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TH17 Versus Treg Cells in Renal Transplant Candidates: Effect of a Previous Transplant
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TH17 Versus Treg Cells in Renal Transplant Candidates: Effect of a Previous Transplant D. San Segundo, M. López-Hoyos, G. Fernández-Fresnedo, M.J. Benito, J.C. Ruiz, A. Benito, E. Rodrigo, and M. Arias ABSTRACT Introduction. The TH1 and TH2 cells were described several years ago. However, this dichotomy has been disrupted by the description of other CD4⫹ T cell subsets: the proinflammatory interleukin (IL)-17-producing T cells (TH17) and regulatory T cells (Tregs). The latter group inhibits the immune responses driven by TH1, TH2, and TH17 cells. IL-6 is involved in TH17 development, down-regulating Treg differentiation. Our hypothesis suggested that an imbalance between TH17 and Tregs enhances immune responses among renal transplant patients. Materials and methods. We studied 26 end-stage renal disease (ESRD) subjects and 10 patients awaiting a second renal transplant after previous graft dysfunction. We assessed the number of CD4⫹CD25⫹Foxp3⫹ cells and serum levels of IL-17, the prototypic interleukin of TH17 cells. Results. We observed a lower number of CD4⫹CD25⫹Foxp3⫹ T cells among patients with previous graft dysfunction than those with ESRD (median 3.37 vs 8.63 cells/mm3, P ⫽ .008). In contrast, IL-17 serum levels were augmented in graft dysfunction (median 4.45 pg/mL) compared with ESRD patients (1.39 pg/mL, P ⫽ .036), suggesting a proinflammatory state in patients awaiting a second renal transplant. Conclusion. The emerging alloresponse from a previous transplant favors the generation of TH17 instead of Treg cells. The enhanced activity of TH17 cells in retransplanted patients may down-regulate Treg cells, producing a proinflammatory environment that favors rejection of the next transplant.
From the Servicios de Inmunología (D.S.S., M.L.-H., M.J.B.) y Nefrología (G.F.-F., J.C.R., A.B., E.R., M.A.), Hospital Universitario Marqués de Valdecilla, Fundación Marqués de ValdecillaIFIMAV, Santander, Spain. This work was partially supported by grants from the Fondo de Investigaciones Sanitarias (PI050047, PI070683, REDINREN 06/16 from ISCIII), from the Fundación Marqués de Valdecilla-
IFIMAV (API07/12), and from the Fundación Mutua-Madrileña. D.S.S. is a recipient of a Lopez-Albo grant (Fundación Marqués de Valdecilla-IFIMAV). Address reprint requests to Marcos López-Hoyos, Servicio de Inmunología, Hospital Universitario Marqués de Valdecilla, 39008 Santander, Spain. E-mail: [email protected]
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.09.043
Transplantation Proceedings, 40, 2885–2888 (2008)
2885
2886
SAN SEGUNDO, LÓPEZ-HOYOS, FERNÁNDEZ-FRESNEDO ET AL
T
HE CELLS INVOLVED in the T-dependent immune responses have classically been divided as TH1 and TH2 cells, which show distinct effector functions in cellular and humoral responses, respectively. These subpopulations display specific cytokine secretion profiles. In the last years, several studies in experimental allergic encephalitis mouse models have demonstrated an interleukin (IL)-17-producing T-cell subpopulation (TH17) with a pathological role in the development of autoimmune disease.1 IL-17 release causes effective immunity against extracellular pathogens by recruitment of neutrophils, provoking a proinflammatory state.2 Differentiation from naïve T cells to TH subpopulations is strictly regulated. It is well known that interferon-␥ (IFN␥), a TH1 cytokine, inhibits TH2 differentiation; in contrast, IL-4, a TH2 cytokine, inhibits the TH1 differentiation. IFN-␥ and IL-4 inhibit TH17 differentiation. All responses exerted by these subpopulations are dampened by regulatory T cells (Tregs), which show inhibitory functions to control autoimmune diseases, infection responses, and alloresponses.3 Some studies have demonstrated the differentiation of Tregs after challenge with transforming growth factor-.4 The links between TH17 and Tregs are not well studied in humans. Based on animal models, which show IL-6 to be necessary for the development of TH17 and to inhibit the differentiation of Tregs, present study was performed to assess the relationship between the TH17 cells and Tregs in the context of the alloresponse.
Fig 1. Quantification of the absolute numbers of CD4⫹CD25high Foxp3⫹ regulatory T cells in peripheral blood and serum interleukin-17 (IL-17) levels in renal transplant patients divided in groups depending on their first transplant and retransplantation. The boxes represent the median and interquartil range. A statistical difference was observed in the absolute numbers of CD4⫹CD25highFoxp3⫹ in white box (P ⫽ .008) and IL-17 levels in gray box (P ⫽ .03) between both groups of patients.
MATERIALS AND METHODS A cohort of 36 renal transplant candidates was divided into two groups: one composed of patients awaiting a second transplant after a previous graft loss (reTx, n ⫽ 10), and another composed of subjects with end-stage renal disease awaiting their first renal transplant (Tx, n ⫽ 26). There was no significant difference between groups in the main demographic and clinical features (Table 1). To quantify blood Tregs, three-color cytometry analysis was performed on peripheral whole blood samples within 2 hours of collection before the transplant. Blood cells were surface-stained with the following monoclonal antibodies (mAbs) from BectonDickinson Biosciences (San Jose, Calif, USA): anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-FITC, anti-CD8-FITC, anti-CD25phycoerythrin, anti-CD3-peridinin chlorophyll protein (PerCP), anti-CD4-PerCP, anti-CD45-PerCP. Additionally, FOXP3 expres-
sion was analyzed by flow cytometry after intracellular staining with an specific mAb (clone PCH101, eBioscience, San Diego, Calif, USA) together with the surface staining with anti-CD4-FITC and anti-CD25-allophycocyanin following the protocol recommended by the manufacturer. To determine the regulatory function, we performed functional assays by mixed lymphocyte reaction with donor splenocytes as stimulator and CD4⫹CD25⫺ as effector cells. Serum levels of IL-17 were measured using an enzyme-linked-immunosorbent-assay (eBioscience) following the recommended protocol. Data were nonparametrically distributed (Kolmogorov-Smirnov fit test) and therefore expressed as median values and interquartile ranges. Differences in the number of CD4⫹CD25highFOXP3⫹ T cells and the serum levels of IL-17 between the groups of patients were analyzed by Mann-Whitney U test.
Table 1. Demographic and Clinical Characteristics of the Two Groups of Renal Transplant Recipients Studied: Waiting for the First Transplant (Tx) and for the Second Transplant (ReTx)
Recipient age (mean ⫾ SD), y Donor age (mean ⫾ SD), y Gender (% female) Peak panel-reactive antibodies (%) Current panel-reactive antibodies (%) HLA mismatches (mean), A/B/DR Acute tubular necrosis (%) Acute rejection episodes (%)
Tx (n ⫽ 26)
ReTx (n ⫽ 10)
P*
50 ⫾ 19.6 52.4 ⫾ 14.8 19.2 1.62 ⫾ 4.55 0.38 ⫾ 1.96 1.3/1.4/1 23.1 26.9
51 ⫾ 15.6 48.8 ⫾ 9.3 10.0 15.2 ⫾ 23.34 7.4 ⫾ 15.06 1/1.2/1.4 10 50.0
.193 .745 .532 .018 .026 .168/.176/.274 .399 .165
*Levels of statistical significance were calculated by the Mann-Whitney U test and the chi-square test for nonparametric quantitative and discrete values, respectively. SD, standard deviation.
TH17 VERSUS TREG CELLS
2887
Fig 2. Representative FACS profile showing the expression of FOXP3 in histograms gated on CD4⫹CD25⫺ cells (R2, lower right), CD4⫹CD25⫹ cells (R3, medium right), and CD4⫹CD25high cells (R4, upper right). FITC, fluorescein isothiocyanate.
RESULTS
Compared with the Tx group, the reTx patients showed a significantly greater peak and current panel-reactive antibodies (P ⫽ .018 and P ⫽ .026, respectively). This observation could be explained by the prior allograft response. No difference in other clinical parameters was observed. The absolute number of CD4⫹CD25highFOXP3⫹ T cells was significantly higher in the Tx group (8.73 cells/mm3
[6.3–12.03]) compared with the ReTx subjects 3.37 cells/mm3 (1.68 –7.38; P ⫽ .008; Fig 1A). The results indicated that a previous rejection affected the absolute numbers of Tregs, although the effect of the previous immunosuppressive regimen not be ignored. More than 90% of CD4⫹CD25high T cells expressed FOXP3 compared with lack of expression by CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells (Fig 2). In addition, the regulatory activity of CD4⫹CD25highFOXP3⫹ T cells
2888
was demonstrated in mixed lymphocyte reactions (data not shown). Although lower IL-17 production by double negative cells5 and invariant natural killer T cells has been described,6 IL-17 is the prototypic interleukin produced by TH17 cells. To test TH17 activation among renal transplant patients, we measured the serum IL-17. The level was significantly higher among the ReTx group (4.46 pg/mL [2.90 –7.32]) compared with the Tx group (1.39 pg/mL [0.77– 4.78]; P ⫽ .030; Figs 1 and 2), reflecting a greater ratio of TH17/Tregs among reTx patients, suggesting a proinflammatory state among patients with previous graft rejection. DISCUSSION
We demonstrated a proinflammatory state in reTx patients that could be maintained by large amounts of IL-17 and associated with low absolute numbers of circulating Tregs. The alloresponse induced by the previous transplant probably favored TH17 cell induction and dampened Tregs. The lack of Tregs in these patients could explain the high acute rejection rate observed in this group (50%). A previous study showed of IL-17 mRNA expression in patients with subclinical acute renal graft rejection.7 Herein, we have shown a link between Tregs and TH17 cells in renal
SAN SEGUNDO, LÓPEZ-HOYOS, FERNÁNDEZ-FRESNEDO ET AL
transplantation, but further studies must address the complex networks of regulatory and inflammatory contexts. ACKNOWLEDGMENTS We thank Iñaki Beares (recipient of a grant from FIS PI070683) for his helpful technical assistance in the flow cytometry analysis.
REFERENCES 1. Batten M, Li J, Kljavin NM, et al: Interleukin-27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing cells. Nat Immunol 7:929, 2005 2. Fossiez F, Braneherean J, Murray R, et al: Interleukin-17. Int Rev Immunol 16:541, 1998 3. Fehérvari Z, Sakaguchi S: CD4⫹ Tregs and immune control. J Clin Invest 114:1209, 2004 4. Chen W, Jin W, Hardegen N, et al: Conversion of peripheral CD4⫹CD25⫺naïve T cells to CD4⫹CD25⫹ regulatory T cells by TGF- induction of transcription factor Foxp3. J Exp Med 198: 1875, 2003 5. Stark MA, Huo Y, Burcin TL, et al: Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity 22:285, 2005 6. Michel ML, Keller AC, Paget C, et al: Identification of an IL-17 producing NK1.1neg iNKT cell population involved in airway neutrophilia. J Exp Med 204:995, 2007 7. Loong CC, Hsieh HG, Lui WY, et al: Evidence for the early involvement of interleukin 17 in human and experimental renal allograft rejection. J Pathol 197:322, 2002
TH17 Versus Treg Cells in Renal Transplant Candidates: Effect of a Previous Transplant D. San Segundo, M. López-Hoyos, G. Fernández-Fresnedo, M.J. Benito, J.C. Ruiz, A. Benito, E. Rodrigo, and M. Arias ABSTRACT Introduction. The TH1 and TH2 cells were described several years ago. However, this dichotomy has been disrupted by the description of other CD4⫹ T cell subsets: the proinflammatory interleukin (IL)-17-producing T cells (TH17) and regulatory T cells (Tregs). The latter group inhibits the immune responses driven by TH1, TH2, and TH17 cells. IL-6 is involved in TH17 development, down-regulating Treg differentiation. Our hypothesis suggested that an imbalance between TH17 and Tregs enhances immune responses among renal transplant patients. Materials and methods. We studied 26 end-stage renal disease (ESRD) subjects and 10 patients awaiting a second renal transplant after previous graft dysfunction. We assessed the number of CD4⫹CD25⫹Foxp3⫹ cells and serum levels of IL-17, the prototypic interleukin of TH17 cells. Results. We observed a lower number of CD4⫹CD25⫹Foxp3⫹ T cells among patients with previous graft dysfunction than those with ESRD (median 3.37 vs 8.63 cells/mm3, P ⫽ .008). In contrast, IL-17 serum levels were augmented in graft dysfunction (median 4.45 pg/mL) compared with ESRD patients (1.39 pg/mL, P ⫽ .036), suggesting a proinflammatory state in patients awaiting a second renal transplant. Conclusion. The emerging alloresponse from a previous transplant favors the generation of TH17 instead of Treg cells. The enhanced activity of TH17 cells in retransplanted patients may down-regulate Treg cells, producing a proinflammatory environment that favors rejection of the next transplant.
From the Servicios de Inmunología (D.S.S., M.L.-H., M.J.B.) y Nefrología (G.F.-F., J.C.R., A.B., E.R., M.A.), Hospital Universitario Marqués de Valdecilla, Fundación Marqués de ValdecillaIFIMAV, Santander, Spain. This work was partially supported by grants from the Fondo de Investigaciones Sanitarias (PI050047, PI070683, REDINREN 06/16 from ISCIII), from the Fundación Marqués de Valdecilla-
IFIMAV (API07/12), and from the Fundación Mutua-Madrileña. D.S.S. is a recipient of a Lopez-Albo grant (Fundación Marqués de Valdecilla-IFIMAV). Address reprint requests to Marcos López-Hoyos, Servicio de Inmunología, Hospital Universitario Marqués de Valdecilla, 39008 Santander, Spain. E-mail: [email protected]
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.09.043
Transplantation Proceedings, 40, 2885–2888 (2008)
2885
2886
SAN SEGUNDO, LÓPEZ-HOYOS, FERNÁNDEZ-FRESNEDO ET AL
T
HE CELLS INVOLVED in the T-dependent immune responses have classically been divided as TH1 and TH2 cells, which show distinct effector functions in cellular and humoral responses, respectively. These subpopulations display specific cytokine secretion profiles. In the last years, several studies in experimental allergic encephalitis mouse models have demonstrated an interleukin (IL)-17-producing T-cell subpopulation (TH17) with a pathological role in the development of autoimmune disease.1 IL-17 release causes effective immunity against extracellular pathogens by recruitment of neutrophils, provoking a proinflammatory state.2 Differentiation from naïve T cells to TH subpopulations is strictly regulated. It is well known that interferon-␥ (IFN␥), a TH1 cytokine, inhibits TH2 differentiation; in contrast, IL-4, a TH2 cytokine, inhibits the TH1 differentiation. IFN-␥ and IL-4 inhibit TH17 differentiation. All responses exerted by these subpopulations are dampened by regulatory T cells (Tregs), which show inhibitory functions to control autoimmune diseases, infection responses, and alloresponses.3 Some studies have demonstrated the differentiation of Tregs after challenge with transforming growth factor-.4 The links between TH17 and Tregs are not well studied in humans. Based on animal models, which show IL-6 to be necessary for the development of TH17 and to inhibit the differentiation of Tregs, present study was performed to assess the relationship between the TH17 cells and Tregs in the context of the alloresponse.
Fig 1. Quantification of the absolute numbers of CD4⫹CD25high Foxp3⫹ regulatory T cells in peripheral blood and serum interleukin-17 (IL-17) levels in renal transplant patients divided in groups depending on their first transplant and retransplantation. The boxes represent the median and interquartil range. A statistical difference was observed in the absolute numbers of CD4⫹CD25highFoxp3⫹ in white box (P ⫽ .008) and IL-17 levels in gray box (P ⫽ .03) between both groups of patients.
MATERIALS AND METHODS A cohort of 36 renal transplant candidates was divided into two groups: one composed of patients awaiting a second transplant after a previous graft loss (reTx, n ⫽ 10), and another composed of subjects with end-stage renal disease awaiting their first renal transplant (Tx, n ⫽ 26). There was no significant difference between groups in the main demographic and clinical features (Table 1). To quantify blood Tregs, three-color cytometry analysis was performed on peripheral whole blood samples within 2 hours of collection before the transplant. Blood cells were surface-stained with the following monoclonal antibodies (mAbs) from BectonDickinson Biosciences (San Jose, Calif, USA): anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-FITC, anti-CD8-FITC, anti-CD25phycoerythrin, anti-CD3-peridinin chlorophyll protein (PerCP), anti-CD4-PerCP, anti-CD45-PerCP. Additionally, FOXP3 expres-
sion was analyzed by flow cytometry after intracellular staining with an specific mAb (clone PCH101, eBioscience, San Diego, Calif, USA) together with the surface staining with anti-CD4-FITC and anti-CD25-allophycocyanin following the protocol recommended by the manufacturer. To determine the regulatory function, we performed functional assays by mixed lymphocyte reaction with donor splenocytes as stimulator and CD4⫹CD25⫺ as effector cells. Serum levels of IL-17 were measured using an enzyme-linked-immunosorbent-assay (eBioscience) following the recommended protocol. Data were nonparametrically distributed (Kolmogorov-Smirnov fit test) and therefore expressed as median values and interquartile ranges. Differences in the number of CD4⫹CD25highFOXP3⫹ T cells and the serum levels of IL-17 between the groups of patients were analyzed by Mann-Whitney U test.
Table 1. Demographic and Clinical Characteristics of the Two Groups of Renal Transplant Recipients Studied: Waiting for the First Transplant (Tx) and for the Second Transplant (ReTx)
Recipient age (mean ⫾ SD), y Donor age (mean ⫾ SD), y Gender (% female) Peak panel-reactive antibodies (%) Current panel-reactive antibodies (%) HLA mismatches (mean), A/B/DR Acute tubular necrosis (%) Acute rejection episodes (%)
Tx (n ⫽ 26)
ReTx (n ⫽ 10)
P*
50 ⫾ 19.6 52.4 ⫾ 14.8 19.2 1.62 ⫾ 4.55 0.38 ⫾ 1.96 1.3/1.4/1 23.1 26.9
51 ⫾ 15.6 48.8 ⫾ 9.3 10.0 15.2 ⫾ 23.34 7.4 ⫾ 15.06 1/1.2/1.4 10 50.0
.193 .745 .532 .018 .026 .168/.176/.274 .399 .165
*Levels of statistical significance were calculated by the Mann-Whitney U test and the chi-square test for nonparametric quantitative and discrete values, respectively. SD, standard deviation.
TH17 VERSUS TREG CELLS
2887
Fig 2. Representative FACS profile showing the expression of FOXP3 in histograms gated on CD4⫹CD25⫺ cells (R2, lower right), CD4⫹CD25⫹ cells (R3, medium right), and CD4⫹CD25high cells (R4, upper right). FITC, fluorescein isothiocyanate.
RESULTS
Compared with the Tx group, the reTx patients showed a significantly greater peak and current panel-reactive antibodies (P ⫽ .018 and P ⫽ .026, respectively). This observation could be explained by the prior allograft response. No difference in other clinical parameters was observed. The absolute number of CD4⫹CD25highFOXP3⫹ T cells was significantly higher in the Tx group (8.73 cells/mm3
[6.3–12.03]) compared with the ReTx subjects 3.37 cells/mm3 (1.68 –7.38; P ⫽ .008; Fig 1A). The results indicated that a previous rejection affected the absolute numbers of Tregs, although the effect of the previous immunosuppressive regimen not be ignored. More than 90% of CD4⫹CD25high T cells expressed FOXP3 compared with lack of expression by CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells (Fig 2). In addition, the regulatory activity of CD4⫹CD25highFOXP3⫹ T cells
2888
was demonstrated in mixed lymphocyte reactions (data not shown). Although lower IL-17 production by double negative cells5 and invariant natural killer T cells has been described,6 IL-17 is the prototypic interleukin produced by TH17 cells. To test TH17 activation among renal transplant patients, we measured the serum IL-17. The level was significantly higher among the ReTx group (4.46 pg/mL [2.90 –7.32]) compared with the Tx group (1.39 pg/mL [0.77– 4.78]; P ⫽ .030; Figs 1 and 2), reflecting a greater ratio of TH17/Tregs among reTx patients, suggesting a proinflammatory state among patients with previous graft rejection. DISCUSSION
We demonstrated a proinflammatory state in reTx patients that could be maintained by large amounts of IL-17 and associated with low absolute numbers of circulating Tregs. The alloresponse induced by the previous transplant probably favored TH17 cell induction and dampened Tregs. The lack of Tregs in these patients could explain the high acute rejection rate observed in this group (50%). A previous study showed of IL-17 mRNA expression in patients with subclinical acute renal graft rejection.7 Herein, we have shown a link between Tregs and TH17 cells in renal
SAN SEGUNDO, LÓPEZ-HOYOS, FERNÁNDEZ-FRESNEDO ET AL
transplantation, but further studies must address the complex networks of regulatory and inflammatory contexts. ACKNOWLEDGMENTS We thank Iñaki Beares (recipient of a grant from FIS PI070683) for his helpful technical assistance in the flow cytometry analysis.
REFERENCES 1. Batten M, Li J, Kljavin NM, et al: Interleukin-27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing cells. Nat Immunol 7:929, 2005 2. Fossiez F, Braneherean J, Murray R, et al: Interleukin-17. Int Rev Immunol 16:541, 1998 3. Fehérvari Z, Sakaguchi S: CD4⫹ Tregs and immune control. J Clin Invest 114:1209, 2004 4. Chen W, Jin W, Hardegen N, et al: Conversion of peripheral CD4⫹CD25⫺naïve T cells to CD4⫹CD25⫹ regulatory T cells by TGF- induction of transcription factor Foxp3. J Exp Med 198: 1875, 2003 5. Stark MA, Huo Y, Burcin TL, et al: Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity 22:285, 2005 6. Michel ML, Keller AC, Paget C, et al: Identification of an IL-17 producing NK1.1neg iNKT cell population involved in airway neutrophilia. J Exp Med 204:995, 2007 7. Loong CC, Hsieh HG, Lui WY, et al: Evidence for the early involvement of interleukin 17 in human and experimental renal allograft rejection. J Pathol 197:322, 2002