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Regulatory function of cytomegalovirus-specific CD4+CD27−CD28− T cells
Virology 398 (2010) 158–167
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Virology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o
Regulatory function of cytomegalovirus-specific CD4+CD27−CD28− T cells Adriana Tovar-Salazar, Julie Patterson-Bartlett, Renee Jesser, Adriana Weinberg ⁎ University of Colorado Denver School of Medicine, Aurora, CO, USA
a r t i c l e
i n f o
Article history: Received 15 October 2009 Returned to author for revision 2 November 2009 Accepted 24 November 2009 Available online 24 December 2009 Keywords: Cytomegalovirus Regulatory T cells HIV infection Cell-mediated immunity
a b s t r a c t CMV infection is characterized by high of frequencies of CD27−CD28− T cells. Here we demonstrate that CMV-specific CD4+CD27−CD28− cells are regulatory T cells (TR). CD4+CD27−CD28− cells sorted from CMVstimulated PBMC of CMV-seropositive donors inhibited de novo CMV-specific proliferation of autologous PBMC in a dose-dependent fashion. Compared with the entire CMV-stimulated CD4+ T-cell population, higher proportions of CD4+CD27−CD28− TR expressed FoxP3, TGFβ, granzyme B, perforin, GITR and PD-1, lower proportions expressed CD127 and PD1-L and similar proportions expressed CD25, CTLA4, Fas-L and GITR-L. CMV-CD4+CD27−CD28− TR expanded in response to IL-2, but not to CMV antigenic restimulation. The anti-proliferative effect of CMV-CD4+CD27−CD28− TR significantly decreased after granzyme B or TGFβ inhibition. The CMV-CD4+CD27−CD28− TR of HIV-infected and uninfected donors had similar phenotypes and anti-proliferative potency, but HIV-infected individuals had higher proportions of CMV-CD4+CD27− CD28− TR. The CMV-CD4+CD27−CD28− TR may contribute to the downregulation of CMV-specific and nonspecific immune responses of CMV-infected individuals. © 2009 Elsevier Inc. All rights reserved.
Introduction The role of natural regulatory T cells (TR) in the modulation of immune responses against self antigens has been well established (Jonuleit and Schmitt, 2003; Rubtsov and Rudensky, 2007). Natural TR were also shown to attenuate immune responses against grafted nonself (Adeegbe et al., 2006; Joffre et al., 2008; Sandner et al., 2005) and microbial antigens (Aandahl et al., 2004; Chougnet and Shearer, 2007; Estes et al., 2006; Haribhai et al., 2007; Kinter et al., 2007; Li et al., 2008; Nilsson et al., 2006; Smyk-Pearson et al., 2008; Suffia et al., 2006) and to hinder clearance of neoplastic proliferation (Beyer et al., 2006; Broderick and Bankert, 2006; Chemnitz et al., 2007; Curiel et al., 2003; Filaci et al., 2007). Natural TR with CD4+CD25+FoxP3+ phenotype are generated in the thymus, but CD4+CD25− cells can also acquire TR phenotypes and function in the periphery (Curotto de Lafaille et al., 2004; Kretschmer et al., 2005; Stephens et al., 2007; VukmanovicStejic et al., 2006; Walker et al., 2005). Although there is growing evidence that adaptive TR may arise as a consequence of antigenic stimulation and specifically target the responses against neoplastic cells or microbial pathogens, the characterization of this process is incomplete (Vlad et al., 2005). TR are functionally characterized by their ability to inhibit proliferation of autologous T lymphocytes in response to antigenic stimulation. Phenotypically, the expression of the forkhead transcription factor FoxP3 has been used as a unique marker of TR (Jonuleit and ⁎ Corresponding author. 12700 E. 19th Ave, Mail Stop 8604, Aurora CO 80045, USA. Fax: + 1 303 724 4485. E-mail address: [email protected] (A. Weinberg). 0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2009.11.038
Schmitt, 2003; Stephens et al., 2007). FoxP3 is not only a marker, but plays an important role in the differentiation, function and survival of CD4+ TR (Allan et al., 2005; Fontenot and Rudensky, 2005; Marson et al., 2007; Tone et al., 2008; Zheng et al., 2007; Zheng and Rudensky, 2007). FoxP3 regulates the expression of several hundreds cellular genes either directly or indirectly by modulating expression of other transcription factors. Both mice and humans with genetic defects in the FoxP3 gene develop aggressive auto-immune disorders that can be controlled in mice by adoptive transfer of CD4+CD25+ TR from healthy animals. Other cell surface markers that are commonly used to identify TR are CTLA4, GITR/ GITR-L, GPR83, PD-1/PD1-L and CD127 (Hansen et al., 2006; Liu et al., 2006; Sandner et al., 2005; Seddiki et al., 2006; Sharpe et al., 2007; Shevach and Stephens, 2006; Weihong et al., 2006). The exact mechanism used by TR to downmodulate immune responses has not been completely elucidated (von Boehmer, 2005). To date, there is strong evidence that TGFβ and IL-10 mediate some TR activities (Jonuleit and Schmitt, 2003; Mekala et al., 2005; Thomas and Massague, 2005). In addition, TGFβ also enhances the expression of FoxP3 and has been implicated in the generation of TR in the periphery (Fantini et al., 2004; Rao et al., 2005; Selvaraj and Geiger, 2007; Yamazaki et al., 2007). In our previous studies we identified a subpopulation of CMVspecific T cells phenotypically defined as CD4+CD27−CD28−, whose frequency inversely correlated with in vitro CMV-specific proliferation of peripheral blood mononuclear cells (PBMC) and positively correlated with CMV-stimulated TGFβ production (Jesser et al., 2006). In this study, we show that CD4+CD27−CD28− T cells purified from CMV-stimulated PBMC inhibit de novo proliferation of autologous PBMC in response to CMV antigenic stimulation, fulfilling the
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Fig. 1. CMV-specific CD4+CD27−CD28− T cells inhibit CMV-stimulated proliferation of autologous PBMC. A: Gating strategy for CD4+CD27−CD28− separation. CMV-stimulated PBMC were stained using the following conjugated mAbs: anti-CD3-AmCyan, anti-CD4-PE Texas Red, anti-CD27-FITC and anti-CD28-FITC surface markers. Cells in the lymphocyte gate delineated by side and forward scatter (left plot) were analyzed for expression of CD3 and CD4 (middle plot) and subsequently combined CD27 and CD28 (right plot). Using a cell sorter, CD4+CD27−CD28− cells were separated from CD4+nonCD27−CD28− cells. B: Data were derived from 6 experiments using PBMC from independent donors. Freshly thawed autologous PBMC were treated with CMV-specific CD4+CD27−CD28− cells at the indicated ratios and stimulated with CMV lysate for 6 days. Percent inhibition of CMV-stimulated proliferation was calculated by the following formula: (1 − median cpm in treated wells / median cpm in untreated wells) ⁎ 100. Bars represent means and SEM. The inhibition of proliferation was dose-dependent as shown by linear trend analysis (p value and r2 depicted in the graph). C: Freshly thawed PBMC from 3 donors were treated with autologous CMV-specific CD4+nonCD27−CD28− cells prior to de novo CMV stimulation. Ratios were similar to the experiments depicted in panel B. Untreated indicates no added cells. Bars represent means and SEM of median cpm for each condition. There was no evidence of a CD4+nonCD27−CD28− inhibitory effect on proliferation of autologous PBMC.
definition of TR. We also describe the phenotypic, growth and effector mechanism characteristics of the CMV-stimulated CD4+ CD27−CD28− TR. Results
individuals (CD4 of 200 to 947 cells/μl plasma HIV RNA b400 copies/ml) with those from 6 healthy subjects by adding the TR to their autologous PBMC at a 1:3 ratio in the presence of CMV lysate for 6 days. Inhibition of in vitro proliferation of PBMC by TR was similar in HIV-infected and uninfected subjects (87 ± 3% vs. 85 ± 5%), indicating that the function of CMV-stimulated CD4+CD27−CD28− did not
Anti-proliferative effect of CMV-stimulated CD4+CD27−CD28− TR To assess the TR activity of CMV-stimulated CD4+CD27−CD28− T cells, PBMC from 6 CMV-seropositive healthy donors were stimulated in vitro for 6 days with CMV lysate, after which CD4+CD27−CD28− cells were isolated by flow cytometry (Fig. 1A). The CMV-stimulated CD4+CD27−CD28− T cells or CD4+non-CD27−CD28− controls were added to 100,000 freshly thawed autologous PBMC in quadruplicate wells at 1:3, 1:10 and 1:30 ratios and allowed to expand for 6 days in the presence of CMV antigen. Proliferation was compared with that of untreated PBMC cultures. The addition of CMV-stimulated CD4+ CD27−CD28− cells decreased CMV-specific proliferation of autologous PBMC in a dose-dependent fashion from mean ± SEM 86 ± 5% at a TR: PBMC ratio of 1:3 to 25 ± 12% at a ratio of 1:30 (r2 = 0.61, p = 0.0001, ANOVA for repeated measures; Fig. 1B). In contrast, the addition of CD4+non-CD27−CD28− T cells, which included CD4+CD27+CD28+, CD4+CD27+CD28− and CD4+CD27−CD28+ cells, tended to increase CMV-induced proliferation of the autologous PBMC (Fig. 1C). These data indicated that downregulation of CMV-stimulated PBMC proliferation was a specific effect of the CD4+CD27−CD28− T cells. Next, we compared the anti-proliferative effect of sorted CMVstimulated CD4+CD27−CD28− TR obtained from 3 HIV-infected
Fig. 2. Comparison of the inhibitory effect of CMV-specific CD4+CD27−CD28− TR with that of CD4+CD25+CD127− natural TR on CMV-stimulated proliferation of autologous PBMC. Light bars represent mean and SEM of 9 experiments that measured the inhibitory effect of CMV-specific CD4+CD27−CD28− TR at the indicated ratios. Dark bars represent mean and SEM of 3 experiments that measured the effect of CD4+CD25+ CD127− natural TR on CMV-specific proliferation. The 2-way ANOVA analysis did not reveal any appreciable differences.
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Specificity of the generation and of the effect of CMV-stimulated CD4+CD27−CD28− TR
Fig. 3. Frequency of CD4+CD27−CD28− TR in CMV-stimulated compared with mockinfected control and C. albicans-stimulated PBMC. Data were derived from 10 CMVseropositive HIV-seropositive (CD4 of 200 to 957 cells/μl; plasma HIV RNA b 400 copies/ml), 7 CMV-seropositive HIV-seronegative, and 4 CMV-seronegative HIVseronegative individuals. The enumeration of CD4+CD27−CD28− was performed after 6 days of in vitro PBMC stimulation with the indicated antigens. CMV stimulation significantly increased the proportion of CD4+CD27−CD28− TR in CMV-seropositive individuals, irrespective of their HIV status, but not in CMV-seronegative donors. Candida stimulation moderately increased CD4+CD27−CD28− TR.
appreciably differ between HIV-infected and uninfected individuals. Based on this observation, some of the subsequent analyses combined data generated using cells from HIV-infected and uninfected individuals as described below. To assess the relative TR potency of CMV-induced CD4+CD27− CD28− T cells, we compared their inhibitory effect on proliferation with that of CD4+CD25+CD127− natural TR. Natural TR selected by flow cytometry from freshly thawed PBMC were added at ratios of 1:3, 1:10 and 1:30 to autologous PBMC, which were subsequently stimulated with CMV lysate for 6 days. The data (Fig. 2) showed that CMV-stimulated CD4+CD27−CD28− TR and freshly thawed CD4+CD25+CD127− TR generated comparable inhibition of CMVspecific proliferation when added to autologous PBMC (p = 0.68, 2way ANOVA).
To determine if CD4+CD27−CD28− TR were specifically generated as an adaptive response to antigenic stimulation, we compared the frequencies of CD4+CD27−CD28− T cells in CMV and control antigenstimulated PBMC cultures obtained from 10 CMV-seropositive HIVinfected, 7 CMV-seropositive HIV-seronegative and 4 CMV-seronegative HIV-seronegative subjects (Fig. 3). CMV stimulation generated a significantly higher frequency of CD4+CD27−CD28− TR compared with mock-infected control stimulation in CMV-seropositive HIVseropositive and in CMV-seropositive HIV-seronegative individuals (p of 0.003 and 0.009, respectively). In contrast, CMV-seronegative individuals had equally low frequencies of CD4+CD27−CD28− cells in CMV lysate- and mock-infected control-stimulated PBMC cultures, and a trend towards higher CD4+CD27−CD28− TR in Candida albicans-stimulated cell preparations (p = 0.06). It is important to note that the frequency of CD4+CD27−CD28− cells in control antigenstimulated cultures was significantly lower in CMV-seronegative compared with CMV-seropositive subjects (p = 0.03) and that among CMV-seropositive donors, the frequency of CMV-stimulated CD4+ CD27−CD28− TR was significantly higher among cells from HIVseropositive compared with HIV-seronegative donors (p = 0.01). The specificity of the anti-proliferative effect of CMV-stimulated CD4+CD27−CD28− TR was assessed by comparing their inhibitory effect on proliferation of autologous PBMC stimulated with CMV vs. C. albicans. CD4+CD27−CD28− TR generated in CMV-stimulated cultures were added at a ratio of 1:10 to autologous PBMC, which were subsequently stimulated with C. albicans antigen or CMV lysate for 6 days. The data generated with PBMC from 3 HIV-infected (median CD4 = 322 cells/μl; median plasma HIV RNA b400 copies/ml) and 7 uninfected individuals showed no difference in inhibitory effect by HIV status. Although both CMV- (Fig. 4A) and C. albicans-stimulated (Fig. 4B) proliferation decreased after the addition of CD4+CD27− CD28− TR (p of 0.002 and 0.02, respectively), the inhibitory effect of the CD4+CD27−CD28− TR on CMV-specific proliferation was N3-fold greater than their effect on C. albicans-stimulated proliferation
Fig. 4. Antigen specificity of the anti-proliferative effect of CD4+CD27−CD28− TR. Data were derived from 10 experiments including PBMC from 3 HIV-infected individuals (CD4 of 506 to 947 cells/μl; plasma HIV RNA b400 copies/ml) and 7 healthy subjects. Panel A shows inhibition of CMV-specific proliferation of PBMC after addition of autologous CMVstimulated CD4+CD27−CD28− TR at a ratio of 1:10. Panel B shows C. albicans-specific proliferation in similar experimental conditions. Incorporated radioactivity was measured in counts per minute (cpm). P values were calculated by paired t test of log10cpm. Panel C shows percent inhibition of C. albicans- or CMV-stimulated PBMC calculated with the following formula: (1 − median cpm in treated wells / median cpm in untreated wells) ⁎ 100. Bars represent means and SEM. P values were obtained by paired t test analysis.
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(mean ± SEM inhibition of 73 ± 10% vs. 22 ± 5% p = 0.0098 paired t test; Fig. 4C). Phenotypic characterization of CMV-stimulated CD4+CD27−CD28− TR We assessed the expression of markers known to be overrepresented in previously described natural and adaptive TR . The expression of the markers of interest on CD4+CD27−CD28− TR was compared with their expression on the parent CD4+ T-cell population after 6 days of in vitro CMV stimulation (Fig. 5A). Compared with the
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entire CMV-stimulated CD4+ T-cell population, a higher frequency of CMV-stimulated CD4+CD27−CD28− TR expressed FoxP3 (p = 0.02), TGFβ (p = 0.03), GITR (p = 0.01), PD-1 (p = 0.003), granzyme B (p = 0.0003), and perforin (p = 0.05) (Fig. 5B). A significantly lower proportion of CMV-stimulated CD4+CD27−CD28− TR expressed CD127 and PD-1L (p b 0.0001 and 0.0005, respectively, Fig. 5C). There were no appreciable differences in the frequencies of CD25+, CTLA-4+, GITR-L+, or Fas-L+ cells (Fig. 5D). The comparison of the phenotypic characteristics of CMVstimulated CD4+CD27−CD28− from HIV-infected and uninfected
Fig. 5. Phenotypic characteristics of the CMV-specific CD4+CD27−CD28− TR. PBMC were stimulated in vitro with CMV lysate for 6 days and stained for the indicated markers. Panel A shows an example of the gating strategy used to measure the expression of TGFβ on the CD4+ populations of interest. PBMC were stimulated for 6 days with CMV lysate. The frequency of cells expressing TGFβ or other markers (not depicted) was measured in the total CD4+ cell population (UL + UR) and in the individualized CD4+CD27−CD28− subpopulation (UL). Panel B shows markers over-expressed by the CD4+CD27−CD28− TR (13 to 23 independent samples for each marker). Panel C shows markers under-expressed by CD4+CD27−CD28− TR (5 and 20 samples for CD127 and PD1-L, respectively). Panel D shows markers whose expression was not appreciably different between CD4+ cells and CD4+CD27−CD28− TR (6 to 17 samples/analyte).
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Table 1 Comparative expression of selective phenotypic markers by CMV-specific CD4+CD27− CD28− TR from HIV-infected and uninfected donors. Marker
HIV-infected
HIV-uninfected
pa
FoxP3 TGFβ Granzyme B PD-1 PD1-L
37.7 ± 9b (Nc = 6) 24.0 ± 10.3 (N = 10) 38.5 ± 9.9 (N = 11) 50.3 ± 12.9 (N = 10) 41.2 ± 14.3 (N = 10)
29.2 ± 10.7 (N = 7) 22.8 ± 8.0 (N = 10) 41.9 ± 8.5 (N = 12) 30.4 ± 9.3 (N = 5) 32.9 ± 8.4 (N = 5)
0.56 0.93 0.79 0.33 0.58
a
Calculated by t test. Numbers represent percent CD4+CD27−CD28− cells expressing the indicated marker. c N indicates the number of donors. b
individuals studied in parallel did not reveal appreciable differences (Table 1). The data showed that the CMV-stimulated CD4+CD27− CD28− TR of HIV-infected and uninfected individuals did not appreciably differ with respect to the frequency of cells expressing FoxP3, TGFβ, granzyme B, PD-1 or PD1-L. To determine if the increased expression of FoxP3, TGFβ, granzyme B and PD-1 occurred on a single unique subpopulation of the CMVspecific CD4+CD27−CD28− cells as opposed to each marker being expressed by distinct subpopulations, further phenotypic characterization was performed on PBMC from 8 subjects using polychromatic flow cytometry (Fig. 6A). The frequency of TGFβ-expressing cells was
higher in CD4+CD27−CD28−FoxP3+ cells compared with CD4+ CD27−CD28−FoxP3− cells (66.1 ± 12.4% vs. 29.8 ± 7.9%; p = 0.003; Fig. 6B). Likewise, compared with CD4+CD27−CD28−TGFβ− cells, a higher proportion of CD4+CD27−CD28−TGFβ+ cells expressed FoxP3 (36.2 ± 9.2% vs. 7.2 ± 2.7%; p = 0.006; Fig. 6C); granzyme B (71.0 ± 7.3% vs. 35.2 ± 3.8%; p = 0.0003; Fig. 6D); and PD-1 (40.0 ± 3.5% vs. 15.3 ± 3.3%; p b 0.0001; Fig. 6E). Taken together, these data supported the co-expression of FoxP3, TGFβ, granzyme B and PD-1 by a unique subpopulation of CD4+CD27−CD28− T cells. Growth characteristics of CMV-specific CD4+CD27−CD28− TR To identify the growth restrictions of CMV-specific CD4+CD27− CD28− TR, we compared the proliferation of the CMV-stimulated CD4+CD27−CD28− TR with that of CD4+nonCD27−CD28− lymphocytes in response to CMV lysate, mock-infected control or IL2 stimulation. CD4+CD27−CD28− and CD4+nonCD27−CD28− cells were sorted from CMV-stimulated PBMC using the gating strategy depicted in Fig. 1A, washed and allowed to expand for 6 days in the presence of CMV lysate, mock-infected control antigen or IL2. Compared with mock-infected control antigen stimulation conditions, the CMV-specific CD4+CD27−CD28− TR proliferated in response to IL2 (p = 0.02; Fig. 7A), but not in response to CMV lysate (p = 0.33; N = 4; Fig. 7A). In parallel experiments, the CMV-stimulated CD4+ nonCD27−CD28− T cells proliferated in response to IL-2 (p = 0.002;
Fig. 6. Co-expression of FoxP3, TGFβ, granzyme B and PD-1 on CMV-specific CD4+CD27−CD28− TR. PBMC from 8 donors were stimulated in vitro with CMV lysate for 6 days and stained for the indicated markers. Panel A shows examples of the gating strategy. In the upper group, gated CD4+CD27−CD28− TR (left plot) were analyzed for TGFβ expression (middle plot). Next, we measured the frequency of cells expressing granzyme B among the CD4+CD27−CD28−TGFβ− and the CD4+CD27−CD28−TGFβ+ subpopulations. In the lower left and right areas, a similar strategy is depicted for measuring expression of TGFβ on granzyme B+ compared with granzyme B− cells and on PD-1+ compared with PD-1− cells, respectively. The bar graphs represent means and SEM of the 8 experiments and show that a higher proportion of CMV-specific CD4+CD27−CD28−FoxP3+ cells express TGFβ compared with CD4+CD27−CD28−FoxP3− cells (B); CMV-specific CD4+CD27−CD28−TGFβ+ cells express FoxP3 (C), granzyme B (D) and PD-1(E) in a higher proportion compared with CD4+CD27−CD28−TGFβ− cells.
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Fig. 6 (continued).
Fig. 7B) and in response to CMV lysate (p = 0.02; Fig. 7B). Taken together, these data indicate that both IL2 and CMV lysate stimulation are able to generate expansion of a mixed CD4+ subpopulation.
However, the CMV-specific TR lose their ability to expand in response to CMV stimulation while retaining responses to IL2. Mediators of the regulatory effect of CMV-stimulated CD4+CD27− CD28− TR The phenotypic characterization of the CMV-stimulated CD4+ CD27−CD28− TR suggested several potential candidates as mediators of their anti-proliferative effect, including TGFβ, granzyme B and PD-1. In order to investigate the role of these putative mediators, CMV-stimulated CD4+CD27−CD28− TR were treated with neutralizing mAbs anti-TGFβ or anti-PD-1 or with a granzyme B inhibitor with combined intra- and extra-cellular activity. The anti-proliferative activity of the treated TR was compared with that of untreated TR in paired experiments by adding them to autologous PBMC at a 1:10 ratio and allowing the cell culture to proliferate in the presence of CMV lysate for 6 days. All 3 blocking agents partially reversed the antiproliferative effect of the TR from 72 ± 8 to 51 ± 9% for anti-TGFβ (N = 7; p = 0.04; Fig. 8A); from 69 ± 9% to 59 ± 11% for anti-PD-1 (N = 6; p = 0.05; Fig. 8B); and from 75 ± 9% to 38 ± 14% for the granzyme B inhibitor (N = 7; p = 0.02; Fig. 8C). Parallel experiments in which CMV-stimulated CD4+CD27−CD28− TR were treated with an irrelevant mouse IgG2b mAb prior to incubation with autologous PBMC, showed no appreciable change in the anti-proliferative effect of the TR (N = 4; p = 0.77; Fig. 8D). Overall, inhibition of granzyme B activity had the most robust downregulatory effect on the antiproliferative activity of the CD4+CD27−CD28− TR. There was no synergistic activity of granzyme B, TGFβ and PD-1 inhibitors (data not shown). Discussion
Fig. 7. Growth characteristics of CMV-specific CD4+CD27−CD28− TR. Sorted CMVspecific CD4+CD27−CD28− (A) and CD4+nonCD27−CD28− (B) were restimulated for 6 days with mock-infected control antigen (mock), CMV lysate (CMV) or IL2. Data points represent 3H-thymidine incorporation (cpm) measured at the end of the restimulation period. P values were calculated by paired t test of log10cpm.
We demonstrated the TR activity of CMV-stimulated CD4+CD27− CD28− T cells from CMV-seropositive donors. CMV infection is known to increase the frequency of circulating CD4+CD27−CD28− T cells (Bekker et al., 2005; Bronke et al., 2007; Hooper et al., 1999; van Leeuwen et al., 2004). Because these cells express granzyme B in abundance, it was postulated that they represented cytotoxic effectors whose targets were CMV-infected cells (Casazza et al., 2006; van
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Fig. 8. Mediators of the anti-proliferative effect of CMV-specific CD4+CD27−CD28− TR. CD4+CD27−CD28− TR sorted from CMV-stimulated PBMC were treated with anti-TGFβ neutralizing mouse mAbs (A), anti-PD-1 blocking mouse mAbs (B), granzyme B inhibitors (C) or an irrelevant control mouse mAb (D) before incubation with autologous PBMC. The bars represent inhibition of CMV-stimulated proliferation of PBMC incubated with treated or untreated TR in paired experiments.
Leeuwen et al., 2004). However, we show that CMV-stimulated CD4+ CD27−CD28− cells from CMV-seropositive donors inhibit CMVspecific proliferation of autologous PBMC and exhibit other TR characteristics, such as expression of FoxP3, GITR and TGFβ and not of CD127; lack of growth in response to antigenic restimulation, but significant expansion in the presence of IL2. Further studies are needed to determine if the CD4+CD27−CD28− TR are generated as TR or evolve from effector cells who acquire different functions over the course of the CMV infection in a similar fashion to the process described in mice infected with lymphocytic choriomeningitis virus (Brooks et al., 2006; Knoechel et al., 2005). We explored the mechanism used by the CD4+CD27−CD28− TR to inhibit proliferation of autologous PBMC using blocking experiments. Treatment of the CMV-specific CD4+CD27−CD28− TR with anti-TGFβ neutralizing antibodies reduced the anti-proliferative effect of the TR by 30%. This effect was consistent both with the anti-proliferative effect of TGFβ on Th1 lymphocytes and with the TR-inducing activity of TGFβ (Broderick and Bankert, 2006; Classen et al., 2007; Rao et al., 2005; Rubtsov and Rudensky, 2007). These two effects of TGFβ are not mutually exclusive and both may operate simultaneously. Inhibition of granzyme B, which is expressed by a high proportion of the CD4+CD27−CD28− TR, reduced the CMV-specific anti-proliferative effect of the CD4+CD27−CD28− TR by 50%. Granzyme B is a traditional mediator of cytotoxicity that can act alone or in concert with perforin. We also showed that perforin was expressed by a higher proportion of CMV-stimulated CD4+CD27−CD28− lymphocytes compared with their parent CD4+ T-cell population. Both granzyme B and perforin have been shown to play a critical role in terminating acute immune responses (Gondek et al., 2005; Grossman et al., 2004; Voskoboinik et al., 2006). Granzyme B and other granzymes induce apoptosis of the target cells, which is a common mechanism used by the immune system to regulate its responses (Mateo et al., 2007; Pardo et al., 2004). PD-1 upregulation has been commonly described in association with TR, although some studies found it expressed on the surface of TR
and others found it sequestered in the cytoplasm (Chemnitz et al., 2007; Raimondi et al., 2006; Sandner et al., 2005). Blocking PD-1 on the CD4+CD27−CD28− TR with anti-PD-1 neutralizing mAbs was associated with a 10% reduction of the TR anti-proliferative effect. Although statistically significant, the effect of blocking PD-1 was small and, therefore, its biological significance remains questionable. The increased frequency of circulating CD27− and CD28− T cells is one of the characteristics of CMV infection (Bekker et al., 2005; Pourgheysari et al., 2007; van de Berg et al., 2008; Yue et al., 2004). Based on their in vitro TR activity, we propose that these cells may play a role in the immunosuppression associated with CMV infection. Other investigators described a negative association between the frequency of CD27− and/or CD28− circulating T cells and the immune repertoire of CMV-seropositive hosts (Almanzar et al., 2005; Sylwester et al., 2005; Vescovini et al., 2007). It was also previously shown that CMV-specific T cells suppress responses to other viruses in young and elderly individuals (Khan et al., 2004; Levin et al., 1979) and it was proposed that this phenomenon may contribute to the association between CMV seropositivity and increased risk of death in octogenarians and nonagenarians (Wikby et al., 2005). The immunosuppressive effect of CMV infection has been well documented in transplant patients who become more susceptible to bacterial and fungal super-infections during CMV reactivations. In HIV-infected patients, CMV reactivation is associated with increased risk of death independent of the development of CMV end-organ disease (Deayton et al., 2004; Wohl et al., 2005). We also showed that CMV stimulation of PBMC from HIV-infected subjects elicits higher frequencies of CD4+CD27−CD28− TR than in HIV-seronegative individuals (Jesser et al., 2006). It is conceivable that the CMV-stimulated CD4+CD27−CD28− TR may attenuate immune responses to other pathogens through a nonspecific effect similar to the one that we described against C. albicans antigeninduced proliferation. The role of CD4+CD27−CD28− as mediators of the immune suppression associated with CMV infection deserves to be further investigated.
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Subjects and methods Subjects
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(Treestar). The gating strategy took into account the distribution of cells with respect to the markers of interest and the fluorescence minus one histograms. Cell sorting
PBMC used in this study were obtained from 15 HIV-infected and 23 uninfected subjects. All subjects were CMV-seropositive. Inclusion criteria for HIV-infected individuals included HAART treatment ≥3 months (3-drug regimen, including at least one protease inhibitor or a non-nucleoside analogue) and CD4+ counts ≥ 100 cells/μl at study entry. At study visits for immunologic evaluations, the median CD4+ numbers of HIV-infected subjects was 506 cells/μl (range of 200 to 947 cells/μl). HIV viral load was b400 copies/ml. The median time on HAART was 20 months.
After 6 days of CMV stimulation at 37 °C in 5% CO2 atmosphere, PBMC were washed twice in 2% FBS in PBS and stained for surface markers as described above. After that, cells were washed in 2% FBS in PBS and sorted using FACSAria using FACSDiva software (BD Bioscience) or MoFlo™ XDP (Dako) using Summit software. Sorted PBMC were collected in stimulation medium and used in functional experiments.
Monoclonal antibodies (MAb) and other reagents
Blocking experiments
The following staining reagents were used in this study: anti-CD3AmCyan, anti-CD27-FITC, anti-CD28-FITC and anti-CTLA-4-APC (BD Biosciences); anti-CD4-PE Texas Red, anti-granzyme B-PE-Cy5.5, antiCD25-APC and anti-Fas-L-APC (Caltag); anti-TGF-β-biotin, streptavidin-PE-Cy7, anti-FoxP3-APC, anti-PD1-L, anti-PD-1-PE, anti-GITR-LAlexa647, anti-GITR-PerCP and streptavidin-APC (eBiosciences); goat anti-mouse IgG-Pacific Blue, goat anti-mouse IgG-Alexa Fluor 750APC and anti-GPR83-APC (Invitrogen); anti-TGF-β-PE (IQ Products); and anti-perforin-biotin (Ancell). The following blocking reagents were used in this study: anti-TGF-β1 and anti-human PD-1 mouse mAb's (R&D Systems, Inc), granzyme B inhibitor II, cell-permeable (Calbiochem).
Purified CD4+CD27−CD28− cells were treated with granzyme B inhibitor II (Calbiochem), neutralizing mAbs anti-TGFβ (R&D Systems, Inc) or anti-PD1 (R&D Systems, Inc), or an irrelevant mouse mAb control (BD Bioscience) for 2 h at 37 °C in 5% CO2 atmosphere in stimulation medium. Treated and untreated CD4+CD27−CD28− cells were added to freshly thawed autologous PBMC in the presence of inactivated CMV-infected cell lysate. After 6 days of incubation in quadruplicate 96-microtiter plate wells/stimulant, cells were pulsed with 3H-thymidine, harvested and counted as above.
Specimen processing and cryopreservation PBMC from heparinized blood, separated by Ficoll-Hypaque density gradient centrifugation (Sigma Chemical Company), were cryopreserved at 107 cells/ml in fetal bovine serum (FBS, Gemini BioProducts) with 10% dimethyl sulfoxide (DMSO, Sigma Chemical Company) and stored in liquid nitrogen tanks until use. Lymphocyte proliferation assay (LPA) PBMC at 106 cells/ml in stimulation medium consisting of RPMI 1640 with L-glutamine (Gibco), 10% human AB serum (GemCell) and 1% penicillin–streptomycin (Gibco) were incubated in quadruplicate wells of 96-well microtiter plates at 100 μl/well with 100 μl of inactivated CMV-infected cell lysate, mock-infected cell control antigen, recombinant human IL-2 at 4 ng/ml (R&D Systems, Inc), C. albicans antigen (Greer), or phytohemagglutinin (Sigma). After 6 days of culture at 37 °C in 5% CO2 atmosphere, cells were pulsed with 1 μCi of 3H-thymidine and their DNA was harvested 6 h later onto Unifilter plates (Perkin Elmer). Radioactivity gathered on the filters was counted with a microplate scintillation counter (Packard) and scintillation fluid (Perkin Elmer). Results were expressed in median counts per minutes (cpm) of the quadruplicate wells. T-cell phenotypic characterization PBMC and separated T-cell subpopulations were allowed to expand in culture tubes containing stimulation medium with CMV lysate, C. albicans or control antigen for 6 days at 37 °C in 5% CO2 atmosphere, after which cells were washed twice in 2% FBS in PBS and incubated for 30 min at room temperature with the surface marker mAbs described above. For intracellular staining, PBMC were then permeabilized and fixed (Cytofix/Cytoperm; BD Pharmingen) for 20 min at 4 °C, washed and incubated with the appropriate mAbs. At the completion of the staining procedure, PBMC were washed in 2% FBS in PBS and analyzed with a FACSCalibur using CellQuest software or FACSAria using FACSDiva software (BD Biosciences), or FlowJo
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Virology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o
Regulatory function of cytomegalovirus-specific CD4+CD27−CD28− T cells Adriana Tovar-Salazar, Julie Patterson-Bartlett, Renee Jesser, Adriana Weinberg ⁎ University of Colorado Denver School of Medicine, Aurora, CO, USA
a r t i c l e
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Article history: Received 15 October 2009 Returned to author for revision 2 November 2009 Accepted 24 November 2009 Available online 24 December 2009 Keywords: Cytomegalovirus Regulatory T cells HIV infection Cell-mediated immunity
a b s t r a c t CMV infection is characterized by high of frequencies of CD27−CD28− T cells. Here we demonstrate that CMV-specific CD4+CD27−CD28− cells are regulatory T cells (TR). CD4+CD27−CD28− cells sorted from CMVstimulated PBMC of CMV-seropositive donors inhibited de novo CMV-specific proliferation of autologous PBMC in a dose-dependent fashion. Compared with the entire CMV-stimulated CD4+ T-cell population, higher proportions of CD4+CD27−CD28− TR expressed FoxP3, TGFβ, granzyme B, perforin, GITR and PD-1, lower proportions expressed CD127 and PD1-L and similar proportions expressed CD25, CTLA4, Fas-L and GITR-L. CMV-CD4+CD27−CD28− TR expanded in response to IL-2, but not to CMV antigenic restimulation. The anti-proliferative effect of CMV-CD4+CD27−CD28− TR significantly decreased after granzyme B or TGFβ inhibition. The CMV-CD4+CD27−CD28− TR of HIV-infected and uninfected donors had similar phenotypes and anti-proliferative potency, but HIV-infected individuals had higher proportions of CMV-CD4+CD27− CD28− TR. The CMV-CD4+CD27−CD28− TR may contribute to the downregulation of CMV-specific and nonspecific immune responses of CMV-infected individuals. © 2009 Elsevier Inc. All rights reserved.
Introduction The role of natural regulatory T cells (TR) in the modulation of immune responses against self antigens has been well established (Jonuleit and Schmitt, 2003; Rubtsov and Rudensky, 2007). Natural TR were also shown to attenuate immune responses against grafted nonself (Adeegbe et al., 2006; Joffre et al., 2008; Sandner et al., 2005) and microbial antigens (Aandahl et al., 2004; Chougnet and Shearer, 2007; Estes et al., 2006; Haribhai et al., 2007; Kinter et al., 2007; Li et al., 2008; Nilsson et al., 2006; Smyk-Pearson et al., 2008; Suffia et al., 2006) and to hinder clearance of neoplastic proliferation (Beyer et al., 2006; Broderick and Bankert, 2006; Chemnitz et al., 2007; Curiel et al., 2003; Filaci et al., 2007). Natural TR with CD4+CD25+FoxP3+ phenotype are generated in the thymus, but CD4+CD25− cells can also acquire TR phenotypes and function in the periphery (Curotto de Lafaille et al., 2004; Kretschmer et al., 2005; Stephens et al., 2007; VukmanovicStejic et al., 2006; Walker et al., 2005). Although there is growing evidence that adaptive TR may arise as a consequence of antigenic stimulation and specifically target the responses against neoplastic cells or microbial pathogens, the characterization of this process is incomplete (Vlad et al., 2005). TR are functionally characterized by their ability to inhibit proliferation of autologous T lymphocytes in response to antigenic stimulation. Phenotypically, the expression of the forkhead transcription factor FoxP3 has been used as a unique marker of TR (Jonuleit and ⁎ Corresponding author. 12700 E. 19th Ave, Mail Stop 8604, Aurora CO 80045, USA. Fax: + 1 303 724 4485. E-mail address: [email protected] (A. Weinberg). 0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2009.11.038
Schmitt, 2003; Stephens et al., 2007). FoxP3 is not only a marker, but plays an important role in the differentiation, function and survival of CD4+ TR (Allan et al., 2005; Fontenot and Rudensky, 2005; Marson et al., 2007; Tone et al., 2008; Zheng et al., 2007; Zheng and Rudensky, 2007). FoxP3 regulates the expression of several hundreds cellular genes either directly or indirectly by modulating expression of other transcription factors. Both mice and humans with genetic defects in the FoxP3 gene develop aggressive auto-immune disorders that can be controlled in mice by adoptive transfer of CD4+CD25+ TR from healthy animals. Other cell surface markers that are commonly used to identify TR are CTLA4, GITR/ GITR-L, GPR83, PD-1/PD1-L and CD127 (Hansen et al., 2006; Liu et al., 2006; Sandner et al., 2005; Seddiki et al., 2006; Sharpe et al., 2007; Shevach and Stephens, 2006; Weihong et al., 2006). The exact mechanism used by TR to downmodulate immune responses has not been completely elucidated (von Boehmer, 2005). To date, there is strong evidence that TGFβ and IL-10 mediate some TR activities (Jonuleit and Schmitt, 2003; Mekala et al., 2005; Thomas and Massague, 2005). In addition, TGFβ also enhances the expression of FoxP3 and has been implicated in the generation of TR in the periphery (Fantini et al., 2004; Rao et al., 2005; Selvaraj and Geiger, 2007; Yamazaki et al., 2007). In our previous studies we identified a subpopulation of CMVspecific T cells phenotypically defined as CD4+CD27−CD28−, whose frequency inversely correlated with in vitro CMV-specific proliferation of peripheral blood mononuclear cells (PBMC) and positively correlated with CMV-stimulated TGFβ production (Jesser et al., 2006). In this study, we show that CD4+CD27−CD28− T cells purified from CMV-stimulated PBMC inhibit de novo proliferation of autologous PBMC in response to CMV antigenic stimulation, fulfilling the
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Fig. 1. CMV-specific CD4+CD27−CD28− T cells inhibit CMV-stimulated proliferation of autologous PBMC. A: Gating strategy for CD4+CD27−CD28− separation. CMV-stimulated PBMC were stained using the following conjugated mAbs: anti-CD3-AmCyan, anti-CD4-PE Texas Red, anti-CD27-FITC and anti-CD28-FITC surface markers. Cells in the lymphocyte gate delineated by side and forward scatter (left plot) were analyzed for expression of CD3 and CD4 (middle plot) and subsequently combined CD27 and CD28 (right plot). Using a cell sorter, CD4+CD27−CD28− cells were separated from CD4+nonCD27−CD28− cells. B: Data were derived from 6 experiments using PBMC from independent donors. Freshly thawed autologous PBMC were treated with CMV-specific CD4+CD27−CD28− cells at the indicated ratios and stimulated with CMV lysate for 6 days. Percent inhibition of CMV-stimulated proliferation was calculated by the following formula: (1 − median cpm in treated wells / median cpm in untreated wells) ⁎ 100. Bars represent means and SEM. The inhibition of proliferation was dose-dependent as shown by linear trend analysis (p value and r2 depicted in the graph). C: Freshly thawed PBMC from 3 donors were treated with autologous CMV-specific CD4+nonCD27−CD28− cells prior to de novo CMV stimulation. Ratios were similar to the experiments depicted in panel B. Untreated indicates no added cells. Bars represent means and SEM of median cpm for each condition. There was no evidence of a CD4+nonCD27−CD28− inhibitory effect on proliferation of autologous PBMC.
definition of TR. We also describe the phenotypic, growth and effector mechanism characteristics of the CMV-stimulated CD4+ CD27−CD28− TR. Results
individuals (CD4 of 200 to 947 cells/μl plasma HIV RNA b400 copies/ml) with those from 6 healthy subjects by adding the TR to their autologous PBMC at a 1:3 ratio in the presence of CMV lysate for 6 days. Inhibition of in vitro proliferation of PBMC by TR was similar in HIV-infected and uninfected subjects (87 ± 3% vs. 85 ± 5%), indicating that the function of CMV-stimulated CD4+CD27−CD28− did not
Anti-proliferative effect of CMV-stimulated CD4+CD27−CD28− TR To assess the TR activity of CMV-stimulated CD4+CD27−CD28− T cells, PBMC from 6 CMV-seropositive healthy donors were stimulated in vitro for 6 days with CMV lysate, after which CD4+CD27−CD28− cells were isolated by flow cytometry (Fig. 1A). The CMV-stimulated CD4+CD27−CD28− T cells or CD4+non-CD27−CD28− controls were added to 100,000 freshly thawed autologous PBMC in quadruplicate wells at 1:3, 1:10 and 1:30 ratios and allowed to expand for 6 days in the presence of CMV antigen. Proliferation was compared with that of untreated PBMC cultures. The addition of CMV-stimulated CD4+ CD27−CD28− cells decreased CMV-specific proliferation of autologous PBMC in a dose-dependent fashion from mean ± SEM 86 ± 5% at a TR: PBMC ratio of 1:3 to 25 ± 12% at a ratio of 1:30 (r2 = 0.61, p = 0.0001, ANOVA for repeated measures; Fig. 1B). In contrast, the addition of CD4+non-CD27−CD28− T cells, which included CD4+CD27+CD28+, CD4+CD27+CD28− and CD4+CD27−CD28+ cells, tended to increase CMV-induced proliferation of the autologous PBMC (Fig. 1C). These data indicated that downregulation of CMV-stimulated PBMC proliferation was a specific effect of the CD4+CD27−CD28− T cells. Next, we compared the anti-proliferative effect of sorted CMVstimulated CD4+CD27−CD28− TR obtained from 3 HIV-infected
Fig. 2. Comparison of the inhibitory effect of CMV-specific CD4+CD27−CD28− TR with that of CD4+CD25+CD127− natural TR on CMV-stimulated proliferation of autologous PBMC. Light bars represent mean and SEM of 9 experiments that measured the inhibitory effect of CMV-specific CD4+CD27−CD28− TR at the indicated ratios. Dark bars represent mean and SEM of 3 experiments that measured the effect of CD4+CD25+ CD127− natural TR on CMV-specific proliferation. The 2-way ANOVA analysis did not reveal any appreciable differences.
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Specificity of the generation and of the effect of CMV-stimulated CD4+CD27−CD28− TR
Fig. 3. Frequency of CD4+CD27−CD28− TR in CMV-stimulated compared with mockinfected control and C. albicans-stimulated PBMC. Data were derived from 10 CMVseropositive HIV-seropositive (CD4 of 200 to 957 cells/μl; plasma HIV RNA b 400 copies/ml), 7 CMV-seropositive HIV-seronegative, and 4 CMV-seronegative HIVseronegative individuals. The enumeration of CD4+CD27−CD28− was performed after 6 days of in vitro PBMC stimulation with the indicated antigens. CMV stimulation significantly increased the proportion of CD4+CD27−CD28− TR in CMV-seropositive individuals, irrespective of their HIV status, but not in CMV-seronegative donors. Candida stimulation moderately increased CD4+CD27−CD28− TR.
appreciably differ between HIV-infected and uninfected individuals. Based on this observation, some of the subsequent analyses combined data generated using cells from HIV-infected and uninfected individuals as described below. To assess the relative TR potency of CMV-induced CD4+CD27− CD28− T cells, we compared their inhibitory effect on proliferation with that of CD4+CD25+CD127− natural TR. Natural TR selected by flow cytometry from freshly thawed PBMC were added at ratios of 1:3, 1:10 and 1:30 to autologous PBMC, which were subsequently stimulated with CMV lysate for 6 days. The data (Fig. 2) showed that CMV-stimulated CD4+CD27−CD28− TR and freshly thawed CD4+CD25+CD127− TR generated comparable inhibition of CMVspecific proliferation when added to autologous PBMC (p = 0.68, 2way ANOVA).
To determine if CD4+CD27−CD28− TR were specifically generated as an adaptive response to antigenic stimulation, we compared the frequencies of CD4+CD27−CD28− T cells in CMV and control antigenstimulated PBMC cultures obtained from 10 CMV-seropositive HIVinfected, 7 CMV-seropositive HIV-seronegative and 4 CMV-seronegative HIV-seronegative subjects (Fig. 3). CMV stimulation generated a significantly higher frequency of CD4+CD27−CD28− TR compared with mock-infected control stimulation in CMV-seropositive HIVseropositive and in CMV-seropositive HIV-seronegative individuals (p of 0.003 and 0.009, respectively). In contrast, CMV-seronegative individuals had equally low frequencies of CD4+CD27−CD28− cells in CMV lysate- and mock-infected control-stimulated PBMC cultures, and a trend towards higher CD4+CD27−CD28− TR in Candida albicans-stimulated cell preparations (p = 0.06). It is important to note that the frequency of CD4+CD27−CD28− cells in control antigenstimulated cultures was significantly lower in CMV-seronegative compared with CMV-seropositive subjects (p = 0.03) and that among CMV-seropositive donors, the frequency of CMV-stimulated CD4+ CD27−CD28− TR was significantly higher among cells from HIVseropositive compared with HIV-seronegative donors (p = 0.01). The specificity of the anti-proliferative effect of CMV-stimulated CD4+CD27−CD28− TR was assessed by comparing their inhibitory effect on proliferation of autologous PBMC stimulated with CMV vs. C. albicans. CD4+CD27−CD28− TR generated in CMV-stimulated cultures were added at a ratio of 1:10 to autologous PBMC, which were subsequently stimulated with C. albicans antigen or CMV lysate for 6 days. The data generated with PBMC from 3 HIV-infected (median CD4 = 322 cells/μl; median plasma HIV RNA b400 copies/ml) and 7 uninfected individuals showed no difference in inhibitory effect by HIV status. Although both CMV- (Fig. 4A) and C. albicans-stimulated (Fig. 4B) proliferation decreased after the addition of CD4+CD27− CD28− TR (p of 0.002 and 0.02, respectively), the inhibitory effect of the CD4+CD27−CD28− TR on CMV-specific proliferation was N3-fold greater than their effect on C. albicans-stimulated proliferation
Fig. 4. Antigen specificity of the anti-proliferative effect of CD4+CD27−CD28− TR. Data were derived from 10 experiments including PBMC from 3 HIV-infected individuals (CD4 of 506 to 947 cells/μl; plasma HIV RNA b400 copies/ml) and 7 healthy subjects. Panel A shows inhibition of CMV-specific proliferation of PBMC after addition of autologous CMVstimulated CD4+CD27−CD28− TR at a ratio of 1:10. Panel B shows C. albicans-specific proliferation in similar experimental conditions. Incorporated radioactivity was measured in counts per minute (cpm). P values were calculated by paired t test of log10cpm. Panel C shows percent inhibition of C. albicans- or CMV-stimulated PBMC calculated with the following formula: (1 − median cpm in treated wells / median cpm in untreated wells) ⁎ 100. Bars represent means and SEM. P values were obtained by paired t test analysis.
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(mean ± SEM inhibition of 73 ± 10% vs. 22 ± 5% p = 0.0098 paired t test; Fig. 4C). Phenotypic characterization of CMV-stimulated CD4+CD27−CD28− TR We assessed the expression of markers known to be overrepresented in previously described natural and adaptive TR . The expression of the markers of interest on CD4+CD27−CD28− TR was compared with their expression on the parent CD4+ T-cell population after 6 days of in vitro CMV stimulation (Fig. 5A). Compared with the
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entire CMV-stimulated CD4+ T-cell population, a higher frequency of CMV-stimulated CD4+CD27−CD28− TR expressed FoxP3 (p = 0.02), TGFβ (p = 0.03), GITR (p = 0.01), PD-1 (p = 0.003), granzyme B (p = 0.0003), and perforin (p = 0.05) (Fig. 5B). A significantly lower proportion of CMV-stimulated CD4+CD27−CD28− TR expressed CD127 and PD-1L (p b 0.0001 and 0.0005, respectively, Fig. 5C). There were no appreciable differences in the frequencies of CD25+, CTLA-4+, GITR-L+, or Fas-L+ cells (Fig. 5D). The comparison of the phenotypic characteristics of CMVstimulated CD4+CD27−CD28− from HIV-infected and uninfected
Fig. 5. Phenotypic characteristics of the CMV-specific CD4+CD27−CD28− TR. PBMC were stimulated in vitro with CMV lysate for 6 days and stained for the indicated markers. Panel A shows an example of the gating strategy used to measure the expression of TGFβ on the CD4+ populations of interest. PBMC were stimulated for 6 days with CMV lysate. The frequency of cells expressing TGFβ or other markers (not depicted) was measured in the total CD4+ cell population (UL + UR) and in the individualized CD4+CD27−CD28− subpopulation (UL). Panel B shows markers over-expressed by the CD4+CD27−CD28− TR (13 to 23 independent samples for each marker). Panel C shows markers under-expressed by CD4+CD27−CD28− TR (5 and 20 samples for CD127 and PD1-L, respectively). Panel D shows markers whose expression was not appreciably different between CD4+ cells and CD4+CD27−CD28− TR (6 to 17 samples/analyte).
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Table 1 Comparative expression of selective phenotypic markers by CMV-specific CD4+CD27− CD28− TR from HIV-infected and uninfected donors. Marker
HIV-infected
HIV-uninfected
pa
FoxP3 TGFβ Granzyme B PD-1 PD1-L
37.7 ± 9b (Nc = 6) 24.0 ± 10.3 (N = 10) 38.5 ± 9.9 (N = 11) 50.3 ± 12.9 (N = 10) 41.2 ± 14.3 (N = 10)
29.2 ± 10.7 (N = 7) 22.8 ± 8.0 (N = 10) 41.9 ± 8.5 (N = 12) 30.4 ± 9.3 (N = 5) 32.9 ± 8.4 (N = 5)
0.56 0.93 0.79 0.33 0.58
a
Calculated by t test. Numbers represent percent CD4+CD27−CD28− cells expressing the indicated marker. c N indicates the number of donors. b
individuals studied in parallel did not reveal appreciable differences (Table 1). The data showed that the CMV-stimulated CD4+CD27− CD28− TR of HIV-infected and uninfected individuals did not appreciably differ with respect to the frequency of cells expressing FoxP3, TGFβ, granzyme B, PD-1 or PD1-L. To determine if the increased expression of FoxP3, TGFβ, granzyme B and PD-1 occurred on a single unique subpopulation of the CMVspecific CD4+CD27−CD28− cells as opposed to each marker being expressed by distinct subpopulations, further phenotypic characterization was performed on PBMC from 8 subjects using polychromatic flow cytometry (Fig. 6A). The frequency of TGFβ-expressing cells was
higher in CD4+CD27−CD28−FoxP3+ cells compared with CD4+ CD27−CD28−FoxP3− cells (66.1 ± 12.4% vs. 29.8 ± 7.9%; p = 0.003; Fig. 6B). Likewise, compared with CD4+CD27−CD28−TGFβ− cells, a higher proportion of CD4+CD27−CD28−TGFβ+ cells expressed FoxP3 (36.2 ± 9.2% vs. 7.2 ± 2.7%; p = 0.006; Fig. 6C); granzyme B (71.0 ± 7.3% vs. 35.2 ± 3.8%; p = 0.0003; Fig. 6D); and PD-1 (40.0 ± 3.5% vs. 15.3 ± 3.3%; p b 0.0001; Fig. 6E). Taken together, these data supported the co-expression of FoxP3, TGFβ, granzyme B and PD-1 by a unique subpopulation of CD4+CD27−CD28− T cells. Growth characteristics of CMV-specific CD4+CD27−CD28− TR To identify the growth restrictions of CMV-specific CD4+CD27− CD28− TR, we compared the proliferation of the CMV-stimulated CD4+CD27−CD28− TR with that of CD4+nonCD27−CD28− lymphocytes in response to CMV lysate, mock-infected control or IL2 stimulation. CD4+CD27−CD28− and CD4+nonCD27−CD28− cells were sorted from CMV-stimulated PBMC using the gating strategy depicted in Fig. 1A, washed and allowed to expand for 6 days in the presence of CMV lysate, mock-infected control antigen or IL2. Compared with mock-infected control antigen stimulation conditions, the CMV-specific CD4+CD27−CD28− TR proliferated in response to IL2 (p = 0.02; Fig. 7A), but not in response to CMV lysate (p = 0.33; N = 4; Fig. 7A). In parallel experiments, the CMV-stimulated CD4+ nonCD27−CD28− T cells proliferated in response to IL-2 (p = 0.002;
Fig. 6. Co-expression of FoxP3, TGFβ, granzyme B and PD-1 on CMV-specific CD4+CD27−CD28− TR. PBMC from 8 donors were stimulated in vitro with CMV lysate for 6 days and stained for the indicated markers. Panel A shows examples of the gating strategy. In the upper group, gated CD4+CD27−CD28− TR (left plot) were analyzed for TGFβ expression (middle plot). Next, we measured the frequency of cells expressing granzyme B among the CD4+CD27−CD28−TGFβ− and the CD4+CD27−CD28−TGFβ+ subpopulations. In the lower left and right areas, a similar strategy is depicted for measuring expression of TGFβ on granzyme B+ compared with granzyme B− cells and on PD-1+ compared with PD-1− cells, respectively. The bar graphs represent means and SEM of the 8 experiments and show that a higher proportion of CMV-specific CD4+CD27−CD28−FoxP3+ cells express TGFβ compared with CD4+CD27−CD28−FoxP3− cells (B); CMV-specific CD4+CD27−CD28−TGFβ+ cells express FoxP3 (C), granzyme B (D) and PD-1(E) in a higher proportion compared with CD4+CD27−CD28−TGFβ− cells.
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Fig. 6 (continued).
Fig. 7B) and in response to CMV lysate (p = 0.02; Fig. 7B). Taken together, these data indicate that both IL2 and CMV lysate stimulation are able to generate expansion of a mixed CD4+ subpopulation.
However, the CMV-specific TR lose their ability to expand in response to CMV stimulation while retaining responses to IL2. Mediators of the regulatory effect of CMV-stimulated CD4+CD27− CD28− TR The phenotypic characterization of the CMV-stimulated CD4+ CD27−CD28− TR suggested several potential candidates as mediators of their anti-proliferative effect, including TGFβ, granzyme B and PD-1. In order to investigate the role of these putative mediators, CMV-stimulated CD4+CD27−CD28− TR were treated with neutralizing mAbs anti-TGFβ or anti-PD-1 or with a granzyme B inhibitor with combined intra- and extra-cellular activity. The anti-proliferative activity of the treated TR was compared with that of untreated TR in paired experiments by adding them to autologous PBMC at a 1:10 ratio and allowing the cell culture to proliferate in the presence of CMV lysate for 6 days. All 3 blocking agents partially reversed the antiproliferative effect of the TR from 72 ± 8 to 51 ± 9% for anti-TGFβ (N = 7; p = 0.04; Fig. 8A); from 69 ± 9% to 59 ± 11% for anti-PD-1 (N = 6; p = 0.05; Fig. 8B); and from 75 ± 9% to 38 ± 14% for the granzyme B inhibitor (N = 7; p = 0.02; Fig. 8C). Parallel experiments in which CMV-stimulated CD4+CD27−CD28− TR were treated with an irrelevant mouse IgG2b mAb prior to incubation with autologous PBMC, showed no appreciable change in the anti-proliferative effect of the TR (N = 4; p = 0.77; Fig. 8D). Overall, inhibition of granzyme B activity had the most robust downregulatory effect on the antiproliferative activity of the CD4+CD27−CD28− TR. There was no synergistic activity of granzyme B, TGFβ and PD-1 inhibitors (data not shown). Discussion
Fig. 7. Growth characteristics of CMV-specific CD4+CD27−CD28− TR. Sorted CMVspecific CD4+CD27−CD28− (A) and CD4+nonCD27−CD28− (B) were restimulated for 6 days with mock-infected control antigen (mock), CMV lysate (CMV) or IL2. Data points represent 3H-thymidine incorporation (cpm) measured at the end of the restimulation period. P values were calculated by paired t test of log10cpm.
We demonstrated the TR activity of CMV-stimulated CD4+CD27− CD28− T cells from CMV-seropositive donors. CMV infection is known to increase the frequency of circulating CD4+CD27−CD28− T cells (Bekker et al., 2005; Bronke et al., 2007; Hooper et al., 1999; van Leeuwen et al., 2004). Because these cells express granzyme B in abundance, it was postulated that they represented cytotoxic effectors whose targets were CMV-infected cells (Casazza et al., 2006; van
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Fig. 8. Mediators of the anti-proliferative effect of CMV-specific CD4+CD27−CD28− TR. CD4+CD27−CD28− TR sorted from CMV-stimulated PBMC were treated with anti-TGFβ neutralizing mouse mAbs (A), anti-PD-1 blocking mouse mAbs (B), granzyme B inhibitors (C) or an irrelevant control mouse mAb (D) before incubation with autologous PBMC. The bars represent inhibition of CMV-stimulated proliferation of PBMC incubated with treated or untreated TR in paired experiments.
Leeuwen et al., 2004). However, we show that CMV-stimulated CD4+ CD27−CD28− cells from CMV-seropositive donors inhibit CMVspecific proliferation of autologous PBMC and exhibit other TR characteristics, such as expression of FoxP3, GITR and TGFβ and not of CD127; lack of growth in response to antigenic restimulation, but significant expansion in the presence of IL2. Further studies are needed to determine if the CD4+CD27−CD28− TR are generated as TR or evolve from effector cells who acquire different functions over the course of the CMV infection in a similar fashion to the process described in mice infected with lymphocytic choriomeningitis virus (Brooks et al., 2006; Knoechel et al., 2005). We explored the mechanism used by the CD4+CD27−CD28− TR to inhibit proliferation of autologous PBMC using blocking experiments. Treatment of the CMV-specific CD4+CD27−CD28− TR with anti-TGFβ neutralizing antibodies reduced the anti-proliferative effect of the TR by 30%. This effect was consistent both with the anti-proliferative effect of TGFβ on Th1 lymphocytes and with the TR-inducing activity of TGFβ (Broderick and Bankert, 2006; Classen et al., 2007; Rao et al., 2005; Rubtsov and Rudensky, 2007). These two effects of TGFβ are not mutually exclusive and both may operate simultaneously. Inhibition of granzyme B, which is expressed by a high proportion of the CD4+CD27−CD28− TR, reduced the CMV-specific anti-proliferative effect of the CD4+CD27−CD28− TR by 50%. Granzyme B is a traditional mediator of cytotoxicity that can act alone or in concert with perforin. We also showed that perforin was expressed by a higher proportion of CMV-stimulated CD4+CD27−CD28− lymphocytes compared with their parent CD4+ T-cell population. Both granzyme B and perforin have been shown to play a critical role in terminating acute immune responses (Gondek et al., 2005; Grossman et al., 2004; Voskoboinik et al., 2006). Granzyme B and other granzymes induce apoptosis of the target cells, which is a common mechanism used by the immune system to regulate its responses (Mateo et al., 2007; Pardo et al., 2004). PD-1 upregulation has been commonly described in association with TR, although some studies found it expressed on the surface of TR
and others found it sequestered in the cytoplasm (Chemnitz et al., 2007; Raimondi et al., 2006; Sandner et al., 2005). Blocking PD-1 on the CD4+CD27−CD28− TR with anti-PD-1 neutralizing mAbs was associated with a 10% reduction of the TR anti-proliferative effect. Although statistically significant, the effect of blocking PD-1 was small and, therefore, its biological significance remains questionable. The increased frequency of circulating CD27− and CD28− T cells is one of the characteristics of CMV infection (Bekker et al., 2005; Pourgheysari et al., 2007; van de Berg et al., 2008; Yue et al., 2004). Based on their in vitro TR activity, we propose that these cells may play a role in the immunosuppression associated with CMV infection. Other investigators described a negative association between the frequency of CD27− and/or CD28− circulating T cells and the immune repertoire of CMV-seropositive hosts (Almanzar et al., 2005; Sylwester et al., 2005; Vescovini et al., 2007). It was also previously shown that CMV-specific T cells suppress responses to other viruses in young and elderly individuals (Khan et al., 2004; Levin et al., 1979) and it was proposed that this phenomenon may contribute to the association between CMV seropositivity and increased risk of death in octogenarians and nonagenarians (Wikby et al., 2005). The immunosuppressive effect of CMV infection has been well documented in transplant patients who become more susceptible to bacterial and fungal super-infections during CMV reactivations. In HIV-infected patients, CMV reactivation is associated with increased risk of death independent of the development of CMV end-organ disease (Deayton et al., 2004; Wohl et al., 2005). We also showed that CMV stimulation of PBMC from HIV-infected subjects elicits higher frequencies of CD4+CD27−CD28− TR than in HIV-seronegative individuals (Jesser et al., 2006). It is conceivable that the CMV-stimulated CD4+CD27−CD28− TR may attenuate immune responses to other pathogens through a nonspecific effect similar to the one that we described against C. albicans antigeninduced proliferation. The role of CD4+CD27−CD28− as mediators of the immune suppression associated with CMV infection deserves to be further investigated.
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Subjects and methods Subjects
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(Treestar). The gating strategy took into account the distribution of cells with respect to the markers of interest and the fluorescence minus one histograms. Cell sorting
PBMC used in this study were obtained from 15 HIV-infected and 23 uninfected subjects. All subjects were CMV-seropositive. Inclusion criteria for HIV-infected individuals included HAART treatment ≥3 months (3-drug regimen, including at least one protease inhibitor or a non-nucleoside analogue) and CD4+ counts ≥ 100 cells/μl at study entry. At study visits for immunologic evaluations, the median CD4+ numbers of HIV-infected subjects was 506 cells/μl (range of 200 to 947 cells/μl). HIV viral load was b400 copies/ml. The median time on HAART was 20 months.
After 6 days of CMV stimulation at 37 °C in 5% CO2 atmosphere, PBMC were washed twice in 2% FBS in PBS and stained for surface markers as described above. After that, cells were washed in 2% FBS in PBS and sorted using FACSAria using FACSDiva software (BD Bioscience) or MoFlo™ XDP (Dako) using Summit software. Sorted PBMC were collected in stimulation medium and used in functional experiments.
Monoclonal antibodies (MAb) and other reagents
Blocking experiments
The following staining reagents were used in this study: anti-CD3AmCyan, anti-CD27-FITC, anti-CD28-FITC and anti-CTLA-4-APC (BD Biosciences); anti-CD4-PE Texas Red, anti-granzyme B-PE-Cy5.5, antiCD25-APC and anti-Fas-L-APC (Caltag); anti-TGF-β-biotin, streptavidin-PE-Cy7, anti-FoxP3-APC, anti-PD1-L, anti-PD-1-PE, anti-GITR-LAlexa647, anti-GITR-PerCP and streptavidin-APC (eBiosciences); goat anti-mouse IgG-Pacific Blue, goat anti-mouse IgG-Alexa Fluor 750APC and anti-GPR83-APC (Invitrogen); anti-TGF-β-PE (IQ Products); and anti-perforin-biotin (Ancell). The following blocking reagents were used in this study: anti-TGF-β1 and anti-human PD-1 mouse mAb's (R&D Systems, Inc), granzyme B inhibitor II, cell-permeable (Calbiochem).
Purified CD4+CD27−CD28− cells were treated with granzyme B inhibitor II (Calbiochem), neutralizing mAbs anti-TGFβ (R&D Systems, Inc) or anti-PD1 (R&D Systems, Inc), or an irrelevant mouse mAb control (BD Bioscience) for 2 h at 37 °C in 5% CO2 atmosphere in stimulation medium. Treated and untreated CD4+CD27−CD28− cells were added to freshly thawed autologous PBMC in the presence of inactivated CMV-infected cell lysate. After 6 days of incubation in quadruplicate 96-microtiter plate wells/stimulant, cells were pulsed with 3H-thymidine, harvested and counted as above.
Specimen processing and cryopreservation PBMC from heparinized blood, separated by Ficoll-Hypaque density gradient centrifugation (Sigma Chemical Company), were cryopreserved at 107 cells/ml in fetal bovine serum (FBS, Gemini BioProducts) with 10% dimethyl sulfoxide (DMSO, Sigma Chemical Company) and stored in liquid nitrogen tanks until use. Lymphocyte proliferation assay (LPA) PBMC at 106 cells/ml in stimulation medium consisting of RPMI 1640 with L-glutamine (Gibco), 10% human AB serum (GemCell) and 1% penicillin–streptomycin (Gibco) were incubated in quadruplicate wells of 96-well microtiter plates at 100 μl/well with 100 μl of inactivated CMV-infected cell lysate, mock-infected cell control antigen, recombinant human IL-2 at 4 ng/ml (R&D Systems, Inc), C. albicans antigen (Greer), or phytohemagglutinin (Sigma). After 6 days of culture at 37 °C in 5% CO2 atmosphere, cells were pulsed with 1 μCi of 3H-thymidine and their DNA was harvested 6 h later onto Unifilter plates (Perkin Elmer). Radioactivity gathered on the filters was counted with a microplate scintillation counter (Packard) and scintillation fluid (Perkin Elmer). Results were expressed in median counts per minutes (cpm) of the quadruplicate wells. T-cell phenotypic characterization PBMC and separated T-cell subpopulations were allowed to expand in culture tubes containing stimulation medium with CMV lysate, C. albicans or control antigen for 6 days at 37 °C in 5% CO2 atmosphere, after which cells were washed twice in 2% FBS in PBS and incubated for 30 min at room temperature with the surface marker mAbs described above. For intracellular staining, PBMC were then permeabilized and fixed (Cytofix/Cytoperm; BD Pharmingen) for 20 min at 4 °C, washed and incubated with the appropriate mAbs. At the completion of the staining procedure, PBMC were washed in 2% FBS in PBS and analyzed with a FACSCalibur using CellQuest software or FACSAria using FACSDiva software (BD Biosciences), or FlowJo
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