Primary HIV and simian immunodeficiency virus infections lead to rapid depletion of CD4+ T cells from the gut mucosa.1-3 The majority of gut mucosal CD4+ T cells exhibit an activated effector memory phenotype and are highly susceptible to HIV infection.4 The depletion of CD4+ T cells from the gut is more pronounced than the depletion in the peripheral blood or lymphoid tissues and may be slower to recover after initiation of combination antiretroviral therapy (ART).5 Loss of these CD4+ T cells may contribute to the disruption of the gut epithelium allowing for translocation of microbial products, which in turn is associated with systemic immune activation in HIV-infected patients.6 Recent studies have shown that prolonged treatment with ART leads to restoration of gut mucosal CD4+ T cells and a decrease in the proportion of T cells in cell cycle.7
Treatment of HIV-infected patients with ART leads to control of viral replication to below the limits of quantification of commercially available assays, restoration of peripheral blood CD4+ T-cell counts, and reduced morbidity and mortality.8,9 However, once begun ART must be taken for life, and lifelong ART imposes multiple burdens such as long-term toxicities, drug-drug interactions, and drug resistance as a result of nonadherence. Therefore, immune based therapies that restore the CD4 T-cell pool have been explored as an adjunct or alternative to ART. One of the best studied of these therapies has been intermittent interleukin-2 (IL-2).
Several phase I and II studies have demonstrated that IL-2 leads to significant and sustained increases in peripheral blood CD4+ T cells in HIV-infected patients.10-13 The increased CD4+ T-cell numbers were primarily due to peripheral expansion and increased survival14 of a subset of CD4+ T cells that express the IL-2 receptor alpha chain (CD25)15 and have a naive or central memory phenotype.14,16 Furthermore, the increased CD4+ T-cell numbers were associated with a long-term decrease in T-cell proliferation.16 Despite these promising findings, 2 large multicenter phase III trials demonstrated recently that the increase in CD4+ T-cell counts does not confer clinical benefit.17
In light of the failure of IL-2 to provide clinical benefit in phase III studies, it is important to understand better how the expanded T cells are distributed and if they are capable of homing after maturation to tissues such as the gut. We conducted this study to measure the degree of immune restoration, the proportion of cycling T cells, and the proportion of CD4+ T cells expressing CD25 in the colon and terminal ileum (TI) of HIV-infected patients who received IL2 and ART and compare them with patients who received ART alone and to healthy uninfected participants.
Biopsies from colon and TI and peripheral blood samples were collected from 11 HIV-infected patients treated with ART and intermittent IL-2 (IL-2), 12 HIV-infected patients treated with combination ART (ART) and 10 HIV-uninfected participants (HIV−). Patients in the IL-2 group had previously received intermittent IL-2 therapy as part of other ongoing or completed protocols. All participants provided informed consent for peripheral blood and gastrointestinal tract sampling under an IRB-approved National Institutes of Health Protocol. Plasma viral loads were determined by ultrasensitive bDNA assay (Versant HIV-1 version 3.0, Siemens Corp, New York City, NY). Results from the ART and the HIV- groups have been reported previously in part.7
Colonic and TI tissue biopsies were collected as previously described.7 Briefly, approximately 30 biopsies were extracted from the gut mucosa at each location, and 16-20 were processed for flow cytometric analysis. The biopsies were weighed, placed in RPMI (Mediatech, Herndon, VA) with 10% heat-inactivated fetal bovine serum, and digested using collagenase (Sigma-Aldrich, St Louis, MO) and DNAse I (Invitrogen, Carlsbad, CA) or benzoase (Novagen, Madison, WI), and then filtered. After washing, the cell suspension was counted using a Beckman Coulter Counter to obtain the number of total viable cells. Absolute numbers of CD4+ and CD8+ T-cell per gram of gut tissue were determined as previously described.7
Immunophenotypic analyses were performed on whole blood and cells extracted from the gut biopsies as described elsewhere.18 The following monoclonal antibodies were used for staining: CD3, CD4, CD8, CD27, CD45RO, CD25, and Ki67 conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin-chlorophyll-protein, or allophycocyanin [Becton Dickinson (BD) Pharmingen, San Jose, CA]. Samples were acquired using a FACSCalibur flow cytometer (BD Pharmingen, San Jose, CA). The data were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA).
Values are expressed as medians with interquartile ranges. Kruskal-Wallis and Mann-Whitney tests were used for between-group comparisons. Given the exploratory nature of the study, all P values <0.05 are shown. Analyses were performed using Prism v5.0 (GraphPad Software, Inc, La Jolla, CA).
Clinical characteristics of the 3 groups are shown in Table 1. Patients in the ART and IL-2 groups had initiated ART during chronic infection. Patients in the IL-2 group had received a median of 7 cycles of IL-2 administered at doses ranging from 3.0 to 7.5 IU twice daily for 5 days by subcutaneous injection, and the median time since the last IL-2 cycle was 4 months. All participants in the ART and IL-2 groups were on regimens containing 3 or more antiretrovirals, at least 1 of which was a protease inhibitor or a nonnucleoside reverse transcriptase inhibitor. Two participants in the ART group were on regimens containing both a protease inhibitor and a nonnucleoside reverse transcriptase inhibitor. No participants were taking entry inhibitors, CCR5 antagonists, or integrase inihibitors. All participants were asymptomatic at the time of biopsy.
Immunologic Response to IL-2 Therapy
The absolute number of peripheral blood CD4+ T cells in the IL-2 group (701 cells/μL) was higher than the ART group (535 cells/μL, P = 0.018) and similar to the HIV−group (773 cells/μL, P = 0.699) (Table 1). In the IL-2 group, CD4+ T-cell counts at the time of sampling were a median of 51% (22%-70%) higher compared with a time just before their most recent IL-2 cycle.
In both the colon and TI, there were no differences in the proportions of CD4+ T cells between the IL-2 group and the ART group (colon: 33 vs 38%, P = 0.559; TI: 16 vs. 30%, P = 0.342) (Fig. 1A). The proportions of CD4+ T cells were lower at both gastrointestinal sites in the IL-2 and ART groups compared with the HIV−group (colon: 54%; P = 0.004 for both comparisons; TI: 38%; vs. ART, P = 0.015; vs. IL-2, P < 0.001) (Fig. 1A). A similar pattern was seen in the peripheral blood.
The proportion of CD8+ T cells was also similar at the gastrointestinal sites of the IL-2 and ART groups (colon: 42 vs. 42%, P = 0.758; TI: 54 vs. 61%, P = 0.597) (Fig. 1B). In the colon, the proportion of CD8+ T cells was lower in the HIV-group compared with both the IL-2 group and the ART group (22%, P = 0.001 for both comparisons). Again a similar pattern was seen in peripheral blood.
There were no differences in the absolute numbers of CD4+ or CD8+ T cells in the gastrointestinal sites between the IL-2 group and the ART group (data not shown).
CD25 Expression and Cell Cycling Following IL-2 Therapy
In the IL-2 group, CD4+ T cells expressed significantly higher levels of CD25 (26%) compared with the ART group or HIV− group (ART: 8.52%, HIV−: 6.13%; P < 0.001 for both comparisons). However, at the gastrointestinal sites there were no differences between groups in CD25 expression on CD4+ T cells (Fig. 1C).
The proportion of cycling (Ki67+) CD4+ T cells in the colon in the IL-2 group (3.9%) was not different compared with the ART group (3.0%; P = 0.086) and higher than the HIV− group (2.2%; P = 0.006). Expression of Ki67 by CD8+ T cells in the colon was higher in the IL-2 group (4.4%) compared with the ART group (2.6%; P = 0.033) and not different from the HIV− group (3.3%; P = 0.397). In the ileum, Ki67 expression by CD4+ T cells was not different in the IL2 group (2.2%) compared with the ART group (3.9%; P = 0.622) or the HIV− group (1.9%; P = 0.065). There were no differences in Ki67 expression by CD8+ T cells among the groups in the ileum. Expression of Ki67 by CD4+ or CD8+ T cells in the peripheral blood was not different between the 3 groups (data not shown).
In contrast to its known effect of increasing CD4+ T-cell counts in the peripheral blood, this study showed no additional effect of IL-2 treatment compared with ART alone, on the proportion of CD4+ T cells in the gut mucosa. In addition, there was no difference in CD25 expression on CD4+ T cells or proportion of cycling CD4+ T cells in the gut.
IL-2, a cytokine secreted by activated lymphocytes, plays a critical role in the homeostasis of lymphocytes by regulating their proliferation, differentiation, and survival.19 Given that IL-2 administration had no effect on the proportion of CD4+ T cells in the gut, it seems that it is not having an impact on CD4+ T-cell homeostasis in that site. Additionally, CD4+ CD25+ T cells do not seem to be homing to the gut or maturing into effectors that can migrate to the gut tissue in response to local stimuli, although it should be noted that the function of these cells was not examined in this study and might also be differentially affected by IL-2. Interestingly, Ki67 expression was higher in CD8+ T cells in the colon in the IL-2 group than the ART group, although the proportions of CD8+ T cells overall were not different. The reasons for this are unclear and warrant further studies, including examination of additional markers of T-cell activation and apoptosis and gut viral load.
In interpreting the recent phase III studies of IL-2 administration in combination with ART, two hypotheses have emerged to explain the lack of clinical benefit of increased peripheral CD4+ T-cell counts: (1) the CD4+ T cells induced by IL-2 are not functional or (2) the CD4+ T cells are functional, but the adverse effects of interleukin-2 negate the benefits of the expanded cells.17 Subsequently it has been shown that IL-2 administration induced transient increases in high sensitivity C-reactive protein and D-dimer suggesting increased inflammation and/or thrombosis as a possible mechanism to explain some of the adverse clinical events seen in these trials.20 It is also possible that expanded CD4+CD25+ T cells were not maturing or differentiating into effectors able to home to gut or other tissue. Although IL-2 has been shown to decrease CD4+ T-cell cycling in the periphery long term,16 in the gut there was no difference in the proportion of cycling CD4+ T cells between the IL-2 treated and ART-only groups.
This study shows that IL-2 administration does not affect the proportion of gut CD4+ T cells nor does it have an effect on the cycling of CD4+ T cells or proportion of CD4+CD25+ T cells in the gut. Given these findings and the lack of clinical benefit of IL-2, it may be worth examining the effects of other immune-based therapies currently being developed on gut or other mucosal T-cell populations to evaluate homing and maturation potential of newly expanded T lymphocytes.
The authors would like to acknowledge the staff of the OP8 clinic and the GI suite at the NIH Clinical Center for their assistance with patient recruitment and care. Special thanks to all study participants who volunteered for research gut biopsy procedures.
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