A proportion of HIV patients receiving antiretroviral therapy (ART) retain low CD4+ T-cell counts, despite optimal suppression of HIV replication. The frequency of persistent CD4+ T-cell deficiency varies from 8% in patients observed over 2 years on ART  to 35.8% over 5 years on ART . A low nadir CD4+ T-cell count increases the risk of persistent CD4+ T-cell deficiency, so that 40% of patients with a nadir CD4+ T-cell count of less than 200 cells/μl have this problem after 10 years on ART . CD4+ T-cell deficiency on ART is associated with a higher rate of mortality and morbidity , which includes atherosclerotic vascular disease [5,6], osteoporosis and fractures  and non-AIDS-defining cancers [8–10].
CD4+ T-cell deficiency on ART is associated with older age, ongoing immune activation [11,12], fibrosis of lymphoid tissue [13,14] and a failure to regenerate naive T cells through thymopoiesis [15–17] and/or peripheral homeostasis. The latter may reflect a reduced capacity of naive T cells to proliferate following stimulation with interleukin-7 (IL-7) and/or T-cell receptor (TCR) ligation . Furthermore, we have shown that the naive T-cell subset is particularly affected by immune activation . CD4+ T-cell deficiency on ART is also associated with activation of the innate immune system, particularly monocytes .
IL-7 is produced by stromal cells of the bone marrow and thymus, and by dendritic cells in secondary lymphoid organs. It has an essential role in thymopoiesis, homeostatic proliferation and survival of naive and memory T cells [21–23] with CD31+ naive T cells being more responsive than long-term resident peripheral T cells . T-cell responses to IL-7 are initiated through the IL-7 receptor (IL-7R) . The IL-7R is expressed by cells of the lymphoid lineage and consists of a unique α chain (IL-7Rα; CD127) and a common cytokine receptor γ chain. IL-7 binding to IL-7R activates Janus kinases, JAK1 and JAK3, and stimulates the intracellular domain of CD127. This leads to phosphorylation of the transcription factor signal transducer and activator of transcription 5 (pSTAT5), which dimerizes and translocates to the nucleus to bind to its target DNA and activate transcription [26,27].
Untreated HIV infection is associated with increased circulating levels of IL-7 and low expression of CD127 on peripheral T cells [28–31]. Elevated levels of IL-7 have been detected in other lymphopenic conditions, such as severe combined immunodeficiency, chemotherapy-induced lymphopenia  and idiopathic CD4+ lymphopenia , so increased IL-7 production may be a compensatory mechanism invoked as a homeostatic response to T-cell depletion.
Serum IL-7 levels decline with increased CD4+ T-cell numbers in HIV patients receiving ART, but CD127 expression on naive, effector and terminally differentiated CD4+ T cells may remain low [31,33,34]. IL-7 binding and IL-7R signalling in naive and memory CD4+ T cells from viremic and aviremic patients appears to be normal, despite elevated levels of pSTAT5 and reduced expression of the survival protein Bcl-2 in naive T cells . However, T cells from HIV patients display impaired access of STAT5 to the nuclear compartment, which may prevent the induction of downstream pro-survival signals such as Bcl-2 .
Recombinant IL-7 therapy can increase CD4+ T-cell counts in HIV patients with CD4+ T-cell deficiency on ART , but it is unclear whether this reflects increased thymopoiesis or homeostatic proliferation of CD4+ T cells. Here, we examine IL-7-induced homeostatic mechanisms in naive CD4+ T cells of HIV patients with good or poor CD4+ T-cell recovery on ART to determine whether they are affected by immune activation and CD4+ T-cell senescence.
Materials and methods
Thirty-nine adult HIV-1-infected patients (37 men) receiving combination ART were recruited in 2007–2008. Criteria for inclusion were nadir CD4+ T-cell counts less than 100 cells/μl, ART for more than 12 months and undetectable plasma HIV RNA (<50 copies/ml) for more than 6 months. Patients were categorized as having normal (n = 20) or low (n = 13) CD4+ T-cell counts based on values of more than 500 or less than 350 cells/μl, respectively. For correlations between the parameters measured, an additional six patients with intermediate CD4+ T-cell counts (350–500 cells/μl) were included. Informed consent was obtained from all individuals, and the study was approved by an institutional ethics committee.
HIV-1 RNA viral load
Plasma HIV RNA levels were assayed by quantitative reverse transcription PCR (Amplicor Version 1.5, Ultrasensitive Protocol, 50–75 000 copies/ml) (Roche Diagnostic Systems, Branchburg, New Jersey, USA).
Total and naive CD4+ and CD8+ T cells were enumerated in EDTA-treated whole blood using the following fluorescently conjugated mAbs: CD45-APC-H7, CD3-PerCP, CD4-APC, CD8-PeCy7, CD45RA-FITC and CD62L-PE (BD Biosciences, San-Jose, California, USA) by the Flow Cytometry Unit at Royal Perth Hospital. Peripheral blood mononuclear cells (PBMCs) were separated from lithium heparin treated whole blood by Ficoll-Paque density centrifugation and washed twice in RPMI 1640. Viable cells were counted by trypan blue exclusion and resuspended in 10% dimethylsulfoxide/90% heat-inactivated fetal calf serum at 107 cells/ml for storage in liquid nitrogen. Thawed PBMCs were stained for surface markers for 15 min. The following fluorescently conjugated mAbs were used for assessment of CD127 expression: CD3-V450, CD4-V500, CD45RA-APC-H7, CD27-PerCP-Cy5.5, CD28-PE, CD31-FITC and CD127-AF674 (BD Biosciences). For assessment of immune activation molecules, PBMCs were incubated with CD3-V450, CD4-V500, CD45RA-APC-H7, CD27-PerCP-Cy5.5, HLA-DR-PE, CD38-APC and CD31-FITC (BD Biosciences). For assessment of immunosenescene molecules, EDTA-treated whole blood was incubated with CD4-PerCP-Cy5.5, CD8-APC-Cy7, CD57-FITC, HLA-DR-APC and Fas-PE for 20 min, followed by 1 ml FACSlyse (BD Biosciences) for a further 15 min. A minimum of 100 000 events per sample were analysed and gates were set using appropriate controls. Lymphocytes were identified by their forward and side light scatter, and subsequently, total CD4+ T cells were identified as CD3+CD4+ while naive CD4+ T cells were identified as CD3+CD4+CD45RA+CD27+. The CD31+ subpopulation of naive CD4+ T cells was also assessed, as this phenotype is thought to mark recent thymic emigrants. All analyses were performed using a FACS Canto II cytometer (BD Biosciences). Files were exported in FCS 3.0 format and visualised using FlowJo software, version 7.6.3 (Tree Star, Ashland, Oregon, USA).
Assessment of STAT5 phosphorylation
Cryopreserved PBMC were thawed, washed and incubated for 15 min at room temperature with the following fluorescently conjugated mAbs: CD3-V450, CD4-V500, CD27-FITC and CD31-PE (BD Biosciences). Following surface staining, cells were cultured in the presence or absence of IL-7 (5 ng/ml) at 37°C for 15 min and then fixed using Cytofix Buffer (BD Biosciences). Cells were then permeabilised using Phosflow Perm Buffer III and stained with fluorescently conjugated mAbs to STAT5-AF647 and CD45RA-APC-H7 (BD Biosciences).
Proliferation assay and Ki67 expression
Thawed PBMCs were cultured (106 cells/ml) in the presence or absence of IL-7 (5 ng/ml) at 37°C for 5 days. Cells were then surface stained with the following fluorescently conjugated mAbs: CD3-V450, CD4-V500, CD45RA-APC-H7, CD27-PerCP-Cy5.5, CD28-APC and CD31-PE (BD Biosciences). Intracellular staining using Ki67-FITC was then performed using the Human FoxP3 Buffer Set (BD Biosciences) according to the manufacture's protocol.
Measurement of plasma levels of sCD14, CXCL9 and CXCL10
Plasma levels of soluble (s) CD14 were assayed by enzyme-linked immunosorbent assay (R&D Systems) as described previously . Plasma levels of (C–X–C motif) ligand 9 (CXCL9) and CXCL10 were assayed by cytometric bead arrays .
Statistical analyses were performed with GraphPad Prism software, version 5.01 (Graph-Pad Software, San Diego, California, USA). The Mann–Whitney test was performed for continuous variables and correlation coefficients were determined by the Spearman's rank correlation test. Fisher's exact test was performed for categorical analyses. For all tests, a P value less than 0.05 was considered to represent a significant difference.
Demographic and clinical data on patients are presented in Table 1. All patients had received ART for more than 12 months, but the duration of therapy was longer in patients with normal CD4+ T-cell counts. However, time on ART did not correlate with proportions of total or naive CD4+ T cells in an analysis that combined both patient groups (r = 0.23–0.24, P = 0.14–0.17, respectively). Furthermore, 5 years after study entry, all participants originally classified as having CD4+ T-cell deficiency had CD4+ T-cell counts less than 340 cells/μl after a median (range) time of 12 (4–17) years on ART. In addition, CD4+ T-cell counts obtained from the start of ART until the time of study entry were previously modelled for each patient and best-fit curves used to estimate a 10-year CD4+ T-cell count . Using these data, all patients classified as having CD4+ T-cell deficiency here had estimated CD4+ T-cell counts of less than 350 cells/μl 10 years after commencing ART.
Interleukin-7-induced proliferation assessed in viable T cells did not differentiate HIV patients with low or normal CD4+ T-cell counts
CD4+ T-cell proliferation induced by IL-7 (5 ng/ml) in 5-day cultures was assessed by subtracting the percentages of CD4+ T cells expressing Ki67 in unstimulated cultures from the percentages expressing Ki67 after stimulation (ΔKi67). Low lymphocyte viability (absent lymphocyte population on flow cytometry plots; Supplemental Figure 1, http://links.lww.com/QAD/A484) precluded analysis of data from cultures in some patients. This was more common in patients with CD4+ T-cell deficiency than patients with normal CD4+ T-cell counts (38 vs. 5%; Fisher's exact test, P = 0.02). Analysis of data from patients with CD4+ T-cell deficiency demonstrated no differences in any demographic characteristics described in Table 1 between those with and without viable cells in culture. When analyses were restricted to samples with good cell viability (n = 26), ΔKi67 was similar in patients with low or normal CD4+ T-cell counts (P = 0.93) (Supplemental Figure 2, http://links.lww.com/QAD/A484). Similarly, ΔKi67 in total, naive and CD31+ naive CD4+ T cells did not correlate with numbers of circulating total, naive or CD31+ naive CD4+ T cells, respectively (data not shown).
HIV patients with low CD4+ T-cell counts exhibited diminished CD127 expression on all CD4+ T-cell populations
To assess whether CD127 expression is perturbed on CD4+ T cells, we measured mean fluorescence intensity (MFI) of CD127 expression at day 0 and following 5-day cultures with and without IL-7. At day 0, CD127 expression on total, naive and CD31+ naive CD4+ T cells was higher in patients with normal CD4+ T-cell counts (P = 0.03–0.04; Fig. 1a). Stimulation with IL-7 for 5 days downregulated CD127 in all CD4+ T-cell populations (P = 0.008 to P < 0.0001), but no differences in CD127 expression were evident when patients with CD4+ T-cell deficiency were compared with those with normal CD4+ T-cell counts (P = 0.15–0.70; Fig. 1b).
To evaluate the relative degree of downregulation of CD127, we subtracted the CD127-MFI of cells incubated in medium alone from that of cells incubated with IL-7 (ΔCD127-MFI). The downregulation of CD127 was reported as an absolute value. The ΔCD127-MFI on total CD4+ T cells was lower in patients with normal CD4+ T-cell counts than in patients with CD4+ T-cell deficiency, although statistical significance was not reached (medians of 245 and 398, respectively; Fig. 1c). Furthermore, the ΔCD127-MFI correlated inversely with total CD4+ T-cell counts (r = −0.37, P = 0.04; Fig. 1d) but not naive CD4+ T-cell counts (r = −0.03, P = 0.89) in all patients (n = 39). Levels of CD127 at day 0 did not correlate with the ΔCD127-MFI (r = 0.27, P = 0.16).
CD4+ T-cell proliferation correlated inversely with downregulation of CD127 expression
In 5-day cultures stimulated with IL-7, the ΔCD127-MFI on total, naive and CD31+ naive CD4+ T-cell subsets correlated with ΔKi67 in total (r = 0.39, P = 0.03; Fig. 2a), naive (r = 0.60, P = 0.002; Fig. 2b) and CD31+ naive (r = 0.59, P = 0.003; Fig. 2c) CD4+ T cells. Levels of CD127 expression at day 0 in total, naive and CD31+ naive CD4+ T-cell subsets did not correlate with IL-7-induced ΔKi67 following 5-day cultures in total, naive or CD31+ naive CD4+ T-cell subsets. Thus, greater downregulation of CD127 was associated with increased proliferation.
Interleukin-7-induced STAT5 phosphorylation in CD4+ T cells correlated with circulating CD4+ T-cell counts
To assess the IL-7R signalling pathway, PBMCs were incubated for 15 min with or without IL-7, and the increase in the proportion of cells positive for pSTAT5 above the background levels (ΔpSTAT5) was determined. ΔpSTAT5 was higher in HIV patients with normal CD4+ T-cell counts than patients with CD4+ T-cell deficiency when assessed in total CD4+ T cells (P = 0.003), naive CD4+ T cells (P = 0.003) and CD31+ naive CD4+ T cells (P = 0.008; Fig. 3a). Accordingly, ΔpSTAT5 in each population correlated directly with numbers of total (r = 0.38, P = 0.03), naive (r = 0.43, P = 0.01) and CD31+ naive (r = 0.35, P = 0.04) CD4+ T cells.
Induction of STAT5 phosphorylation by interleukin-7 correlated with CD127 expression on CD4+ T cells but not with proliferation
IL-7 induced ΔpSTAT5 in total, naive and CD31+ naive CD4+ T cells correlated with levels of CD127 expression at day 0 in total (r = 0.62, P = 0.0002; Fig. 3b), naive (r = 0.54, P = 0.001; Fig. 3c) and CD31+ naive (r = 0.37, P = 0.03; Fig. 3d) CD4+ T cells. However, ΔpSTAT5 did not correlate with ΔKi67 (r = −0.08 to −0.16, P = 0.46–0.68) or ΔCD127-MFI (r = −0.002 to −0.18, P = 0.36–0.9) in any CD4+ T-cell subset.
Interleukin-7 induced STAT5 phosphorylation in CD4+ T cells correlated inversely with T-cell activation and senescence
Finally, we examined the relationship between ΔpSTAT5 in CD4+ T cells and markers of CD4+ T-cell activation and senescence and innate immune system activation. As previously demonstrated , patients with low CD4+ T-cell counts had higher expression of both HLA-DR (P = 0.0007) and CD57 (P = 0.01; Fig. 4a) on total CD4+ T cells than patients with normal CD4+ T-cell counts.
Expression of CD57 on total CD4+ T cells correlated inversely with ΔpSTAT5 in total (r = −0.62, P < 0.001; Fig. 4b), naive (r = −0.61, P = 0.002; Fig. 4c) and CD31+ naive CD4+ T cells (r = −0.65, P < 0.001; Fig. 4d). Furthermore, inverse correlations were observed between CD57 expression on total CD4+ T cells and expression of CD127 (at day 0) on total (r = −0.54, P = 0.008; Fig. 4e), naive (r = −0.40, P = 0.02; Fig. 4f) and CD31+ naive CD4+ T cells (r = −0.64, P = 0.03; Fig. 4g). We also observed inverse correlations between the expression of HLA-DR on CD4+ T cells and ΔpSTAT5 in total (r = −0.37, P = 0.03) and naive CD4+ T cells (r = −0.36, P = 0.03), but not CD31+ naive CD4+ T cells (r = −0.17, P = 0.32). However, no correlations were evident between HLA-DR expression on total CD4+ T cells and expression of CD127 (at day 0) on total (r = −0.11, P = 0.54), naive (r = −0.19 P = 0.27) and CD31+ naive CD4+ T cells (r = −0.21, P = 0.22).
In contrast to the findings for T-cell activation and senescence, ΔpSTAT5 in CD4+ T cells was not clearly associated with plasma markers of innate immune system activation. Although ΔpSTAT5 in total CD4+ T cells showed a weak inverse correlation with plasma levels of CXCL9 (r = −0.35, P = 0.04), there was no association with sCD14 (r = −0.08, P = 0.63) or CXCL10 (r = 0.11, P = 0.50) (data not shown).
Substantial disruption of the IL-7/IL-7R pathway has been described in patients with HIV infection receiving effective ART, contributing to impaired naive T-cell homeostasis and persistent CD4+ T-cell deficiency. A greater understanding of these defects may assist in identifying patients likely to benefit from recombinant IL-7 therapy.
Although low cell viability precluded analysis of data from cell cultures in 38% of patients with persistent CD4+ T-cell deficiency, we demonstrated that IL-7-induced proliferation of CD4+ T cells (Ki67 expression) was not associated with numbers of circulating total, naive or CD31+ naive CD4+ T cells. Similarly, Bazdar and Sieg  showed that TCR responsiveness is diminished in naive CD4+ T cells from viremic HIV-infected patients, whereas responsiveness to IL-7-induced stimulation is relatively preserved and IL-7 enhances responses after TCR stimulation. Hence, diminished TCR responsiveness may be more important than impaired responsiveness to IL-7 in causing decreased CD4+ T-cell proliferation in patients with persistent CD4+ T-cell deficiency.
Our findings of lower CD127 expression at day 0 on total, naive and CD31+ naive CD4+ T cells in patients with persistent CD4+ T-cell deficiency are in accord with those of previous studies [31,33,34]. Bai et al.  examined immunological nonresponders and found that low CD127 expression on CD4+ T cells was the only marker associated with incomplete CD4+ T-cell recovery. Furthermore, CD4+ T-cell recovery during ART is associated with genetic polymorphism of CD127 . Hence, low CD4+ T-cell counts may be attributed to the lack of IL-7-induced signalling caused by decreased CD127 availability, resulting in reduced survival of these cells . Our study furthers the work of Bai et al.  by examining the effects of IL-7 stimulation on IL-7R expression on, and induction of pSTAT5 in, naive CD4+ T cells, as well as the relationship with immune activation.
We found that total, naive and CD31+ naive CD4+ T cells from all patients were responsive to IL-7 and able to modulate CD127 expression (Fig. 1b), which occurred to a greater degree in total CD4+ T cells of patients with CD4+ T-cell deficiency and negatively correlated, albeit weakly, with CD4+ T-cell counts (Fig. 1c, d). It is established that transcription and expression of IL-7Rα is suppressed by IL-7 and other pro-survival cytokines [24,25,40,41]. The transient downregulation of IL-7Rα on T cells that have recently received an IL-7 signal ensures that they will not compete with unstimulated T cells for any remaining IL-7, thus increasing T-cell survival.
STAT5 activation is crucial in signalling pathways controlling CD4+ T-cell survival and proliferation, through the induction of anti-apoptotic molecules such as Bcl-2, and through the phosphatidylinositol 3-kinase/Akt pathway [42,43]. We therefore examined IL-7-induced pSTAT5 in CD4+ T-cell subsets. This was greater in total, naive and CD31+ naive CD4+ T cells from patients with normal CD4+ T-cell counts. In these patients, levels of pSTAT5 were relatively uniform (50–95%) and never fell below 50% in any CD4+ T-cell subset. In contrast, levels of pSTAT5 were more variable (8–87%) in patients with CD4+ T-cell deficiency (Fig. 3a). Our findings further those of Camargo et al.  who showed that levels of pSTAT5 in total T cells were higher in patients with CD4+ T-cell counts more than 500 cells/μl than in patients with counts less than 500 cells/μl.
Lower IL-7-induced pSTAT5 expression in CD4+ T-cell of patients with CD4+ T-cell deficiency on ART may have a negative impact on cell survival through reduced induction of anti-apoptotic factors such as Bcl-2 [35,45,46]. We demonstrated that IL-7-induced pSTAT5 expression in total, naive and CD31+ naive CD4+ T cells correlated with CD127 expression on the corresponding CD4+ T-cell subset, as has previously been observed for total T cells . However, IL-7-induced pSTAT5 was not correlated with proliferation in any CD4+ T-cell subsets. Furthermore, when analysis was divided into CD31+ and CD31- naive CD4+ T-cell subsets, the same associations between CD127, pSTAT5 and Ki67 expression were observed (data not shown). The discordance between pSTAT5 and proliferation may have arisen because pSTAT5 was measured following a 15-min incubation with IL-7, whilst Ki67 expression was assessed 5 days post-stimulation with IL-7. In addition, CD4+ T cells from HIV patients receiving ART may be capable of phosphorylating STAT5, but have a reduced ability to translocate STAT5 to the nucleus . Therefore, the link between IL-7-induced pSTAT5 and proliferation may not be linear.
Interestingly, HIV patients with the lowest levels of pSTAT5 (less than 20%) had nonviable cells after stimulation with IL-7 for 5 days (data not shown). The level of CD127 expression has been shown to correlate with IL-7-induced Bcl-2 and CD25 in healthy donors whilst IL-7-induced Bcl-2 and CD25 expression was reduced in untreated viremic HIV patients, who exhibited low CD127 expression . Colle et al.  examined CD4+ T cells following 3–6 days of culture with and without IL-7, and found lower induction of Bcl-2 in treated patients with CD4+ T-cell counts of less than 250 cells/μl than in patients with counts of more than 400 cells/μl. These results suggest that successful ART can partially correct IL-7R signalling defects, but this is least effective in patients with lower CD4+ T-cell counts.
We and others have associated CD4+ T-cell deficiency on ART with immune activation [12,19]. T-cell activation (assessed by HLA-DR or CD38 expression) correlates with reduced CD127 expression on CD4+ T cells in HIV patients [33,47,48], and loss of CD127 expression in HIV infection may be driven by immune activation [49,50]. A notable finding of our study was that CD4+ T-cell activation (HLA-DR+) and senescence (CD57+) correlated inversely with IL-7-induced pSTAT5 in CD4+ T cells and that increased CD4+ T-cell senescence was associated with lower expression of CD127 at day 0 on total, naive and CD31+ naive CD4+ T cells. Hence, HIV patients who have a greater accumulation of senescent CD4+ T cells may be less responsive to IL-7. These findings raise the possibility that IL-7-induced CD4+ T-cell homeostasis in HIV patients receiving ART might be improved by therapies that reduce T-cell activation and senescence. Furthermore, the proportion of CD57+CD4+ T cells should be investigated as a simple predictor of response to recombinant IL-7 therapy.
The association of immunosenescence (CD57+) in total CD4+ T cells with defects of IL-7/IL-7R signalling in naive CD4+ T cells is notable, as naive T cells express very low amounts of CD57. We propose that CD4+ T-cell senescence on the one hand, and low pSTAT5 expression within, and CD127 expression upon, naive CD4+ T cells on the other hand, result from a common cause of T-cell activation. One possible cause is persistent inflammation in lymphoid tissue [13,14].
Although activation of the innate immune system, particularly monocytes, persists in HIV patients receiving ART , we did not demonstrate a clear relationship between plasma markers of innate immune system activation and defects in the IL-7 signalling pathway of naive CD4+ T cells.
Limitations to our study include the low number of patients with CD4+ T-cell deficiency on ART, particularly for studies after PBMC cultures. Also, patients with CD4+ T-cell deficiency had received ART for a shorter period of time. However, we undertook very rigorous analyses to demonstrate the stability of CD4+ T- cell deficiency over time. Particular strengths of our study were the inclusion criterion of a nadir CD4+ T-cell count of less than 100 cells/μl, as low nadir CD4+ T-cell counts are a strong predictor of poor immune reconstitution on ART [51–53], and our definition of CD4+ T-cell deficiency (<350 cells/μl) and normal CD4+ T-cell counts (>500 cells/μl), as these values predict mortality  and, arguably, are more relevant than those used in other studies [39,44,55].
In summary, our findings provide evidence that impaired IL-7/IL-7R signalling, which leads to poor immune reconstitution in HIV patients receiving ART, is associated with activation and senescence of CD4+ T cells. Enhancement of IL-7-induced CD4+ T-cell homeostasis might therefore be achieved by resolution of CD4+ T-cell activation and senescence. Furthermore, assessment of CD4+ T-cell senescence may identify patients who are most likely to benefit from IL-7 therapy for persistent CD4+ T-cell deficiency.
The work was supported by a program grant (510448) from the National Health and Medical Research Council of Australia. This is manuscript number 2013-06 for the Department of Clinical Immunology, Royal Perth Hospital.
M.F. conceived and developed the study, and recruited patients for the study. S.F. and S.T. designed the study. S.T. performed the experiments, conducted data analysis and interpretation, and wrote the manuscript. S.F., P.P. and M.F. were involved in the interpretation of data along with review of the manuscript. All authors have read and approved the text as submitted to AIDS.
Conflicts of interest
The authors certify that they do not have a commercial or other association that might pose a conflict of interest.
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