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Circulating CD8 T Cells Show Increased Interferon-γ mRNA Expression in HIV Infection
CELLULAR IMMUNOLOGY ARTICLE NO.
178, 91–98 (1997)
CI971115
Circulating CD8 T Cells Show Increased Interferon-g mRNA Expression in HIV Infection1 Elizabeth Crabb Breen,*,2 Jesus F. Salazar-Gonzalez,† Lu Ping Shen,‡ Janice A. Kolberg,‡ Mickey S. Urdea,‡ Otoniel Martinez-Maza,*,§ and John L. Fahey*,§ *Department of Microbiology and Immunology and §Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095-1747; †Facultad de Ciencias Quimicas/CIEP, Universidad Auto´noma de San Luis Potosı´, San Luis Potosı´, Mexico; and ‡Chiron Corporation, Emeryville, California Received February 5, 1997; accepted March 17, 1997
The immune deficiency associated with HIV infection is the result of the loss of the number and function of CD4 T helper cells. These cells also have been the subject of great scrutiny in HIV infection because of their contribution to cytokine production. CD4 T cells can generally be divided into two types (type 1 and type 2) according to the pattern of cytokines secreted (6, 7). It has been suggested that a shift away from the production of type 1 cytokines could play a role in the progression of HIV disease (7, 8). Recently, similar patterns of cytokine production were described in CD8 T cells (9, 10). Type 1 cells typically produce IL-2 and IFN-g and are stimulated by IL-12. Type 2 cells produce and/ or respond to IL-4, IL-5, IL-6, and IL-10 and are downregulated by IL-12. It must be remembered, however, that contributions to type 1 and 2 responses can be made by other non-T cell types such as monocyte/macrophages and natural killer (NK) cells. Thus, in an examination of contributions to immune activation/cytokine production, all cell types should be considered. IFN-g is a cytokine that can be produced by multiple cell types, and is considered to enhance cellular immune responses by activation of monocytes and macrophages (2). It is one of the type 1 cytokines that is thought to contribute to effective cell-mediated immunity. IFN-g drives the production of neopterin, an activation marker detectable in serum/plasma which is elevated in HIV infection that is useful as a predictor of disease progression (5, 11). IFN-g itself can be difficult to measure in serum, so its production is often assessed by examining mRNA expression in cells and/or tissue, or measuring IFN-g protein secreted into cell culture supernatants. Much of the data regarding the putative shift from type 1 to type 2 responses in HIV infection has been obtained using cloned T cells, peripheral blood mononuclear cells (PBMC), and/or separated cell subpopulations that have been stimulated in vitro. Many of these studies reported reduced expression or production of IFN-g in association with HIV disease (12–15). This
IFN-g mRNA levels were measured in unstimulated PBMC and purified cell subpopulations, utilizing branched DNA assays, to characterize the cell type(s) that contribute to the in vivo increase in IFN-g gene expression seen in HIV infection. PBMC and CD8 T cells from HIV-seropositive subjects (HIV/) showed 2.5-fold increases in mean IFN-g mRNA levels compared to HIV-uninfected subjects (HIV0). Within individuals, CD8 T cells showed the highest IFN-g expression regardless of HIV status, which suggests that HIV infection enhances the IFN-g gene expression in CD8 T cells rather than inducing a shift to and/or increasing expression of IFN-g mRNA in other cell types. HIV/ subjects with increased PBMC IFN-g mRNA had elevated plasma levels of HIV RNA, neopterin, and b2microglobulin. No differences in IFN-g mRNA levels were seen among HIV/ stratified by CD4 T cell number. Increased IFN-g may result from or be a contributing factor to increased viral load. q 1997 Academic Press
INTRODUCTION Paradoxically, immune dysfunction in HIV infection has been recognized to be a combination of immune deficiency and activation (1). There is ample evidence of immune activation associated with HIV infection, as seen in the overproduction of cytokines such as IFNg, IL-1, IL-6, IL-10, and TNFa, and elevated levels of surrogate markers of immune activation like b2-microglobulin (B2M) and neopterin (NPT) (1–5). 1 This work was supported by AI36086, CA 01588 (NCI RCDA, O.M.M). E.C.B. is a postdoctoral fellow of the UCLA HIV Pathogenesis Institutional Training Grant program (T32AI07388). J.F.S.-G. was supported by an UCLA – Fogarty postdoctoral fellowship (TW00003). 2 To whom correspondence should be addressed at Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90095-1747. Fax: (310) 206-1318. E-mail: [email protected].
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0008-8749/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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has been contradicted in other studies, including our own, which have reported increased serum IFN-g levels, or increased IFN-g expression or production in unstimulated cells from HIV-infected individuals (16– 22). It is likely that this discrepancy is due to differences between freshly isolated, unstimulated cells, which presumably reflect the in vivo state of activation and cytokine gene expression, and cells which are stimulated in vitro, which are indicative of the capacity of cells to respond ex vivo. The work reported here was designed to characterize which cell type(s) contribute to increased IFN-g gene expression in vivo, and to determine whether the predominant IFN-g-producing cell type in HIV-infected individuals is different from that seen in noninfected individuals. MATERIALS AND METHODS Collection of Blood Whole blood was collected by venipuncture into EDTAcontaining sterile vacuum tubes from known HIV-seropositive (HIV/) and HIV-seronegative (HIV0) participants in the UCLA Multicenter AIDS Cohort Study (MACS). All study participants were homosexual men who have had blood samples drawn and detailed histories taken at approximately 6-month intervals since 1984 (23). Plasma samples were collected and stored at 0707C. PBMC were isolated from 36 HIV/ (mean CD4 T cells/mm3 Å 290) and 28 HIV0 subjects (mean CD4 T cells/mm3 Å 891) by centrifugation over sterile 60% Percoll gradients, and aliquots of cells were set aside for flow cytometric and mRNA analyses. When the quantity of PBMC was sufficient, additional separation of subpopulations was performed as described below. Separation and Collection of Immune Cell Subpopulations
(CD4/, depleted of monocytes), followed by a 10-min rotation with CD8 beads and collection of CD8/ cells. In light of preliminary data showing virtually no IFN-g mRNA in B cells and high levels in CD8 cells, a slightly different sequence of positive selections was used to facilitate the collection of NK cells instead of B cells. Following collection of monocytes, PBMC–CD14 were rotated with anti-CD16 monoclonal antibody (mouse IgG1 , PharMingen) at a concentration of 0.08 mg antibody/106 cells for 30–45 min. An anti-CD16 antibody was chosen for use in positive selection of NK cells in order to retrieve CD16//CD560 cells, which can be a significant percentage of NK cells in HIV infection (24). Cells were washed to remove unbound antibody and then rotated with immunobeads conjugated with goat anti-mouse IgG antibody (Dynal) at an estimated bead:target ratio of 7.5:1. NK cells (CD140, CD16/) were then collected using the MPC as with other cell subpopulations. Approximately half of monocytes are CD16/, but since they were removed by CD14 selection first, the positively selected CD16/ cells were typically ú90% NK cells by flow cytometric analysis following removal of the immunobeads by overnight culture (data not shown). The number of positively selected cells obtained was determined microscopically using a hemocytometer, and the purity was calculated by counting the number of cells with and without immunobeads bound to the surface. Typically, the positively selected cells collected using the MPC were greater than 90% pure. PBMC and cell subpopulations were pelleted by centrifugation, and the pellets were stored at 0707C until used for mRNA analyses. Immunobeads present in positively selected cell pellets did not to interfere with mRNA preparation procedures (data not shown). Isolation of Cellular mRNA Cellular mRNA was obtained from cell pellets as previously described (25). Briefly, pellets containing a known number of PBMC or purified subpopulation cells were homogenized in 8 M guanidine/HCl, and cellular RNA was precipitated in 50% ethanol at 0207C overnight. RNA pellets were obtained by centrifugation, washed with 70% ethanol, and then dissolved for 30 min at 537C in a buffer containing cytokine-specific capture and label probes as described below. No further RNA purification was necessary prior to performing cytokine mRNA assays.
PBMC were further separated into cell subpopulations using immunomagnetic beads (M-450, Dynal). All PBMC and subpopulations were resuspended and/or separated in PBS containing 2% human AB serum, with all steps performed at 47C to minimize cellular activation. In preliminary studies, positive selections were performed sequentially using directly conjugated CD14, CD19, CD4, and CD8-specific immunobeads at a bead-to-target ratio of 5:1, according to the manufacturer’s recommendations. Briefly, PBMC were continuously mixed by rotation with CD14 immunobeads for 1 hr, and then monocytes (CD14/) were collected by five rounds of exposure and washing using a magnetic particle concentrator (MPC, Dynal). The remaining cells (PBMC–CD14) were rotated with CD19 beads for 20 min, and B cells (CD19/) collected. CD4 T cells were collected next after a 30-min rotation with CD4 beads
Quantitation of IFN-g-specific mRNA in the extracts of PBMC and subpopulation cell pellets was performed utilizing a branched DNA (bDNA) signal amplification assay (Chiron Corp., Emeryville, CA), similar to those used for the quantitation of HIV-1 and hepatitis C virus RNA (26, 27). In addition, bDNA assays for TNFa, IL-
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Quantification of IFN-g mRNA by bDNA Analysis
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2, and IL-6 were performed on some PBMC and purified cells. The cytokine bDNA assays were performed directly on the RNA solutions obtained as described above, without reverse transcription or amplification of the target sequence. Aliquots of mRNA solutions containing cellular RNA plus capture and label probes were added to wells of a 96-well microtiter plate, which were coated with a synthetic oligonucleotide complementary to one end of the capture probe (28). The capture probes are designed to hybridize to target sequences within the specific cytokine mRNA of interest, and then capture the cytokine mRNA in the microwell by binding to the oligonucleotide which coats the well. The plate was incubated overnight at 537C and then washed. bDNA amplifiers were added to the wells, and the plate was incubated at 537C for 30 min to hybridize amplifiers to target–probe complexes in the microwells. Following incubation, the plate was washed, and alkaline phosphatase probes were added and allowed to hybridize to amplifier–target–probe complexes for 15 min at 537C. Finally, a chemiluminescent substrate for alkaline phosphatase was added to the wells and incubated for 30 min at 377C, which generated a light signal which was directly proportional to the amount of cytokine-specific mRNA present in the original sample. IFN-g and other cytokine mRNA results were expressed as relative luminescence units (RLU)/106 cells and so represent cytokine gene expression on a per cell basis. Determination of Plasma Levels of HIV RNA, IFN-g, and Activation Markers
FIG. 1. Mean IFN-g mRNA levels in PBMC and purified subpopulations. Mean IFN-g mRNA levels in unstimulated PBMC and purified subpopulations from HIV0 subjects (open bars) and HIV/ subjects (solid bars), expressed in relative luminescence units (RLU)/106 cells. MO, monocytes (CD14/); B, B cells (CD19/); NK, natural killer cells (CD16/); CD4 T, CD4/ T cells; CD8 T, CD8/ T cells. Error bars indicate standard error of the mean (SEM). P values for statistically significant differences between HIV0 and HIV/ as shown; all others P ú 0.05.
Statistical comparisons of results between HIV0 and HIV/ subjects, and between different groups within the HIV/ subjects were performed using a paired t test, linear regression, and/or correlation, all of which are components of the Excel software program (Microsoft). RESULTS
The rate of loss of CD4 T cells (CD4 slope) was determined by calculating the slope of the line defined by plotting the log of absolute CD4 T cell number for each visit vs time over the 3 years up to and including the visit used for mRNA analysis. Subjects were included in this analysis only if there were data available for a minimum of four visits.
IFN-g mRNA levels in freshly isolated, unstimulated PBMC were elevated in HIV/ subjects when compared to HIV0 subjects, with a 2.5-fold increase in the mean level of IFN-g mRNA (Fig. 1). Although this is a modest increase, it was highly statistically significant (P Å 0.000015), due to the number of subjects (HIV/, n Å 36; HIV0, n Å 28) and the small standard error, as shown in Fig. 1. It is also important to recognize that because the PBMC were not stimulated, the mRNA data reflect in vivo gene expression, which is not likely to be as great as in cells stimulated ex vivo. The increase in IFN-g gene expression in PBMC of HIV/ subjects was seen in parallel with significantly elevated circulating levels of plasma IFN-g, and elevated plasma NPT, which is considered a surrogate marker for IFN-g activity (5, 11) (Table 1). Although all three of these measurements were significantly elevated in HIV/ subjects as a group, there were no direct correlations between PBMC IFN-g mRNA levels and either plasma IFN-g or NPT levels on an individual basis (data not shown). The CD8 T cell subpopulation was the only purified cell subpopulation which showed increased levels of IFN-g gene expression in HIV/ subjects (Fig. 1). HIV/ subjects (n Å 18) showed a 2.6-fold higher mean level of IFN-g mRNA compared to HIV0 subjects (n Å 11), which is similar in magnitude to the increase seen in
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HIV RNA bDNA assays were performed on EDTA plasma samples to determine viral load, with results expressed as viral kiloequivalents per milliliter of plasma (26). The level of detection of the assay was 0.5 keq/ml, which is approximately equivalent to 500 copies of HIV RNA/ml plasma. Plasma levels of IFN-g were measured using a modified ELISA protocol (T Cell Sciences) which was capable of detecting 50 units/liter. Plasma levels were also determined for three surrogate markers of immune activation: b2-microglobulin (IMx Microparticle Enzyme Immunoassay, Abbott), neopterin (HENNINGtest, Henning Berlin GMBH), and soluble tumor necrosis factor receptor type II (TNFaRII, Quantikine EIA, R&D Systems). Calculations and Statistics
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TABLE 1 Mean Cell Counts and Plasma Values in HIV-Seronegative and Seropositive Subjectsa
HIV serostatus
Absolute CD4 T cells/ml
Absolute CD8 T cells/ml
Plasma HIV RNA, keq/ml
Plasma IFN-g, U/liter
Plasma neopterin, nM
Plasma b2-microglobulin, mg/liter
Plasma sTNF-RII, ng/ml
0, n Å 28 /, n Å 36 P valueb
891 (50) 290 (34) 3 1 10012
583 (40) 945 (89) 0.0005
õ0.5 (n/ac) 96 (34) 0.008
111 (11) 325 (64) 0.002
5.7 (0.3) 19.4 (2.5) 4 1 1006
1.2 (0.04) 2.7 (0.2) 7 1 10010
2.1 (0.1) 4.5 (0.4) 3 1 1006
a b c
All data shown are mean value (standard error of the mean). P value from two-tailed t test for comparison of all HIV0 and HIV/ subjects. Not applicable (SEM cannot be calculated due to mean value õ0.5).
PBMC. As in the PBMC, the differences in IFN-g mRNA between HIV/ and HIV0 CD8 T cells were clearly statistically significant (P Å 0.003), even with fewer subjects and slightly larger standard errors. No statistically significant differences between HIV/ and HIV0 subjects were seen in IFN-g mRNA levels in either monocytes or B cells, which typically showed little or no IFN-g gene expression. Likewise, there were no significant differences in HIV/ and HIV0 IFN-g mRNA levels in either NK cells or CD4 T cells. This was not due to a lack of IFN-g expression or inability to detect expression because both of these subpopulations usually showed moderate levels of IFN-g mRNA, similar to those seen in unfractionated PBMC. Because the bDNA result is determined on a per cell basis, the increase seen in the purified CD8 T cells is not attributable to an increased absolute number of CD8 T cells seen in HIV/ subjects, but to an overall increase in the amount of gene expression in CD8 T cells. This is consistent with the result that there was no correlation between the absolute number of circulating CD8 T cells and the IFN-g mRNA level in either PBMC or CD8 T cells in HIV/ individuals (data not shown). However, there was a significant positive correlation between PBMC IFN-g mRNA and CD8 T cell IFN-g mRNA levels within HIV/ individuals (r Å 0.54, P Å 0.02, data not shown). This observation strongly suggests that increased IFN-g gene expression in the CD8 T cells is the driving force behind the overall increase in IFN-g mRNA levels seen in HIV/ PBMC. CD8 T cells consistently showed the highest IFN-g mRNA level when compared to PBMC and CD4 T cells of the same individual, regardless of HIV status. In 23 of 24 subjects for whom PBMC, CD4, and CD8 IFN-g mRNA data were available, the CD8 T cells had the highest IFN-g mRNA level (data not shown). In a subset of 14 individuals who had a complete set of PBMC, NK, CD4, and CD8 T cell data (HIV/, n Å 8; HIV0, n Å 6), 11/14 had the highest IFN-g mRNA levels in CD8/ cells (Fig. 2). Two of the remaining three (one HIV/, one HIV0) showed slightly higher mRNA levels in the NK cells than the CD8 T cells, and one (HIV/) had CD4 T cell IFN-g mRNA levels marginally higher
than CD8 T cells. Therefore, even when the CD8 T cells did not have the highest IFN-g mRNA levels, they expressed levels very similar to the highest IFN-g-expressing cell type. The consistent observation of CD8 T cells as major contributors to IFN-g expression in both HIV/ and HIV0 subjects shows that the type of cell responsible for much of the IFN-g gene expression generally does not change as a result of HIV infection. Rather, the level of gene expression rises on a per cell basis within the CD8 T cells in HIV-infected persons. We were interested to see if stratification of the HIV/ subjects, according to various measurements of disease status and/ or progression, would be reflected in the level of IFN-g expression in PBMC and/or CD8 T cells. HIV/ subjects were stratified according to various results obtained from the same blood draw (NPT, B2M, HIV viral load, CD4 T cell number) or on the basis of longitudinal data available from the MACS database (time since seroconversion, rate of loss of CD4 T cells), and levels of IFN-g mRNA in different groups were then compared. For NPT and B2M, subjects were divided into normal (5th to 95th percentile) and elevated (ú95th percentile) based on adult reference ranges established by the Clinical Immunology Research Laboratories at UCLA, where the assays were performed. For plasma viral load (HIV RNA), subjects were divided into above and below 10 keq/ml, based on reports which showed a strong correlation between HIV RNA ú 10 keq/ml and development of AIDS and/or poor survival (29, 30). Length of time of seropositivity was selected to group subjects into recent seroconverters (6–18 months postseroconversion) and longer-term seropositives (ú3 years, including those who were seropositive upon entry into the MACS). IFN-g gene expression was significantly increased in the PBMC of HIV/ subjects with elevated markers of immune activation (NPT or B2M, Figs. 3A and 3B), higher HIV viral load (HIV RNA, Fig. 3C), or who had been seropositive ú3 years (Fig. 3D), when compared to HIV/ subjects with lower values or who had been HIV seropositive 6–18 months (P õ 0.05). When each subgroup of HIV/ subjects was compared to HIV0 sub-
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FIG. 2. Individual IFN-g mRNA levels in PBMC and purified subpopulations. (A) IFN-g mRNA levels in all HIV0 subjects with data from PBMC, NK, CD4 T, and CD8 T cells (n Å 6). (B) IFN-g mRNA levels in all HIV/ subjects with data from PBMC, NK, CD4 T, and CD8 T cells (n Å 8). Open shapes indicate subjects 6–18 months post-HIV seroconversion; filled shapes indicate subjects ú3 years postHIV seroconversion.
jects, the HIV/ subjects with normal plasma NPT or B2M, low HIV RNA, or who were 6–18 months post seroconversion consistently showed PBMC IFNg mRNA levels which were indistinguishable from the HIV0 subjects (P ú 0.05). In contrast, the HIV/ subjects with high plasma NPT, B2M, or HIV RNA or who were longer-term seroconverters showed highly significant increases in PBMC IFN-g mRNA (P õ 0.0002). No statistically significant differences were seen when PBMC IFN-g mRNA levels were compared between HIV/ subjects stratified on the basis of CD4 T cell number at the time of the visit or rate of loss of CD4 T cells over the previous 3 years (data not shown). However, HIV/ subjects with 200–500 or õ200 CD4 T cells/mm3 did show elevated PBMC IFN-g mRNA when compared to HIV0 subjects. IFN-g mRNA was also significantly increased in the CD8 T cells of HIV/ subjects with higher plasma levels of NPT, B2M, or HIV RNA, when compared to HIV0 subjects (P £ 0.02, data not shown). As with the PBMC, HIV/ subjects with normal NPT or B2M or lower HIV RNA had CD8 IFN-g mRNA levels which were indistinguishable from HIV0 subjects. The only stratification which resulted in significant differences in CD8 IFN-g mRNA levels between groups of HIV/ subjects was time since seroconversion, with longer-term seroconverters showing elevated CD8 IFN-g mRNA levels compared to two short-term seroconverters for whom
CD8 data were available (P Å 0.007, data not shown). In spite of this difference, both short- and long-term seroconverters showed significantly elevated CD8 IFNg mRNA levels when compared to HIV0 subjects (P Å 0.02 and 0.001, respectively). In addition to IFN-g mRNA, data were obtained for TNFa, IL-2, and IL-6 mRNA by bDNA analysis on a subset of PBMC and purified subpopulation samples. As expected, the major cell subpopulation expressing TNFa mRNA in both HIV0 and HIV/ subjects was the monocytes, with very small amounts of TNFa mRNA detected in CD4 or CD8 T cells (data not shown). There were no statistically significant differences seen in levels of TNFa mRNA from the PBMC or monocytes of HIV0 and HIV/ subjects (data not shown). In spite of the lack of differences in TNFa expression at the mRNA level, there was a highly significant elevation in the plasma levels of the soluble TNFa receptor II in HIV/ subjects (Table 1). In preliminary studies with five HIV0 and five HIV/ subjects, no statistically significant differences in PBMC IL-2 or IL-6 mRNA between HIV0 and HIV/ subjects were seen (data not shown). It was intriguing to note, however, that three of the five seropositives had undetectable levels of IL2 mRNA and the remaining two were barely above background, while all five of the seronegatives had measurable levels clearly above background. Further studies are planned to extend these initial observations
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FIG. 3. PBMC IFN-g mRNA levels in HIV0 subjects, and HIV/ subjects stratified by various criteria. Comparison of PBMC IFN-g mRNA levels in HIV0 subjects, and HIV/ subjects stratified by (A) neopterin (NPT), (B) b2-microglobulin (B2M), or (C) HIV viral load (HIV RNA), determined on plasma from same blood draw as mRNA analysis, or (D) time since HIV seroconversion. P values are shown for comparisons of IFN-g mRNA levels of each HIV/ group to HIV0 subjects.
utilizing bDNA technology to investigate the expression of these additional cytokines in unstimulated cells.
The results presented here confirm previous reports of increased expression and/or production of IFN-g in unstimulated mononuclear cells from the circulation and/or lymph nodes of HIV-infected subjects (17–22). More importantly, we have extended these observations to elucidate the cell type responsible for IFN-g gene expression in peripheral blood in HIV/ persons, and to determine if it is different from the predominant cell type in HIV0 individuals. By quantitating and comparing IFN-g mRNA in positively selected cell subpopulations that represent highly enriched monocytes, B, NK, CD4 T, and CD8 T cells, we have shown that the major contributors to the overall level of IFN-g expression appear to be the CD8 T cells in both HIV/ and HIV0 individuals. Although Emilie et al. have reported IFN-g-expressing CD8 T cells in HIV/ lymph nodes (17, 18), and others have described greater expression or secretion of IFN-g in stimulated or cloned peripheral CD8 T cells compared to CD4 T cells (10, 31, 32), it has not been previously shown which cell type was the predominant one expressing IFN-g in peripheral blood.
By examining freshly isolated, unstimulated PBMC and subpopulations, our data reflect the in vivo state of IFN-g expression in the circulation, rather than the ability of cells to respond to ex vivo stimulation. It clearly shows that the increased IFN-g expression in HIV infection is primarily attributable to the same type of cell, CD8 T cells (CD8-positive, depleted of NK cells), that is the major contributor to the lower levels of IFNg mRNA seen in HIV0 persons. In other words, HIV infection does not cause a shift to and/or increased expression of IFN-g mRNA in other cell types which are known to make IFN-g, i.e., CD8-positive NK cells or type 1 CD4 T cells (33, 34), but instead enhances or exaggerates the IFN-g response by the CD8 T cells. It is important to note that the observed increase in HIV/ PBMC IFN-g mRNA is not due to the increased absolute numbers of CD8 T cells associated with HIV infection, since results are expressed as IFN-g gene expression per million CD8 T cells analyzed. In light of the recent descriptions of subtypes of cytokine-secreting CD8 T cells, TC1 and TC2, similar to CD4 TH1 and TH2 subsets (9, 10), the increase in IFN-g mRNA in CD8 T cells would suggest either that existing TC1 cells have been hyperactivated to dramatically upregulate IFNg expression or there is an increase in the proportion of CD8 T cells which have differentiated into
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DISCUSSION
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the IFN-g-secreting TC1 type. Ex vivo stimulated cells from HIV-infected persons show increased percentages of cells making IFN-g (32), which favors the latter possibility, but it is not known if the same would be seen in unstimulated cells. This also raises the question of the possibility of differential IFN-g gene expression among subsets of CD8 T cells identified by cell surface phenotype. Since the phenotypic distribution of CD8 T cells has been shown to change in HIV infection, with increases in CD38/DR/ and decreases in CD28/ cells (35–40), determination of IFN-g mRNA levels in CD8 T cell subsets in HIV/ and HIV0 persons might shed some light on the relationship between phenotypically defined and TC1/TC2 subsets. Additional CD8 T cell studies are planned or underway to address these questions. Our observations of increased levels of IFN-g mRNA in unstimulated circulating cells, as well as increased levels of plasma IFN-g and NPT in HIV/ individuals as a group indicate that there is an in vivo increase in IFN-g gene expression and production in HIV infection. However, not all HIV/ subjects exhibited increased IFN-g mRNA compared to HIV0 subjects. When HIV/ subjects were stratified on the basis of criteria other than CD4 T cell number, elevated IFN-g expression was clearly and consistently associated with results indicative of more advanced HIV disease, i.e., higher plasma HIV RNA, longer time of infection, and increased immune activation (Fig. 3). This suggests that the hypothesis that the loss of type 1 cytokine production is associated with the development of HIV disease (7, 8) may not be applicable to all type 1 cytokines. The hypothesis may be supported by numerous reports of decreased production of type 1 cytokines such as IL-2 and IL-12 in HIV infection and disease (7, 8, 41–43), but is not consistent with our observations of increased, not decreased, expression of one of the other type 1 cytokines, IFN-g. Virtually all elevated PBMC or CD8 IFN-g mRNA levels (compared to HIV0 subjects) were found in HIV/ subjects with plasma HIV RNA ú 10 keq/ml, a viral load which has been strongly associated with development of AIDS and/or poor survival (29, 30). While there was not a direct correlation between viral load and PBMC or CD8 IFN-g mRNA levels within individuals (data not shown), the consistent finding of elevated IFN-g expression in those HIV/ subjects with higher viral load suggests that these two parameters may be influencing one another. It is interesting to note that two of the three HIV/ who were 6–18 months postHIV seroconversion (short-term seroconverters) had HIV RNA levels above 10 keq/ml and yet had PBMC IFN-g mRNA levels comparable to HIV0 subjects. However, even at this relatively early time point in HIV disease, IFN-g mRNA levels appear to be rising in subpopulations because short-term seroconverters showed elevated CD8 T cell levels (data not shown) and ac-
counted for two of the three highest NK cell levels detected (Fig. 2). IFN-g gene expression may begin to increase early in disease course, but requires additional time before any increases can be detected in PBMC. Increased IFN-g mRNA was also seen in those HIV/ subjects with increased immune activation, as indicated by abnormally high levels of either plasma NPT or B2M. Elevated levels of one or both of these markers are strong prognostic indicators of more rapid progression of HIV disease (5). Since NPT reflects IFN-g activity, it is not surprising that HIV/ individuals with high NPT levels showed elevated IFN-g expression. However, the additional observation of elevated IFN-g expression in HIV/ subjects with increased B2M, which is a more broad indicator of immune activation, shows that increased IFN-g expression is associated with immune activation in general during HIV infection. Overall, these results suggest that increased IFN-g gene expression may be influencing HIV viral load and/or immune activation over time and therefore could be a direct contributor to the pathogenesis of HIV disease. Alternatively, increased IFN-g gene expression may reflect the effects of increasing viral loads and immune dysregulation in the course of HIV disease. These issues are not addressed directly in this cross-sectional study, but are under investigation in longitudinal studies of individual MACS subjects utilizing cryopreserved PBMC. The results presented here demonstrate the utility of the bDNA assay to detect differences in IFN-g mRNA in freshly isolated, unstimulated PBMC of HIVinfected and uninfected homosexual men. bDNA technology for the measurement of cytokine mRNA is capable of determining IFN-g, TNFa, IL-2, and IL-6 expression in unstimulated PBMC from most individuals, regardless of HIV status. Evaluation of cytokine gene expression in freshly isolated, unstimulated cells is preferable to measurements in cells which have been stimulated in vitro because this better reflects the status of cytokine expression in vivo. Although the bDNA procedure may require greater cell numbers than reverse transcription–polymerase chain reaction assays, the bDNA methodology may be better suited for truly quantitative determination of cytokine mRNA levels, since it does not rely on either reverse transcription or amplification of the target mRNA (26).
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ACKNOWLEDGMENTS We gratefully acknowledge the dedication of the men who are participants at UCLA in the Multicenter AIDS Cohort Study, without whom this and many other studies would never have been possible. We also thank Dr. R. Detels and Dr. P. Nishanian for facilitating collection of blood samples, Dr. S. O. Derzic for technical discussions, N. Aziz for performing plasma assays, M. Gorre for flow cytometry analyses, and M. McDonald for providing laboratory support.
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REFERENCES 1. Pantaleo, G., and Fauci, A. S., Annu. Rev. Immunol. 113, 487, 1995. 2. Poli, G., Biswas, P., and Fauci, A. S., Antiviral Res. 24, 221, 1994. 3. Miedema, F., Meyaard, L., Koot, M., Klein, M. R., Roos, M. T., Groenink, M., Fouchier, R. A., Van’t Wout, A. B., Tersmette, M., Schellekens, P. T., and Schuitemaker, H., Immunol. Rev. 140, 35, 1994. 4. Breen, E. C., Rezai, A. R., Nakajima, K., Hirano, T., Beall, G. N., Mitsuyasu, R. T., Kishimoto, T., and Martinez-Maza, O., J. Immunol. 144, 480, 1990. 5. Fahey, J. L., Taylor, J. M. G., Detels, R., Hofmann, B., Melmed, R. N., Nishanian, P., and Giorgi, J. V., N. Engl. J. Med. 322, 166, 1990. 6. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., and Coffman, R. L., J. Immunol. 136, 2345, 1986. 7. Clerici, M., and Shearer, G. M., Immunol. Today 15, 575, 1994. 8. Clerici, M., and Shearer, G. M., Immunol. Today 14, 107, 1993. 9. Sad, S., Marcotte, R., and Mosmann, T. R., Immunity 2, 271, 1995. 10. Noble, A., Macary, P. A., and Kemeny, D. M., J. Immunol. 155, 2928, 1995. 11. Melmed, R. N., Taylor, J. M. G., Detels, R., Bozorgmehri, M., and Fahey, J. L., J. Acquired Immune Defic. Syndr. 2, 70, 1989. 12. Barcellini, W., Rizzardi, G. P., Borghi, M. O., Fain, C., Lazzarin, A., and Meroni, P. L., AIDS 8, 757, 1994. 13. Maggi, E., Mazzetti, M., Ravina, A., Annunziato, F., de Carli, M., Piccinni, M. P., Manetti, R., Carbonari, M., Pesce, A. M., del Prete, G., and Romagnani, S., Science 265, 244, 1994. 14. Diaz-Mitoma, F., Kumar, A., Karimi, S., Kryworuchko, M., Daftarian, M. P., Creery, W. D., Filion, L. G., and Cameron, W., Clin. Exp. Immunol. 102, 31, 1995. 15. Vigano, A., Principi, N., Villa, M. L., Riva, C., Crupi, L., Trabattoni, D., Shearer, G. M., and Clerici, M., J. Pediatr. 126, 368, 1995. 16. Fuchs, D., Hausen, A., Reibnegger, G., Werner, E. R., WernerFelmayer, G., Dierich, M. P., and Wachter, H., J. Acquired Immune Defic. Syndr. 2, 158, 1989. 17. Emilie, D., Peuchmaur, M., Maillot, M. C., Crevon, M. C., Brousse, N., Delfraissy, J. F., Dormont, J., and Galanaud, P., J. Clin. Invest. 86, 148, 1990. 18. Emilie, D., Fior, R., Llorente, L., Marfaing-Koka, A., Peuchmaur, M., Devergne, O., Jarrousse, B., Wijdenes, J., Boue, F., and Galanaud, P., Immunol. Rev. 140, 5, 1994. 19. Boyle, M. J., Berger, M. F., Tschuchnigg, M., Valentine, J. E., Kennedy, B. G., Divjak, M., Cooper, D. A., Turner, J. J., Penny, R., Sewell, W. A., Clin. Exp. Immunol. 92, 100, 1993. 20. Fan, J., Bass, H. Z., and Fahey, J. L., J. Immunol. 151, 5031, 1993. 21. Graziosi, C., Pantaleo, G., Gantt, K. R., Fortin, J. P., Demarest, J. F., Cohen, O. J., Sekaly, R. P., and Fauci, A. S., Science 265, 248, 1994.
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22. Navikas, V., Link, J., Wahren, B., Persson, C., and Link, H., Clin. Exp. Immunol. 96, 59, 1994. 23. Kaslow, R. A., Ostrow, D. G., Detels, R., Phair, J. P., Polk, B. F., and Rinaldo, C. R., Am. J. Epidemiol. 126, 126, 1987. 24. Hu, P. F., Hultin, L. E., Hultin, P., Hausner, M. A., Hirji, K., Jewett, A., Bonavida, B., Detels, R., and Giorgi, J. V., J. Acquired Immune Defic. Syndr. 10, 331, 1995. 25. Dailey, P. J., Wilber, J. C., Collins, M. C., Allumbaugh, T., and Urdea, M. S., ‘‘Interscience Conference on Antimicrobial Agents and Chemotherapy,’’ Orlando, FL, 1994. 26. Pachl, C., Todd, J. A., Kern, D. G., Sheridan, P. J., Fong, S. J., Stempien, M., Hoo, B., Besemer, D., Yeghiazarian, T., Irvine, B., Kolberg, J., Kokka, R., Neuwald, P., and Urdea, M. S., J. Acquired Immune Defic. Syndr. 8, 446, 1995. 27. Detmer, J., Lagier, R., Flynn, J., Zayati, C., Kolberg, J., Collins, M., Urdea, M., and Sanchez-Pescador, R., J. Clin. Microbiol. 34, 901, 1996. 28. Running, J. A., and Urdea, M. S., BioTechniques 8, 276, 1990. 29. Mellors, J. W., Kingsley, L. A., Rinaldo, C. R., Todd, J. A., Hoo, B. S., Kokka, R. P., and Gupta, P., Ann. Intern. Med. 122, 573, 1995. 30. Mellors, J. W., Rinaldo, C. R., Gupta, P., White, R. M., Todd, J. A., and Kingsley, L. A., Science 272, 1167, 1996. 31. Fong, T. A., and Mosmann, T. R., J. Immunol. 144, 1744, 1990. 32. Caruso, A., Canaris, A. D., Licenziati, S., Cantalamessa, A., Folghera, S., Lonati, M. A., de Panfilis, G., Garotta, G., and Turano, A., J. Acquired Immune Defic. Syndr. 10, 462, 1995. 33. Toth, F. D., Mosborg-Petersen, P., Kiss, J., Aboagye-Mathiesen, G., Zdravkovic, M., Hager, H., and Ebbesen, P., J. Virol. 67, 5879, 1993. 34. Swain, S., J. Leukocyte Biol. 57, 795, 1995. 35. Giorgi, J. V., Liu, Z., Hultin, L. E., Cumberland, W. G., Hennessey, K., and Detels, R., J. Acquired Immune Defic. Syndr. 6, 904, 1993. 36. Ho, H. N., Hultin, L. E., Mitsuyasu, R. T., Matud, J. L., Hausner, M. A., Bockstoce, D., Chou, C. C., O’Rourke, S., Taylor, J. M., and Giorgi, J. V., J. Immunol. 150, 3070, 1993. 37. Caruso, A., Cantalamessa, A., Licenziati, S., Peroni, L., Prati, E., Martinelli, F., Canaris, A. D., Folghera, S., Gorla, R., Balsari, A., et al., Scand. J. Immunol. 40, 485, 1994. 38. Choremi-Papadopoulou, H., Viglis, V., Gargalianos, P., Kordossis, T., Iniotaki-Theodoraki, A., and Kosmidis, J., J. Acquired Immune Defic. Syndr. 7, 245, 1994. 39. Brinchmann, J. E., Dobloug, J. H., Heger, B. H., Haaheim, L. L., Sannes, M., and Egeland, T., J. Infect. Dis. 169, 730, 1994. 40. Vingerhoets, J. H., Vanham, G. L., Kestens, L. L., Penne, G. G., Colebunders, R. L., Vandenbruaene, M. J., Goeman, J., Gigase, P. L., De Boer, M., and Ceuppens, J. L., Clin. Exp. Immunol. 100, 425, 1995. 41. Kinter, A., and Fauci, A. S., Immunol. Res. 15, 1, 1996. 42. Chehimi, J., and Trinchieri, G., J. Clin. Immunol. 14, 149, 1994. 43. Chougnet, C., Wynn, T. A., Clerici, M., Landay, A. L., Kessler, H. A., Rusnak, J., Melcher, G. P., Sher, A., and Shearer, G. M., J. Infect. Dis. 174, 46, 1996.
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Circulating CD8 T Cells Show Increased Interferon-g mRNA Expression in HIV Infection1 Elizabeth Crabb Breen,*,2 Jesus F. Salazar-Gonzalez,† Lu Ping Shen,‡ Janice A. Kolberg,‡ Mickey S. Urdea,‡ Otoniel Martinez-Maza,*,§ and John L. Fahey*,§ *Department of Microbiology and Immunology and §Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095-1747; †Facultad de Ciencias Quimicas/CIEP, Universidad Auto´noma de San Luis Potosı´, San Luis Potosı´, Mexico; and ‡Chiron Corporation, Emeryville, California Received February 5, 1997; accepted March 17, 1997
The immune deficiency associated with HIV infection is the result of the loss of the number and function of CD4 T helper cells. These cells also have been the subject of great scrutiny in HIV infection because of their contribution to cytokine production. CD4 T cells can generally be divided into two types (type 1 and type 2) according to the pattern of cytokines secreted (6, 7). It has been suggested that a shift away from the production of type 1 cytokines could play a role in the progression of HIV disease (7, 8). Recently, similar patterns of cytokine production were described in CD8 T cells (9, 10). Type 1 cells typically produce IL-2 and IFN-g and are stimulated by IL-12. Type 2 cells produce and/ or respond to IL-4, IL-5, IL-6, and IL-10 and are downregulated by IL-12. It must be remembered, however, that contributions to type 1 and 2 responses can be made by other non-T cell types such as monocyte/macrophages and natural killer (NK) cells. Thus, in an examination of contributions to immune activation/cytokine production, all cell types should be considered. IFN-g is a cytokine that can be produced by multiple cell types, and is considered to enhance cellular immune responses by activation of monocytes and macrophages (2). It is one of the type 1 cytokines that is thought to contribute to effective cell-mediated immunity. IFN-g drives the production of neopterin, an activation marker detectable in serum/plasma which is elevated in HIV infection that is useful as a predictor of disease progression (5, 11). IFN-g itself can be difficult to measure in serum, so its production is often assessed by examining mRNA expression in cells and/or tissue, or measuring IFN-g protein secreted into cell culture supernatants. Much of the data regarding the putative shift from type 1 to type 2 responses in HIV infection has been obtained using cloned T cells, peripheral blood mononuclear cells (PBMC), and/or separated cell subpopulations that have been stimulated in vitro. Many of these studies reported reduced expression or production of IFN-g in association with HIV disease (12–15). This
IFN-g mRNA levels were measured in unstimulated PBMC and purified cell subpopulations, utilizing branched DNA assays, to characterize the cell type(s) that contribute to the in vivo increase in IFN-g gene expression seen in HIV infection. PBMC and CD8 T cells from HIV-seropositive subjects (HIV/) showed 2.5-fold increases in mean IFN-g mRNA levels compared to HIV-uninfected subjects (HIV0). Within individuals, CD8 T cells showed the highest IFN-g expression regardless of HIV status, which suggests that HIV infection enhances the IFN-g gene expression in CD8 T cells rather than inducing a shift to and/or increasing expression of IFN-g mRNA in other cell types. HIV/ subjects with increased PBMC IFN-g mRNA had elevated plasma levels of HIV RNA, neopterin, and b2microglobulin. No differences in IFN-g mRNA levels were seen among HIV/ stratified by CD4 T cell number. Increased IFN-g may result from or be a contributing factor to increased viral load. q 1997 Academic Press
INTRODUCTION Paradoxically, immune dysfunction in HIV infection has been recognized to be a combination of immune deficiency and activation (1). There is ample evidence of immune activation associated with HIV infection, as seen in the overproduction of cytokines such as IFNg, IL-1, IL-6, IL-10, and TNFa, and elevated levels of surrogate markers of immune activation like b2-microglobulin (B2M) and neopterin (NPT) (1–5). 1 This work was supported by AI36086, CA 01588 (NCI RCDA, O.M.M). E.C.B. is a postdoctoral fellow of the UCLA HIV Pathogenesis Institutional Training Grant program (T32AI07388). J.F.S.-G. was supported by an UCLA – Fogarty postdoctoral fellowship (TW00003). 2 To whom correspondence should be addressed at Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90095-1747. Fax: (310) 206-1318. E-mail: [email protected].
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has been contradicted in other studies, including our own, which have reported increased serum IFN-g levels, or increased IFN-g expression or production in unstimulated cells from HIV-infected individuals (16– 22). It is likely that this discrepancy is due to differences between freshly isolated, unstimulated cells, which presumably reflect the in vivo state of activation and cytokine gene expression, and cells which are stimulated in vitro, which are indicative of the capacity of cells to respond ex vivo. The work reported here was designed to characterize which cell type(s) contribute to increased IFN-g gene expression in vivo, and to determine whether the predominant IFN-g-producing cell type in HIV-infected individuals is different from that seen in noninfected individuals. MATERIALS AND METHODS Collection of Blood Whole blood was collected by venipuncture into EDTAcontaining sterile vacuum tubes from known HIV-seropositive (HIV/) and HIV-seronegative (HIV0) participants in the UCLA Multicenter AIDS Cohort Study (MACS). All study participants were homosexual men who have had blood samples drawn and detailed histories taken at approximately 6-month intervals since 1984 (23). Plasma samples were collected and stored at 0707C. PBMC were isolated from 36 HIV/ (mean CD4 T cells/mm3 Å 290) and 28 HIV0 subjects (mean CD4 T cells/mm3 Å 891) by centrifugation over sterile 60% Percoll gradients, and aliquots of cells were set aside for flow cytometric and mRNA analyses. When the quantity of PBMC was sufficient, additional separation of subpopulations was performed as described below. Separation and Collection of Immune Cell Subpopulations
(CD4/, depleted of monocytes), followed by a 10-min rotation with CD8 beads and collection of CD8/ cells. In light of preliminary data showing virtually no IFN-g mRNA in B cells and high levels in CD8 cells, a slightly different sequence of positive selections was used to facilitate the collection of NK cells instead of B cells. Following collection of monocytes, PBMC–CD14 were rotated with anti-CD16 monoclonal antibody (mouse IgG1 , PharMingen) at a concentration of 0.08 mg antibody/106 cells for 30–45 min. An anti-CD16 antibody was chosen for use in positive selection of NK cells in order to retrieve CD16//CD560 cells, which can be a significant percentage of NK cells in HIV infection (24). Cells were washed to remove unbound antibody and then rotated with immunobeads conjugated with goat anti-mouse IgG antibody (Dynal) at an estimated bead:target ratio of 7.5:1. NK cells (CD140, CD16/) were then collected using the MPC as with other cell subpopulations. Approximately half of monocytes are CD16/, but since they were removed by CD14 selection first, the positively selected CD16/ cells were typically ú90% NK cells by flow cytometric analysis following removal of the immunobeads by overnight culture (data not shown). The number of positively selected cells obtained was determined microscopically using a hemocytometer, and the purity was calculated by counting the number of cells with and without immunobeads bound to the surface. Typically, the positively selected cells collected using the MPC were greater than 90% pure. PBMC and cell subpopulations were pelleted by centrifugation, and the pellets were stored at 0707C until used for mRNA analyses. Immunobeads present in positively selected cell pellets did not to interfere with mRNA preparation procedures (data not shown). Isolation of Cellular mRNA Cellular mRNA was obtained from cell pellets as previously described (25). Briefly, pellets containing a known number of PBMC or purified subpopulation cells were homogenized in 8 M guanidine/HCl, and cellular RNA was precipitated in 50% ethanol at 0207C overnight. RNA pellets were obtained by centrifugation, washed with 70% ethanol, and then dissolved for 30 min at 537C in a buffer containing cytokine-specific capture and label probes as described below. No further RNA purification was necessary prior to performing cytokine mRNA assays.
PBMC were further separated into cell subpopulations using immunomagnetic beads (M-450, Dynal). All PBMC and subpopulations were resuspended and/or separated in PBS containing 2% human AB serum, with all steps performed at 47C to minimize cellular activation. In preliminary studies, positive selections were performed sequentially using directly conjugated CD14, CD19, CD4, and CD8-specific immunobeads at a bead-to-target ratio of 5:1, according to the manufacturer’s recommendations. Briefly, PBMC were continuously mixed by rotation with CD14 immunobeads for 1 hr, and then monocytes (CD14/) were collected by five rounds of exposure and washing using a magnetic particle concentrator (MPC, Dynal). The remaining cells (PBMC–CD14) were rotated with CD19 beads for 20 min, and B cells (CD19/) collected. CD4 T cells were collected next after a 30-min rotation with CD4 beads
Quantitation of IFN-g-specific mRNA in the extracts of PBMC and subpopulation cell pellets was performed utilizing a branched DNA (bDNA) signal amplification assay (Chiron Corp., Emeryville, CA), similar to those used for the quantitation of HIV-1 and hepatitis C virus RNA (26, 27). In addition, bDNA assays for TNFa, IL-
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2, and IL-6 were performed on some PBMC and purified cells. The cytokine bDNA assays were performed directly on the RNA solutions obtained as described above, without reverse transcription or amplification of the target sequence. Aliquots of mRNA solutions containing cellular RNA plus capture and label probes were added to wells of a 96-well microtiter plate, which were coated with a synthetic oligonucleotide complementary to one end of the capture probe (28). The capture probes are designed to hybridize to target sequences within the specific cytokine mRNA of interest, and then capture the cytokine mRNA in the microwell by binding to the oligonucleotide which coats the well. The plate was incubated overnight at 537C and then washed. bDNA amplifiers were added to the wells, and the plate was incubated at 537C for 30 min to hybridize amplifiers to target–probe complexes in the microwells. Following incubation, the plate was washed, and alkaline phosphatase probes were added and allowed to hybridize to amplifier–target–probe complexes for 15 min at 537C. Finally, a chemiluminescent substrate for alkaline phosphatase was added to the wells and incubated for 30 min at 377C, which generated a light signal which was directly proportional to the amount of cytokine-specific mRNA present in the original sample. IFN-g and other cytokine mRNA results were expressed as relative luminescence units (RLU)/106 cells and so represent cytokine gene expression on a per cell basis. Determination of Plasma Levels of HIV RNA, IFN-g, and Activation Markers
FIG. 1. Mean IFN-g mRNA levels in PBMC and purified subpopulations. Mean IFN-g mRNA levels in unstimulated PBMC and purified subpopulations from HIV0 subjects (open bars) and HIV/ subjects (solid bars), expressed in relative luminescence units (RLU)/106 cells. MO, monocytes (CD14/); B, B cells (CD19/); NK, natural killer cells (CD16/); CD4 T, CD4/ T cells; CD8 T, CD8/ T cells. Error bars indicate standard error of the mean (SEM). P values for statistically significant differences between HIV0 and HIV/ as shown; all others P ú 0.05.
Statistical comparisons of results between HIV0 and HIV/ subjects, and between different groups within the HIV/ subjects were performed using a paired t test, linear regression, and/or correlation, all of which are components of the Excel software program (Microsoft). RESULTS
The rate of loss of CD4 T cells (CD4 slope) was determined by calculating the slope of the line defined by plotting the log of absolute CD4 T cell number for each visit vs time over the 3 years up to and including the visit used for mRNA analysis. Subjects were included in this analysis only if there were data available for a minimum of four visits.
IFN-g mRNA levels in freshly isolated, unstimulated PBMC were elevated in HIV/ subjects when compared to HIV0 subjects, with a 2.5-fold increase in the mean level of IFN-g mRNA (Fig. 1). Although this is a modest increase, it was highly statistically significant (P Å 0.000015), due to the number of subjects (HIV/, n Å 36; HIV0, n Å 28) and the small standard error, as shown in Fig. 1. It is also important to recognize that because the PBMC were not stimulated, the mRNA data reflect in vivo gene expression, which is not likely to be as great as in cells stimulated ex vivo. The increase in IFN-g gene expression in PBMC of HIV/ subjects was seen in parallel with significantly elevated circulating levels of plasma IFN-g, and elevated plasma NPT, which is considered a surrogate marker for IFN-g activity (5, 11) (Table 1). Although all three of these measurements were significantly elevated in HIV/ subjects as a group, there were no direct correlations between PBMC IFN-g mRNA levels and either plasma IFN-g or NPT levels on an individual basis (data not shown). The CD8 T cell subpopulation was the only purified cell subpopulation which showed increased levels of IFN-g gene expression in HIV/ subjects (Fig. 1). HIV/ subjects (n Å 18) showed a 2.6-fold higher mean level of IFN-g mRNA compared to HIV0 subjects (n Å 11), which is similar in magnitude to the increase seen in
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HIV RNA bDNA assays were performed on EDTA plasma samples to determine viral load, with results expressed as viral kiloequivalents per milliliter of plasma (26). The level of detection of the assay was 0.5 keq/ml, which is approximately equivalent to 500 copies of HIV RNA/ml plasma. Plasma levels of IFN-g were measured using a modified ELISA protocol (T Cell Sciences) which was capable of detecting 50 units/liter. Plasma levels were also determined for three surrogate markers of immune activation: b2-microglobulin (IMx Microparticle Enzyme Immunoassay, Abbott), neopterin (HENNINGtest, Henning Berlin GMBH), and soluble tumor necrosis factor receptor type II (TNFaRII, Quantikine EIA, R&D Systems). Calculations and Statistics
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TABLE 1 Mean Cell Counts and Plasma Values in HIV-Seronegative and Seropositive Subjectsa
HIV serostatus
Absolute CD4 T cells/ml
Absolute CD8 T cells/ml
Plasma HIV RNA, keq/ml
Plasma IFN-g, U/liter
Plasma neopterin, nM
Plasma b2-microglobulin, mg/liter
Plasma sTNF-RII, ng/ml
0, n Å 28 /, n Å 36 P valueb
891 (50) 290 (34) 3 1 10012
583 (40) 945 (89) 0.0005
õ0.5 (n/ac) 96 (34) 0.008
111 (11) 325 (64) 0.002
5.7 (0.3) 19.4 (2.5) 4 1 1006
1.2 (0.04) 2.7 (0.2) 7 1 10010
2.1 (0.1) 4.5 (0.4) 3 1 1006
a b c
All data shown are mean value (standard error of the mean). P value from two-tailed t test for comparison of all HIV0 and HIV/ subjects. Not applicable (SEM cannot be calculated due to mean value õ0.5).
PBMC. As in the PBMC, the differences in IFN-g mRNA between HIV/ and HIV0 CD8 T cells were clearly statistically significant (P Å 0.003), even with fewer subjects and slightly larger standard errors. No statistically significant differences between HIV/ and HIV0 subjects were seen in IFN-g mRNA levels in either monocytes or B cells, which typically showed little or no IFN-g gene expression. Likewise, there were no significant differences in HIV/ and HIV0 IFN-g mRNA levels in either NK cells or CD4 T cells. This was not due to a lack of IFN-g expression or inability to detect expression because both of these subpopulations usually showed moderate levels of IFN-g mRNA, similar to those seen in unfractionated PBMC. Because the bDNA result is determined on a per cell basis, the increase seen in the purified CD8 T cells is not attributable to an increased absolute number of CD8 T cells seen in HIV/ subjects, but to an overall increase in the amount of gene expression in CD8 T cells. This is consistent with the result that there was no correlation between the absolute number of circulating CD8 T cells and the IFN-g mRNA level in either PBMC or CD8 T cells in HIV/ individuals (data not shown). However, there was a significant positive correlation between PBMC IFN-g mRNA and CD8 T cell IFN-g mRNA levels within HIV/ individuals (r Å 0.54, P Å 0.02, data not shown). This observation strongly suggests that increased IFN-g gene expression in the CD8 T cells is the driving force behind the overall increase in IFN-g mRNA levels seen in HIV/ PBMC. CD8 T cells consistently showed the highest IFN-g mRNA level when compared to PBMC and CD4 T cells of the same individual, regardless of HIV status. In 23 of 24 subjects for whom PBMC, CD4, and CD8 IFN-g mRNA data were available, the CD8 T cells had the highest IFN-g mRNA level (data not shown). In a subset of 14 individuals who had a complete set of PBMC, NK, CD4, and CD8 T cell data (HIV/, n Å 8; HIV0, n Å 6), 11/14 had the highest IFN-g mRNA levels in CD8/ cells (Fig. 2). Two of the remaining three (one HIV/, one HIV0) showed slightly higher mRNA levels in the NK cells than the CD8 T cells, and one (HIV/) had CD4 T cell IFN-g mRNA levels marginally higher
than CD8 T cells. Therefore, even when the CD8 T cells did not have the highest IFN-g mRNA levels, they expressed levels very similar to the highest IFN-g-expressing cell type. The consistent observation of CD8 T cells as major contributors to IFN-g expression in both HIV/ and HIV0 subjects shows that the type of cell responsible for much of the IFN-g gene expression generally does not change as a result of HIV infection. Rather, the level of gene expression rises on a per cell basis within the CD8 T cells in HIV-infected persons. We were interested to see if stratification of the HIV/ subjects, according to various measurements of disease status and/ or progression, would be reflected in the level of IFN-g expression in PBMC and/or CD8 T cells. HIV/ subjects were stratified according to various results obtained from the same blood draw (NPT, B2M, HIV viral load, CD4 T cell number) or on the basis of longitudinal data available from the MACS database (time since seroconversion, rate of loss of CD4 T cells), and levels of IFN-g mRNA in different groups were then compared. For NPT and B2M, subjects were divided into normal (5th to 95th percentile) and elevated (ú95th percentile) based on adult reference ranges established by the Clinical Immunology Research Laboratories at UCLA, where the assays were performed. For plasma viral load (HIV RNA), subjects were divided into above and below 10 keq/ml, based on reports which showed a strong correlation between HIV RNA ú 10 keq/ml and development of AIDS and/or poor survival (29, 30). Length of time of seropositivity was selected to group subjects into recent seroconverters (6–18 months postseroconversion) and longer-term seropositives (ú3 years, including those who were seropositive upon entry into the MACS). IFN-g gene expression was significantly increased in the PBMC of HIV/ subjects with elevated markers of immune activation (NPT or B2M, Figs. 3A and 3B), higher HIV viral load (HIV RNA, Fig. 3C), or who had been seropositive ú3 years (Fig. 3D), when compared to HIV/ subjects with lower values or who had been HIV seropositive 6–18 months (P õ 0.05). When each subgroup of HIV/ subjects was compared to HIV0 sub-
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FIG. 2. Individual IFN-g mRNA levels in PBMC and purified subpopulations. (A) IFN-g mRNA levels in all HIV0 subjects with data from PBMC, NK, CD4 T, and CD8 T cells (n Å 6). (B) IFN-g mRNA levels in all HIV/ subjects with data from PBMC, NK, CD4 T, and CD8 T cells (n Å 8). Open shapes indicate subjects 6–18 months post-HIV seroconversion; filled shapes indicate subjects ú3 years postHIV seroconversion.
jects, the HIV/ subjects with normal plasma NPT or B2M, low HIV RNA, or who were 6–18 months post seroconversion consistently showed PBMC IFNg mRNA levels which were indistinguishable from the HIV0 subjects (P ú 0.05). In contrast, the HIV/ subjects with high plasma NPT, B2M, or HIV RNA or who were longer-term seroconverters showed highly significant increases in PBMC IFN-g mRNA (P õ 0.0002). No statistically significant differences were seen when PBMC IFN-g mRNA levels were compared between HIV/ subjects stratified on the basis of CD4 T cell number at the time of the visit or rate of loss of CD4 T cells over the previous 3 years (data not shown). However, HIV/ subjects with 200–500 or õ200 CD4 T cells/mm3 did show elevated PBMC IFN-g mRNA when compared to HIV0 subjects. IFN-g mRNA was also significantly increased in the CD8 T cells of HIV/ subjects with higher plasma levels of NPT, B2M, or HIV RNA, when compared to HIV0 subjects (P £ 0.02, data not shown). As with the PBMC, HIV/ subjects with normal NPT or B2M or lower HIV RNA had CD8 IFN-g mRNA levels which were indistinguishable from HIV0 subjects. The only stratification which resulted in significant differences in CD8 IFN-g mRNA levels between groups of HIV/ subjects was time since seroconversion, with longer-term seroconverters showing elevated CD8 IFN-g mRNA levels compared to two short-term seroconverters for whom
CD8 data were available (P Å 0.007, data not shown). In spite of this difference, both short- and long-term seroconverters showed significantly elevated CD8 IFNg mRNA levels when compared to HIV0 subjects (P Å 0.02 and 0.001, respectively). In addition to IFN-g mRNA, data were obtained for TNFa, IL-2, and IL-6 mRNA by bDNA analysis on a subset of PBMC and purified subpopulation samples. As expected, the major cell subpopulation expressing TNFa mRNA in both HIV0 and HIV/ subjects was the monocytes, with very small amounts of TNFa mRNA detected in CD4 or CD8 T cells (data not shown). There were no statistically significant differences seen in levels of TNFa mRNA from the PBMC or monocytes of HIV0 and HIV/ subjects (data not shown). In spite of the lack of differences in TNFa expression at the mRNA level, there was a highly significant elevation in the plasma levels of the soluble TNFa receptor II in HIV/ subjects (Table 1). In preliminary studies with five HIV0 and five HIV/ subjects, no statistically significant differences in PBMC IL-2 or IL-6 mRNA between HIV0 and HIV/ subjects were seen (data not shown). It was intriguing to note, however, that three of the five seropositives had undetectable levels of IL2 mRNA and the remaining two were barely above background, while all five of the seronegatives had measurable levels clearly above background. Further studies are planned to extend these initial observations
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FIG. 3. PBMC IFN-g mRNA levels in HIV0 subjects, and HIV/ subjects stratified by various criteria. Comparison of PBMC IFN-g mRNA levels in HIV0 subjects, and HIV/ subjects stratified by (A) neopterin (NPT), (B) b2-microglobulin (B2M), or (C) HIV viral load (HIV RNA), determined on plasma from same blood draw as mRNA analysis, or (D) time since HIV seroconversion. P values are shown for comparisons of IFN-g mRNA levels of each HIV/ group to HIV0 subjects.
utilizing bDNA technology to investigate the expression of these additional cytokines in unstimulated cells.
The results presented here confirm previous reports of increased expression and/or production of IFN-g in unstimulated mononuclear cells from the circulation and/or lymph nodes of HIV-infected subjects (17–22). More importantly, we have extended these observations to elucidate the cell type responsible for IFN-g gene expression in peripheral blood in HIV/ persons, and to determine if it is different from the predominant cell type in HIV0 individuals. By quantitating and comparing IFN-g mRNA in positively selected cell subpopulations that represent highly enriched monocytes, B, NK, CD4 T, and CD8 T cells, we have shown that the major contributors to the overall level of IFN-g expression appear to be the CD8 T cells in both HIV/ and HIV0 individuals. Although Emilie et al. have reported IFN-g-expressing CD8 T cells in HIV/ lymph nodes (17, 18), and others have described greater expression or secretion of IFN-g in stimulated or cloned peripheral CD8 T cells compared to CD4 T cells (10, 31, 32), it has not been previously shown which cell type was the predominant one expressing IFN-g in peripheral blood.
By examining freshly isolated, unstimulated PBMC and subpopulations, our data reflect the in vivo state of IFN-g expression in the circulation, rather than the ability of cells to respond to ex vivo stimulation. It clearly shows that the increased IFN-g expression in HIV infection is primarily attributable to the same type of cell, CD8 T cells (CD8-positive, depleted of NK cells), that is the major contributor to the lower levels of IFNg mRNA seen in HIV0 persons. In other words, HIV infection does not cause a shift to and/or increased expression of IFN-g mRNA in other cell types which are known to make IFN-g, i.e., CD8-positive NK cells or type 1 CD4 T cells (33, 34), but instead enhances or exaggerates the IFN-g response by the CD8 T cells. It is important to note that the observed increase in HIV/ PBMC IFN-g mRNA is not due to the increased absolute numbers of CD8 T cells associated with HIV infection, since results are expressed as IFN-g gene expression per million CD8 T cells analyzed. In light of the recent descriptions of subtypes of cytokine-secreting CD8 T cells, TC1 and TC2, similar to CD4 TH1 and TH2 subsets (9, 10), the increase in IFN-g mRNA in CD8 T cells would suggest either that existing TC1 cells have been hyperactivated to dramatically upregulate IFNg expression or there is an increase in the proportion of CD8 T cells which have differentiated into
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DISCUSSION
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the IFN-g-secreting TC1 type. Ex vivo stimulated cells from HIV-infected persons show increased percentages of cells making IFN-g (32), which favors the latter possibility, but it is not known if the same would be seen in unstimulated cells. This also raises the question of the possibility of differential IFN-g gene expression among subsets of CD8 T cells identified by cell surface phenotype. Since the phenotypic distribution of CD8 T cells has been shown to change in HIV infection, with increases in CD38/DR/ and decreases in CD28/ cells (35–40), determination of IFN-g mRNA levels in CD8 T cell subsets in HIV/ and HIV0 persons might shed some light on the relationship between phenotypically defined and TC1/TC2 subsets. Additional CD8 T cell studies are planned or underway to address these questions. Our observations of increased levels of IFN-g mRNA in unstimulated circulating cells, as well as increased levels of plasma IFN-g and NPT in HIV/ individuals as a group indicate that there is an in vivo increase in IFN-g gene expression and production in HIV infection. However, not all HIV/ subjects exhibited increased IFN-g mRNA compared to HIV0 subjects. When HIV/ subjects were stratified on the basis of criteria other than CD4 T cell number, elevated IFN-g expression was clearly and consistently associated with results indicative of more advanced HIV disease, i.e., higher plasma HIV RNA, longer time of infection, and increased immune activation (Fig. 3). This suggests that the hypothesis that the loss of type 1 cytokine production is associated with the development of HIV disease (7, 8) may not be applicable to all type 1 cytokines. The hypothesis may be supported by numerous reports of decreased production of type 1 cytokines such as IL-2 and IL-12 in HIV infection and disease (7, 8, 41–43), but is not consistent with our observations of increased, not decreased, expression of one of the other type 1 cytokines, IFN-g. Virtually all elevated PBMC or CD8 IFN-g mRNA levels (compared to HIV0 subjects) were found in HIV/ subjects with plasma HIV RNA ú 10 keq/ml, a viral load which has been strongly associated with development of AIDS and/or poor survival (29, 30). While there was not a direct correlation between viral load and PBMC or CD8 IFN-g mRNA levels within individuals (data not shown), the consistent finding of elevated IFN-g expression in those HIV/ subjects with higher viral load suggests that these two parameters may be influencing one another. It is interesting to note that two of the three HIV/ who were 6–18 months postHIV seroconversion (short-term seroconverters) had HIV RNA levels above 10 keq/ml and yet had PBMC IFN-g mRNA levels comparable to HIV0 subjects. However, even at this relatively early time point in HIV disease, IFN-g mRNA levels appear to be rising in subpopulations because short-term seroconverters showed elevated CD8 T cell levels (data not shown) and ac-
counted for two of the three highest NK cell levels detected (Fig. 2). IFN-g gene expression may begin to increase early in disease course, but requires additional time before any increases can be detected in PBMC. Increased IFN-g mRNA was also seen in those HIV/ subjects with increased immune activation, as indicated by abnormally high levels of either plasma NPT or B2M. Elevated levels of one or both of these markers are strong prognostic indicators of more rapid progression of HIV disease (5). Since NPT reflects IFN-g activity, it is not surprising that HIV/ individuals with high NPT levels showed elevated IFN-g expression. However, the additional observation of elevated IFN-g expression in HIV/ subjects with increased B2M, which is a more broad indicator of immune activation, shows that increased IFN-g expression is associated with immune activation in general during HIV infection. Overall, these results suggest that increased IFN-g gene expression may be influencing HIV viral load and/or immune activation over time and therefore could be a direct contributor to the pathogenesis of HIV disease. Alternatively, increased IFN-g gene expression may reflect the effects of increasing viral loads and immune dysregulation in the course of HIV disease. These issues are not addressed directly in this cross-sectional study, but are under investigation in longitudinal studies of individual MACS subjects utilizing cryopreserved PBMC. The results presented here demonstrate the utility of the bDNA assay to detect differences in IFN-g mRNA in freshly isolated, unstimulated PBMC of HIVinfected and uninfected homosexual men. bDNA technology for the measurement of cytokine mRNA is capable of determining IFN-g, TNFa, IL-2, and IL-6 expression in unstimulated PBMC from most individuals, regardless of HIV status. Evaluation of cytokine gene expression in freshly isolated, unstimulated cells is preferable to measurements in cells which have been stimulated in vitro because this better reflects the status of cytokine expression in vivo. Although the bDNA procedure may require greater cell numbers than reverse transcription–polymerase chain reaction assays, the bDNA methodology may be better suited for truly quantitative determination of cytokine mRNA levels, since it does not rely on either reverse transcription or amplification of the target mRNA (26).
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ACKNOWLEDGMENTS We gratefully acknowledge the dedication of the men who are participants at UCLA in the Multicenter AIDS Cohort Study, without whom this and many other studies would never have been possible. We also thank Dr. R. Detels and Dr. P. Nishanian for facilitating collection of blood samples, Dr. S. O. Derzic for technical discussions, N. Aziz for performing plasma assays, M. Gorre for flow cytometry analyses, and M. McDonald for providing laboratory support.
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