Read, Sarah W. MD*; Higgins, Jeanette BS†; Metcalf, Julia A. BA*; Stevens, Randy A. BA†; Rupert, Adam BS†; Nason, Martha C. PhD*; Lane, H. Clifford MD*; Sereti, Irini MD*
Interleukin-7 (IL-7) is a cytokine with critical functions during lymphoid development, and its potential as an immunorestorative agent is currently being investigated in phase I studies in immunodeficient hosts.1 IL-7 is produced by stromal tissues, including epithelial cells in the thymus and bone marrow, and acts through stimulation of thymopoiesis by providing survival signals to early thymocyte progenitors.2,3 IL-7 is also essential for the proliferation and survival of naive T cells,4,5 and has been shown to play a key role in development and survival of memory T cells.6-8 Increased levels of circulating IL-7 have been demonstrated in patients with CD4 lymphopenia resulting from HIV infection or chemotherapy.9,10 The mechanism responsible for this increase in IL-7 level is unknown; it may be because of increased production of IL-7 as a homeostatic response, or levels may be elevated as a result of a reduction in cells expressing IL-7 receptor, leading to a decrease in receptor-mediated clearance. Whatever the mechanism, it is clear that IL-7 plays an important role in T-cell homeostasis, and therefore, it has been postulated that exogenous IL-7 could potentially be used to reconstitute T cells in T-cell-deficient patients, as has been demonstrated in animal models.11,12
The IL-7 receptor complex consists of 2 chains: the IL-7 receptor α chain (CD127) that binds IL-7 and the γ chain that is shared by the IL-2 receptor and other cytokine receptors such as IL-4, IL-9, IL-15, and IL-21.13 In humans, CD127 can be found on immature B cells through early pre-B stage, on thymocytes, and on mature T cells. Decreased CD127 expression has been demonstrated on both CD4 and CD8 T cells of untreated HIV-infected patients as compared with uninfected controls.14,15
Several randomized controlled trials have shown that treatment with intermittent IL-2 in HIV-infected patients leads to a sustained increase in number and percent of CD4 T cells.16-18 Recent studies have shown this to be predominantly the result of expansion of naive and central memory CD4+/CD25+ T cells with prolonged survival and decreased turnover.19-21 The effect of IL-2 on IL-7 levels is of interest because both are prosurvival cytokines currently being studied in the context of therapy for immunodeficiencies. Two studies have reported increased IL-7 levels after administration of IL-2,22,23 and no differences in CD127 expression on CD4 or CD8 T cells after IL-2 therapy have been found.24 However, these studies included small numbers of patients, all of whom were classified as "immunologic nonresponders" who had not achieved CD4 counts above 200 cells/μL despite virologic control. Furthermore, the changes reported occurred immediately after administration of an IL-2 cycle or after only short-term follow-up. Hence, these findings may not be applicable to other patient populations and do not reflect the long-term effect of IL-2 on IL-7 levels and CD127 expression.
The goal of this study was to further characterize the relationship between IL-7 levels, IL-7 receptor expression, and clinical parameters in HIV-infected patients and to determine what effect treatment with IL-2 has on the IL-7 system. Because IL-7 levels have been found to be increased and CD127 expression on T cells decreased in HIV infection, we hypothesized that other changes involved in the pathogenesis of HIV infection such as a decrease in CD4 T cell count, decrease in proportion of naive T cells, and increase in activation would be associated with these changes in the IL-7 system. Because treatment with IL-2 leads to increased proliferation and survival of CD4 T cells, we further speculated that treatment with exogenous IL-2 would lead to a decrease in serum IL-7 levels. Furthermore, in vitro experiments have shown a suppressive effect of cytokines such as IL-2 on lymphocyte CD127 expression.25,26 We predicted the same effect would be seen in vivo with decreased CD127 expression on T cells in patients who have received exogenous IL-2.
We measured serum IL-7 levels and CD127 expression on CD4 and CD8 T cells in a large cohort of HIV-infected patients, including a cohort who had previously received treatment with IL-2, and HIV-negative controls. In addition, we investigated a number of patient characteristics and T-cell phenotypic markers to determine those that are associated with IL-7 serum levels and CD127 expression on CD4 and CD8 T cells. Because treatment with IL-2 is known to cause an increase in the number of CD4 T cells that express CD25 (IL-2 receptor α),16,27,28 we examined CD127 expression specifically on those cells as well.
MATERIALS AND METHODS
Blood samples were obtained from patients who were seen consecutively in the National Institutes of Allergy and Infectious Diseases intramural AIDS research clinic between November 17 and December 16, 2003, as well as between January 5 and January 23, 2004. All patients were enrolled on Institutional Review Board-approved study protocols and had given informed consent. If a patient was seen more than once in this period, only the first time point was included. Patients were excluded if they were coinfected with hepatitis B or C or were receiving IL-2 at the time of the visit. They were also excluded if they did not have serum samples available for measurement of IL-7. Patient demographic information and other characteristics such as date of birth, year of HIV infection, whether currently receiving highly active antiretroviral therapy (HAART), and hepatitis B and C serostatus were obtained from the clinic database.
Cryopreserved peripheral blood mononuclear cells were identified from 14 HIV-infected patients and 9 patients who had received treatment with 3 consecutive cycles of recombinant human IL-2 (3-9 million IU, SC, BID, for 5 days every 8 weeks). Samples from the IL-2-treated patients from just before therapy and 8 weeks after the third cycle were used in these experiments. All patients were enrolled on Institutional Review Board-approved study protocols and had given informed consent.
Serum IL-7 Levels
A commercially available enzyme-linked immunosorbent assay kit (R&D, Minneapolis, Minn) was used according to the manufacturer's protocol for the quantitative detection of IL-7 from frozen stored serum samples. The lower limit of sensitivity for the assay was 0.25 pg/mL.
Flow Cytometric Analysis
Phenotypic analysis using 3 or 4 color immunofluorescence was performed on cryopreserved peripheral blood mononuclear cells or whole blood collected in an EDTA tube. Whole blood was processed within 4 hours from the time of the blood draw. The whole-blood lysis technique for surface staining was used28 (Immunocytometry, BD Biosciences, San Jose, Calif). Combinations of the following antibodies were used: CD45 fluorescein isothiocyanate (FITC) (clone 2D1), CD3 FITC or allophycocyanin (APC) (clone SK7), CD27 FITC (clone M-T271 or L128), CD25 FITC or APC (clone 2A3), CD45RO FITC (clone UCHL1), CD25 PE (clone 2A3), CD38 PE (clone HB7), CD14 PE (clone Mϕ Pq), immunoglobulin G1 PE-Cy5 (clone G18-145), CD4 PE-Cy5 (clone RPA-T4), CD8 PE-Cy5 (clone RPA-T8), CD4 PerCP (clone SK-3), CD38 APC (clone HB-7) (all from BD Biosciences, Pharmingen, San Diego, Calif, or Immunocytometry) and CD127 PE (clone R34.34) (Beckman Coulter, Hialeah, FL). Whole blood samples were analyzed on an Epics XL flow cytometer (Beckman Coulter), and cryopreserved samples were analyzed on a FACS Calibur (Immunocytometry, BD). Approximately 2 × 105 total events with a minimum of 5000 events in the CD4+ or CD8+ cell gate were collected per sample. Flow cytometry data were analyzed using FloJo software (Tree Star, San Carlos, CA). CD3+/CD4+ gating and CD3+/CD8+ gating were used for CD4+ and CD8+ T cells, respectively. Percent CD127 expression was then determined for each population, and median fluorescence intensity (MFI) was measured on gated CD127+ cells. Naive and memory phenotypes were studied with CD45RO and CD27 staining. Naive cells were defined as CD45RO−/CD27+. Memory cells were defined as CD45RO+/CD27+ (central memory), CD45RO+/CD27− (effector memory), or CD45RO−/CD27− (effector). The effector population was seen exclusively in CD8+ T cells.
To study expression of CD127 on CD4+CD25+ cells, gating was first set on the CD4+ population based on side scatter. The percentage of CD4+ T cells positive for CD25 was based upon a CD25 histogram (both CD25 intermediate and CD25 high were included in the CD25+ population). The percent expression of CD127 and MFI for CD127 was then measured on the CD4+CD25+ and CD4+CD25− subsets.
Variables with substantial skew were log10-transformed. IL-7 serum levels, CD127 expression, and CD127 MFI were compared between groups (HIV negative, HIV positive, and IL 2 treated) using analysis of variance and t tests. Within each group, univariate regression models were used to describe the relationship between each demographic and phenotypic marker and both IL-7 serum levels and CD127 expression on CD4 and CD8 T cells. Colinearity of predictor variables was assessed using scatterplots and pairwise correlations. Finally, a multiple regression model within each cohort was used to investigate the relationship between the variables and highlight independent predictors of IL-7 serum levels and CD127 expression. Because of the number of models considered, a stronger statistical significance threshold of 0.01 was used.
Blood samples were obtained from 315 patients who presented consecutively to the National Institute of Allergy and Infectious Diseases (NIAID) intramural AIDS clinic during a 2-month period. After excluding patients with hepatitis coinfection, current cytokine therapy, or unavailable serum samples, 270 patients were included in the analyses; 36 were HIV negative, 151 were HIV positive and had never received IL-2 (referred to as "HIV positive" in this report), and 83 were HIV positive and had previously received at least 3 cycles of IL-2 (referred to as "IL-2-treated group" in this report). Participant characteristics are outlined in Table 1. The CD4 T-cell count was higher in the HIV-negative group (805 cells/μL) compared with the HIV-positive group (511 cells/μL, P < 0.001) and similar to the IL-2-treated group (760 cells/μL, P = NS). Likewise, the naive CD4 T-cell count was also higher in the HIV-negative group (265 cell/μL) compared with the HIV-positive group (156 cells/μL, P = 0.007) and similar to the IL-2-treated group (197 cells/μL, P = 0.09).
IL-7 Serum Levels Were Higher in HIV-infected Patients
HIV-positive patients had a mean IL-7 serum level (11.58 pg/mL) that was higher than both HIV-negative (8.73 pg/mL, P = 0.022) and IL-2-treated patients (9.68 pg/mL, P = 0.012) (Fig. 1A). Because of the chosen threshold for significance of P < 0.01, these differences were not statistically significant but showed a trend that was similar to previously published data.9,10 IL-2-treated patients had similar serum IL-7 levels compared with HIV-negative volunteers (P = 0.381).
Multivariate regression analysis was performed to examine relationships between patient variables and IL-7 serum levels. Variables included were HIV viral load, CD4 and CD8 T cells counts, and whether currently being treated with HAART. In HIV-infected patients, CD4 T-cell count was inversely associated with IL-7 serum level (P < 0.001). This was also true in IL-2-treated patients, although not at a level of statistical significance (P = 0.028). No other variables were associated with IL-7 levels.
IL-7Rα Expression on CD4 and CD8 T Cells Is Lower in HIV-infected Patients Compared with HIV-negative Volunteers
In HIV-negative volunteers, 90.6% of CD4 T cells expressed CD127 (Fig. 1B). When HIV-positive and IL-2-treated patients were compared with HIV-negative volunteers, significantly lower expression of CD127 was noted in the CD4 T cells (HIV-positive group, 84.0%, P = 0.002; IL-2-treated group, 83.4%, P = 0.004). Although the difference in CD127 expression between the HIV-positive and IL-2-treated groups was not statistically significant, the MFI of CD127 expression was significantly lower in the IL-2-treated group (4.80) compared with both the HIV-negative and HIV-positive groups (HIV-negative group, 6.03, P = 0.002; HIV-positive group, 5.66, P < 0.001) (Fig. 1C).
The percentage of CD8 T cells from HIV-negative volunteers that expressed CD127 was 62.1% (Fig. 1B). Both the HIV-positive patients and the IL-2-treated patients had a significantly lower percentage of CD8 T cells that expressed CD127 (HIV-positive group, 37.6%, P = <0.001; IL-2-treated group, 45.7%, P = <0.001). The MFI of CD127 on CD8 T cells was similar in all groups (HIV-negative group, 5.10; HIV-positive group, 4.24; IL-2-treated group, 4.43) (Fig. 1C).
Expression of CD127 on CD4 and CD8 T Cells Is Directly Associated with Naive Phenotype and Inversely Associated with Effector/Memory Phenotype in HIV-infected Patients
Univariate and multivariate regression models were performed to explore whether any relationships existed between patient characteristics or cell phenotypic markers and expression of CD127 on CD4 and CD8 T cells. Patient characteristics that were incorporated included age and current use of HAART, total CD4 and CD8 T cell counts, viral load, and serum IL-7 levels. Naive and memory phenotypes were included as well as activation status (CD38 expression) and IL-2Rα (CD25) expression. Only patients with complete data were included in the analyses: 18 HIV-negative, 151-HIV positive, and 83 IL-2-treated patients.
In HIV-negative volunteers, CD127 expression on CD4 T cells was inversely associated with the proportion of effector memory CD4 T cells (P = 0.002) and effector CD8 T cells (P = 0.008), and CD127 expression on CD8 T cells was inversely associated with the proportion of effector CD8 T cells (P = 0.008) in the univariate models only. No parameters were associated with CD127 expression on either CD4 or CD8 T cells in the multivariate models.
In the regression models for HIV-positive patients, CD127 expression on CD4 T cells was directly associated with CD4 T-cell count and the proportion of naive CD4 T cells, and inversely associated with the proportion of CD4 effector memory cells, HIV viral load, and serum IL-7 levels (Table 2A). Similarly, CD127 expression on CD8 T cells was directly associated with current use of HAART and the proportion of naive CD8 T cells and inversely associated with HIV viral load, serum IL-7 levels, the proportion of CD4 and CD8 T cells expressing CD38, and the proportion of CD8 T cells with effector or effector memory phenotype (Table 2B). Figure 2 shows the direct correlation between CD127 expression on CD8 T cells and the proportion of naive CD8 T cells. Flow data from a representative patient illustrate this relationship and the inverse relationship between CD127 expression and activated and effector memory CD 8 T cells (Fig. 3).
Results from the regression models for the IL-2-treated patients were similar to those for HIV-positive patients. In the univariate models for IL-2-treated patients, CD127 expression on both CD4 T cells and CD8 T cells was directly associated with CD4 T cell count (P = 0.002 and P = 0.005) and inversely associated with the percent effector memory CD4 T cells (<0.001; <0.001) and effector memory CD8 T cells (P = 0.001 and P < 0.001). Serum IL-7 levels were also inversely associated with CD127 expression on CD8 T cells in the univariate model (P = 0.005). Additionally, in both the univariate and multivariate models, CD127 expression on CD8 T cells was directly associated with the proportion of naive CD8 T cells (P < 0.001 for both models) and inversely associated with the proportion of CD8 T cells expressing CD38 (P < 0.001 for both models).
IL-7Rα Expression on CD4 T Cells that Express CD25 Is Lower After Treatment with IL-2
Blood samples were collected from an additional 77 patients who presented consecutively to the clinic and were analyzed for CD127 expression on subsets of CD4 T cells with or without CD25 expression. In an effort to decrease the number of variables potentially affecting CD127 expression, only patients who were on HAART with a viral load less than 50 copies/mL were included. CD4+ and CD8+ T-cell counts and number of IL-2 cycles were similar to the patient cohort listed in Table 1 (data not shown). HIV-negative volunteers (n = 20) and HIV-positive patients (n = 34) had similar percentages of CD4 T cells that expressed CD25 (15.9% vs 15.1%, P = NS), whereas IL-2-treated patients (n = 23) had a significantly higher percentage of CD4 T cells that expressed CD25 (33.5%, P < 0.0001). The proportion of CD4+CD25− T cells expressing CD127 was lower in both the HIV-positive (89.5%) and IL−2-treated groups (87.2%) compared with the HIV-negative group (95.7%, P < 0.001 for both comparisons) (data not shown). Likewise, the proportion of CD4+CD25+ T cells expressing CD127 in HIV-positive patients was lower compared with HIV-negative patients, although the difference did not reach statistical significance (74.2% vs 80.5%, P = 0.038) (data not shown). However, the proportion of CD4+CD25+ T cells expressing CD127 in IL-2-treated patients (84.2%) was significantly higher compared with both HIV-negative (P < 0.001) and HIV-positive patients (P = 0.009), whereas the MFI of CD127 on CD4+CD25+ T cells was lower in the IL-2-treated group (MFI 3.4) compared with both the HIV-negative group (MFI 8.1, P < 0.001) and the HIV-positive group (MFI 6.3, P < 0.001) (Fig. 4A) because of expansion of a population of cells with low expression of CD127.
To verify these findings, samples were examined from 9 IL-2-treated patients who had peripheral blood mononuclear cells cryopreserved before (baseline) and 8 weeks after 3 cycles of rhIL-2 treatment. These patients had received no IL-2 before the baseline time point. After rhIL-2 treatment, CD4 T cells expressing both CD25 and CD127 expanded, but this expansion was most prominent in those cells with low MFI for CD127 (P < 0.001). Table 3 summarizes changes in CD4+CD127+ T-cell populations after 3 cycles of rhIL-2. Figure 4B illustrates the expansion of the CD25+CD127-low population as seen in samples from a representative patient before and after IL-2 treatment.
In this study, the serum levels of IL-7 and expression of IL-7 receptor were evaluated in a large cohort of HIV-infected patients, including a group treated long term with IL-2. Serum IL-7 levels were elevated, and IL-7 receptor expression on T cells was low compared with HIV-negative volunteers despite antiretroviral therapy. IL-2 immunotherapy, which leads to higher CD4 T cell counts, was associated with lower IL-7 levels in the presence of persistent decreased IL-7 receptor expression on T cells. Similar findings of elevated IL-7 levels and inverse relationship with CD4 T-cell counts have been previously reported in HIV-infected patients.9,10,29-31
Significantly lower CD127 expression on CD4 and CD8 T cells was seen in this cohort of HIV-infected patients compared with healthy controls, consistent with previously published reports.14,15,32 Parameters such as CD4 T cell count and use of HAART in the univariate models and naive phenotype of T cells in the multivariate models were directly correlated with CD127 expression on T cells. These parameters are all markers of controlled disease and immune reconstitution. Thus, the lack of complete reconstitution of the naive T cell pool with HAART may explain the lower CD127 expression in HIV-infected patients despite therapy. Inverse correlates of CD127 expression in the univariate models included CD38 expression, HIV viral load, and serum IL-7 levels. In the multivariate model, CD127 expression is also inversely correlated with the proportion of effector/memory T cells. Given the higher proportion of activated, effector memory cells seen in HIV infection, it follows that infected patients would have lower CD127 expression. Expansion during HIV infection of the effector memory CD8 T cells that do not express CD127 has recently been reported,33 and decreased CD127 expression in these activated cells may explain their poor survival and function.
Patients treated with IL-2, despite higher CD4 T-cell counts and increased proportion of naive cells, did not have increased percent CD127 expression on CD4 T cells compared with HIV-infected patients who had not received IL-2. Additionally, the MFI on CD4 T cells was significantly lower. This is consistent with reported in vitro data showing that IL-2, IL-4, IL-7, and IL-9 can down-regulate CD12725,26 and may represent a means of distributing prosurvival cytokines. The decreased CD127 expression, despite increase in naive CD4 T cells, is because of an expansion of a pool of CD4+CD25+ naive T cells expressing low levels of CD127 that is seen after IL-2 therapy.20 This idea is further supported by the fact that in this study, CD4 T cells expressing CD25 in IL-2-treated patients had significantly lower CD127 MFI than either HIV-negative controls or HIV-positive patients not treated with IL-2. In contrast, CD8 T cells, which are not expanded in vivo with IL-2, did not show a decrease in either percent CD127 expression or MFI.
This study shows that despite treatment with HAART, increased IL-7 levels and decreased CD127 expression on T cells persists in HIV-infected patients. These abnormalities are likely because of ongoing immune activation and incomplete restoration of CD4 T-cell subpopulations such as the naive T-cell pool. Therapy with IL-2, which leads to increases in total and naive CD4 T-cell pools, results in overall lower IL-7 levels, but CD127 expression on CD4+ T cells decreases further. Thus, regardless of therapy with HAART or IL-2, defects remain in the IL-7 system. A subset of HIV-infected patients described as "CD4 exploders," with very low nadir CD4 T-cell counts and a rapid rise in CD4 T-cell count (>600 cells/μL) after initiation of antiretroviral therapy, have been shown to have elevated IL-7 levels and increased CD127 expression on CD4 T cells both before and after starting therapy.32 The exploders' IL-7 levels and CD127 expression were elevated compared with both HIV-seronegative controls and HIV-infected patients who had normal response to therapy. This suggests that the entire IL-7/IL-7R system is probably up-regulated in these patients and may contribute to an efficient immune reconstitution.
In light of these findings, it is possible that increased availability of IL-7 could play a role in restoring T-cell homeostasis in HIV-infected patients who have experienced T-cell depletion. In studies of both uninfected and simian immunodeficiency virus-infected cynomolgus monkeys as well as simian immunodeficiency virus-infected macaques, administration of exogenous rhIL-7 has resulted in substantial increases in T-cell numbers.11,34 These results in nonhuman primates offer promise that IL-7 could be used as an immunorestorative agent in humans as well.
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