flow cytometric intracellular cytokine detection techniques described in this report, as well as the widespread availability of appropriate flow cytometers and cell surface directed antibodies in clinical laboratories, suggests the possibility that this technique could be broadly applicable to the clinical evaluation of immune status. Since any cell type can be identified with this approach, responses to a variety of clinically relevant stimuli in virtually any leukocyte subset can be evaluated including monocyte responses to LPS, and T cell responses to mitogens and a variety of bacterial and viral antigens. The significance of measuring low frequency antigen-specific responses with respect to clinical significance in assessing immune status in a variety of clinical conditions and determining efficacy or immunotoxicity of drugs and vaccine antigens is discussed.

Rapid assessment of antigen induced cytokine expression in memory T cells by flow cytometry.

Methods for analysis of T cell responses to specific antigen have traditionally relied on measurements of proliferation or cytokine expression in bulk cultures of PBMC in long term incubations with putative antigen. These techniques suffer from the drawback that they do not enable analysis of single cell responses in the context of unselected cellular backgrounds. It is increasingly important to not only identify cells on the basis of expression of unique surface antigens but also to determine functional and molecular parameters of individual cells in response to a variety of stimuli. We have recently developed methodologies to rapidly assess T-cell subset responses to polyclonal activators and specific antigen in whole blood. These procedures determine the percentages of activated cells and the identification of leucocyte subsets capable of expressing various cytokines and cell surface antigens. Multiparameter analysis of CD69+ /cytokine + expression in T cells in response to specific antigen (e.g. CMV, mumps) demonstrated a range of frequencies from 0.05% to 5.0% within 6 h. Frequencies of responding T cells were consistent but varied depending upon the antigen. Antigen specific T cell responses were host specific and both positively and negatively regulated by antibodies to co-receptors involved in APC-T cell interactions. These technologies will be discussed in terms of application to problems of measuring immune function parameters in disease. The modified technique described in this report is compatible with simple and rapid analysis of clinical samples and provides a means to directly examine the effects of in vivo drug concentrations on T cell immunity. Studies are in progress to examine the sensitivity of this cellular assay to drugs and other therapeutic modalities in clinical samples.

Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry.

We have recently described a highly sensitive flow cytometric technique, based on the ability to detect single cell expression of cytokines, to simultaneously quantitate and phenotypically characterize antigen-specific memory/effector T cells in PBMC cultures. In this report, we describe a simplified procedural modification which enables the rapid detection of low frequency memory CD4+ and CD8+ T cells expressing cytokines in response to soluble antigen in whole blood. When compared with T cell responses in PBMC cultures, whole blood cultures demonstrated similar but slightly higher percentages of T cells responsive to specific antigen. In addition, T cell responses to cytomegalovirus in whole blood were observed only in sensitized (seropositive) individuals, and CD4+ T cell responses could be blocked by anti-class II MHC antibodies. This procedure may provide a means to examine direct effects of pharmacological drug concentrations on T cell immunity in clinical samples.

A 15-year follow-up of AJCC stage III malignant melanoma patients treated postsurgically with Newcastle disease virus (NDV) oncolysate and determination of alterations in the CD8 T cell repertoire.

BACKGROUND: The development of effective adjuvant therapies for the treatment of high-risk melanoma patients is critical for the prevention of metastatic disease and improvement of patient survival. Active specific immunotherapy has been tested as an adjuvant treatment in numerous clinical trials with overall limited, but occasionally promising, success rates. Newcastle disease virus (NDV) oncolysate has been utilized as an adjunctive immunotherapeutic agent in the postsurgical management of these patients. A phase II study initiated in 1975 using adjuvant vaccine therapy composed of allogeneic and autologous human melanoma cells infected with live NDV (NDV oncolysate) in patients with AJCC stage III melanoma following therapeutic lymph node dissection has shown >60% survival rate at 10 years with no adverse effects. Continued long-term analysis of trials with promising early results as well as assessment of immunologic responses generated in these patients may result in improved therapeutic decisions for clinical trials in the future. MATERIALS AND METHODS: We analyzed the 15-year survival of patients treated postsurgically with NDV oncolysate in the phase II study described above. In an attempt to understand the immunological effects of this treatment, we have also carried out a comprehensive analysis of the peripheral blood T cell repertoire in these patients. RESULTS: The overall 15-year survival of this group of patients is 55%. Previous studies have suggested that improved outcome in patients undergoing immunotherapy is correlated with increased numbers of CD8(+)CD57(+) cells. In surviving patients, we observed a striking oligoclonality in the CD8(+) T cell population in peripheral blood, which reflects clonal expansions in the CD8(+)CD57(+) subset. CONCLUSIONS: The data suggest that adjuvant vaccination with NDV oncolysates is associated with prolonged survival of patients with lymph node-positive malignant melanoma and that CD8(+) T cells may be an important component of therapeutic efficacy.