- Email: [email protected]
Human Peripheral Blood CD8+ CD28− T Cells of Renal Allograft Recipients Do Not Express FOXP3 Protein
Human Peripheral Blood CD8ⴙ CD28ⴚ T Cells of Renal Allograft Recipients Do Not Express FOXP3 Protein A. Korecka-Polak, A. Duszota, P. Wierzbicki, M. Niemczyk, K. Bocian, D. Kłosowska, L. Pa¸czek, A. Górski, and G. Korczak-Kowalska ABSTRACT Introduction. In recent studies, the FOXP3 molecule has been suggested to be a marker of a suppressor subset of human CD8⫹ CD28⫺ T cells based on correlations between the level of its mRNA and allograft function. Because this transcriptional factor produces a protein, we suggest that these correlations should focus on the FOXP3 protein. The aim of our study was to evaluate whether FOXP3 protein was present in cells of the CD8⫹ CD28⫺ population in the peripheral blood of renal allograft recipients and whether the level of CD8⫹ CD28⫺ FOXP3⫹ cells correlated with allograft function. Methods. The study was performed on 30 renal allograft recipients with uneventful stable courses (n ⫽ 18) or biopsy-proven chronic rejection (n ⫽ 12). The immunosuppression was based on cyclosporine (n ⫽ 12) or rapamycin (n ⫽ 9). Peripheral blood mononuclear cells isolated from recipient blood samples were labeled with anti-CD8 and anti-CD28 MAbs conjugated with fluorochromes. After incubation, washing, and labeling using a PE anti-human FOXP3 Kit, we determined the percentage of cells by flow cytometry. Results. FOXP3 protein expression was not observed either in the CD8⫹ CD28⫺ population, or the whole populations of CD8⫹ or CD28⫺ cells among patient groups. Conclusions. The expression of FOXP3 protein in CD8⫹ CD28⫺ cells seems to be of a questionable value as a diagnostic tool for allograft function, it is probably not a marker for the CD8⫹ CD28⫺ T cell subset. ANY studies have observed an increased level of CD8⫹ CD28⫺ cells in the peripheral blood of organ transplantation patients.1 Some researchers have suggested that the higher level is associated with stable graft function, allowing a reduction in immunosuppressive drug doses,2 whereas others have observed a correlation between the increased CD8⫹ CD28⫺ cells and chronic graft rejection.3 This divergence may be connected to the heterogeneity of the CD8⫹ CD28⫺ population, which contains both suppressor (Ts) and cytotoxic (Tc) T cells.1 The Ts subset suppresses activation of alloreactive CD4⫹ cells, whereas the Tc subset mediates cytotoxicity against allografts.1 Therefore, allograft function may depend on the Ts:Tc ratio in the CD8⫹ CD28⫺ population. The phenotype of the Ts subpopulation has not been identified explicitly. Some researchers have suggested that the transcriptional factor, FOXP3, which is characteristic of CD4⫹CD25high T regulatory cells also represents a pheno-
M
typic marker for the Ts subset of CD8⫹ CD28⫺ cells.4,5 Some studies have shown that CD8⫹ CD28⫺ T cells with suppressive properties bear the mRNA of the transcription factor FOXP3.4 – 6 The level of FOXP3 mRNA in periphFrom the Department of Immunology (A.K.-P., A.D., K.B., G.K.-K.), Faculty of Biology, University of Warsaw, the Department of Clinical Immunology (A.D., P.W., D.K., A.G., G.K.-K.), Transplantation Institute, Medical University of Warsaw and the Department of Immunology (M.W., L.P.), Transplant Medicine and Internal Diseases, Transplantation Institute, Medical University of Warsaw, Poland. This work is supported in part by grant nos. 3P05B 07425, N N402 268036, and N303 015 32/0671 from the Ministry of Science and Higher Education. Address reprint requests to Grazyna Korczak-Kowalska, Department of Immunology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw/Poland. E-mail: [email protected]
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2011.08.016
Transplantation Proceedings, 43, 2917–2921 (2011)
2917
2918
eral blood CD8⫹ CD28⫺ cells of allograft recipients seems to negatively correlate with chronic allograft rejection.3,5,7 Therefore quantitative analysis of the expression of FOXP3 gene may be helpful for a noninvasive diagnosis of allograft rejection. However, the transcription factor FOXP3 encodes a protein that should be more extensively examined. Since one published study described the expression of FOXP3 protein in CD8⫹ T cells in the peripheral blood of allograft recipients. In that study Segundo et al8 did not detect any expression of FOXP3 protein. We suggest that the search for correlations between the FOXP3 marker and allograft function should focus on the FOXP3 protein. The aim of our study was to evaluate whether the FOXP3 protein was present in the CD8⫹ CD28⫺ peripheral blood population of renal allograft recipients and its level correlates with allograft function. MATERIALS AND METHODS The study was performed on 30 renal allograft recipients with a male/female ratio of 2/1. One cohort of subjects had uneventful stable courses (S, n ⫽ 18) and another biopsy-proven chronic rejection (CR, n ⫽ 12). The immunosuppressive protocol was based on cyclosporine (CsA; n ⫽ 21) or rapamycin (RAPA; n ⫽ 9). The median age of the patients in the CR group was 48.5 years (range, 31 to 65 years) and in the S group, 52 years (range, 25 to 64 years). The median time after transplantation was 47.5 months (range, 12 to 172 months) in the CR and 44.5 months (range, 20 to 187 months) in the S group. Control blood samples were obtained from 10 healthy volunteers with the median age of 32.5 years (range, 23 to 47 years). Peripheral blood mononuclear cells (PBMCs) were isolated from the recipient blood samples by density gradient centrifugation using Ficoll-Isopaque. After washing with CellWash (Becton Dickinson, New Jersey) and labeling with mouse anti-human MAbs, the cells were conjugated with the fluorochromes anti-CD8 MAb and anti-CD28 MAb (Pharmingen). The samples were incubated in the dark for 30 minutes, washed twice, and labeled using a PE anti-human FOXP3 Kit (eBioscence) according to the manufacturer’s protocol. The percentage of cells was determined by flow cytometry using a FACSCalibur instrument and Cell Quest software (BD Biosciences). The Mann-Whitney U test was used to assess differences between groups. Correlations were calculated with the Spearman rank order correlation test. A P ⱕ .05 was considered statistically significant.
RESULTS
The mean percentage of CD8⫹ CD28⫺ cells in the peripheral blood lymphocyte population was higher among the CR group than the S group: CR, 25.16 ⫾ 11.98% versus S, 17.93 ⫾ 9.79% (P ⬍ .05). The difference was also significant for the subgroup of patients who underwent immunosuppressive therapy based on CsA: CsA-CR, 26.43 ⫾ 12.83% (n ⫽ 11) versus CsA-S, 17.99 ⫾ 7.78% (n ⫽ 10, P ⬍ .05). There was no correlation between the percentage of CD8⫹ CD28⫺ cells in lymphocytes and the age of patients or their time after transplantation (data not shown). We did not observe FOXP3 protein expression either in the peripheral blood CD8⫹ CD28⫺ cells of renal allograft recipients (Fig 1, Fig 2C) or in the whole peripheral blood populations of
KORECKA-POLAK, DUSZOTA, WIERZBICKI ET AL
CD8⫹ (Fig 2B) or CD28⫺ cells (Fig 2A) from the either the CR or S groups. We noted the presence of FOXP3⫹ cells in the CD8⫺ and CD28⫹ populations of lymphocytes (Fig 2A,B). The Few CD8⫹ CD28⫺ cells in the peripheral blood of healthy volunteers showed a low expression of FOXP3 protein (Fig 1). DISCUSSION
Our data suggested that the increased percentage of CD8⫹ CD28⫺ cells in the peripheral blood of renal recipients was associated with graft rejection among subjects receiving immunosuppressive therapy with CsA but not with RAPA, possibly because of the small number of patients treated with the latter drug. Our results were consistent with the study by Baeten et al.3 which observed a higher percentage of CD8⫹ CD28⫺ cells in the peripheral blood of renal allograft recipients with CR who were treated with calcineurin inhibitors (cyclosporine or tacrolimus) than drugfree tolerant patients (DF-Tol) with stable graft function. We did not observe cells with FOXP3 protein either among the CD8⫹ CD28⫺ subpopulation nor the whole CD8⫹ or CD28⫺ populations in the peripheral blood of the renal allograft recipients. One study by Segundo et al.8 did not detect expression of FOXP3 protein in CD8⫹ T cells in the peripheral blood of cardiac transplant recipients receiving immunosuppressive therapy based either on rapamycin or on calcineurin inhibitors. The inhibition of CD8⫹ cell activation by immunosuppressive drugs is probably the reason for the lack of FOXP3 protein in these cells. Some studies have suggested that FOXP3 is not restricted to regulatory elements and can be a marker of cell activation.9,10 The question is why immunosuppressive drugs inhibit the expression of FOXP3 protein in CD8⫹ cells but not in CD8⫺ cells and why there is no difference in outcomes of immunosuppressive therapies based on rapamycin versus cyclosporine, as indicated in the case of CD4⫹ CD25high regulatory cells.8,11,12 The correlation between T cell activation and FOXP3 expression seems to be confirmed by the lack of this marker in CD28⫺ cells because as the CD28 molecule is a costimulator for the activation of T cells.13 This observation suggests a role for the CD28 signal in the induction of FOXP3 expression in human lymphocytes. Although it is generally believed that stable FOXP3 expression occurs in human CD4⫹ T cells after TCR, triggering it has also been shown that CD28 can induce FOXP3 expression in CD4⫹ T cells.14 This suggests questions about the role of the CD28 signal in FOXP3 expression in human T cells and whether the expression of FOXP3 protein is also possible in CD8⫹ CD28⫺ T cells. Most studies have considered the presence of FOXP3 mRNA in CD8⫹ CD28⫺ cells to correlate with stable graft function. For example, Manavalan et al.7 showed the presence of FOXP3 mRNA in CD8⫹ CD28⫺ cells in the peripheral blood of rejection-free heart transplant recipients. These cells triggered the transcription of ILT3 and
T CELLS AND ALLOGRAFT FUNCTION
2919
Fig 1. Lack of expression of FOXP3 protein in the CD8⫹CD28⫺ from the peripheral blood of renal allograft recipients. Abbreviations: CsA, immunosuppressive protocol based on cyclosporine A; RAPA, immunosuppressive protocol based on rapamycin; CR, biopsy-proven chronic rejection; S, uneventful stable course; healthy Ctrl, healthy volunteers control. Plots are representative of 10 healthy volunteers and all 30 patients with both CR and S graft function and CsA and RAPA immunosuppressive protocols.
ILT4 in ECS sharing HLA class I antigens with the donor. The authors considered this to be proof of the suppressive properties of these cells. However, CD8⫹ CD28⫺ FOXP3⫹ cells were in the peripheral blood of 12 of 15 rejection-free patients who were 10 to 12 months after post-transplantation and 7 of 10 rejection-free patients who were 3 years post-transplantation. All patients were treated with standard immunosuppressive therapy (cyclosporine, steroids, and azathioprine). There is no suggestion in the article about any possible differences between the rejection-
free patients with versus without FOXP3 mRNA in their CD8⫹ CD28⫺ cells. However, as mentioned above, the transcriptional factor FOXP3 acts as a protein, so it seems that such studies should focus on examining the level of FOXP3 protein, not its mRNA. In contrast, some researchers have suggested a lack of correlation between FOXP3 mRNA levels in peripheral blood T cells and immunological processes in the graft. Dijke et al15 examined FOXP3 mRNA expression patterns among PBMCs of heart transplant patients during quies-
2920
KORECKA-POLAK, DUSZOTA, WIERZBICKI ET AL
Fig 2. Lack of expression of FOXP3 protein in the CD28⫺ (A), CD8⫹(B), and CD8⫹CD28⫺ populations (C) in the peripheral blood of a renal allograft recipient. Plots are from the analysis of the CsA-CR patient’s blood sample and are representative of all 30 patients with both CR and S graft function and CsA and RAPA immunosuppressive protocols.
cence versus rejection compared with those in endomyocardial biopsy specimens. Cardiac allograft rejection was only associated with high FOXP3 mRNA levels in the graft, but not with those in the peripheral blood. Therefore, the
phenotype of peripheral blood cells may not be involved in allograft function. To sum up, we did not detect cells with FOXP3 protein among the CD8⫹ CD28⫺ T cell population in the periph-
T CELLS AND ALLOGRAFT FUNCTION
eral blood of renal allograft recipients displaying different graft function and under different immunosuppressive protocols. The expression of this marker in CD8⫹ CD28⫺ cells seems to be useless as a diagnostic tool; FOXP3 protein is probably not a marker of the Ts subset of CD8⫹ CD28⫺ cells. ACKNOWLEDGMENT We thank Lidia Malchar and Marcin Cichocki for technical assistance.
REFERENCES 1. Arosa FA: CD8⫹CD28⫺ T cells: certainties and uncertainties of a prevalent human T-cell subset. Immunol Cell Biol 80:1, 2002 2. Cortesini R, Renna-Molajoni E, Cinti P, at al: Tailoring of immunosuppression in renal and liver allograft recipients displaying donor specific T-suppressor cells. Hum Immunol 63:1010, 2002 3. Baeten D, Louis S, Braud C, et al: Phenotypically and functionally distinct CD8⫹ lymphocyte populations in long-term drug-free tolerance and chronic rejection in human kidney graft recipients. J Am Soc Nephrol 17:294, 2006 4. Suciu-Foca N, Manavalan JS, Scotto L, et al: Molecular characterization of allospecific T suppressor and tolerogenic dendritic cells: review. Int Immunopharmacol 5:7, 2005 5. Meloni F, Morosini M, Solari N, et al: Foxp3 expressing CD4⫹CD25⫹ and CD8⫹CD28⫺ T regulatory cells in the peripheral blood of patients with lung cancer and pleural mesothelioma. Hum Immunol 67:1, 2006 6. Zhou H, Wang ZD, Zhu X, et al: CD8⫹FOXP3⫹T cells from renal transplant recipients in quiescence induce immunoglobulin-
2921 like transcripts-3 and -4 on dendritic cells from their respective donors. Transplant Proc 39:3065, 2007 7. Manavalan JS, Kim-Schulze S, Scotto L, et al: Alloantigen specific CD8⫹CD28⫺FOXP3⫹ T suppressor cells induce ILT3⫹ ILT4⫹ tolerogenic endothelial cells, inhibiting alloreactivity. Int Immunol 16:1055, 2004 8. Segundo DS, Ruiz JC, Izquierdo M, et al: Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4⫹CD25⫹ FOXP3⫹ regulatory T cells in renal transplant recipients. Transplantation 82:550, 2006 9. Morgan ME, van Bilsen JH, Bakker AM, et al: Expression of FOXP3 mRNA is not confined to CD4⫹CD25⫹ T regulatory cells in humans. Hum Immunol 66:13, 2005 10. Pillai V, Ortega SB, Wang CK, et al: Transient regulatory T-cells: a state attained by all activated human T-cells. Clin Immunol 123:18, 2007 11. Coenen JJ, Koenen HJ, van Rijssen E, et al: Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27⫹ subset of human CD4⫹CD25⫹ regulatory T cells. Blood 107:1018, 2006 12. Korczak-Kowalska G, Wierzbicki P, Bocian K, et al: The influence of immuosuppressive therapy on the development of CD4⫹CD25⫹ T cells after renal transplantation. Transplant Proc 39:2721, 2007 13. Korecka A, Duszota A, Korczak-Kowalska G. [The role of the CD28 molecule in immunological tolerance]. Postepy Hig Med Dosw 61:74, 2007 14. Scotta C, Soligo M, Camperio C, et al: FOXP3 induced by CD28/B7 interaction regulates CD25 and anergic phenotype in human CD4⫹CD25⫺ T lymphocytes. J Immunol 181:1025, 2008 15. Dijke IE, Caliskan K, Korevaar SS, et al: FOXP3 mRNA expression analysis in the peripheral blood and allograft of heart transplant patients. Transpl Immunol 18:250, 2008
M
typic marker for the Ts subset of CD8⫹ CD28⫺ cells.4,5 Some studies have shown that CD8⫹ CD28⫺ T cells with suppressive properties bear the mRNA of the transcription factor FOXP3.4 – 6 The level of FOXP3 mRNA in periphFrom the Department of Immunology (A.K.-P., A.D., K.B., G.K.-K.), Faculty of Biology, University of Warsaw, the Department of Clinical Immunology (A.D., P.W., D.K., A.G., G.K.-K.), Transplantation Institute, Medical University of Warsaw and the Department of Immunology (M.W., L.P.), Transplant Medicine and Internal Diseases, Transplantation Institute, Medical University of Warsaw, Poland. This work is supported in part by grant nos. 3P05B 07425, N N402 268036, and N303 015 32/0671 from the Ministry of Science and Higher Education. Address reprint requests to Grazyna Korczak-Kowalska, Department of Immunology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw/Poland. E-mail: [email protected]
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2011.08.016
Transplantation Proceedings, 43, 2917–2921 (2011)
2917
2918
eral blood CD8⫹ CD28⫺ cells of allograft recipients seems to negatively correlate with chronic allograft rejection.3,5,7 Therefore quantitative analysis of the expression of FOXP3 gene may be helpful for a noninvasive diagnosis of allograft rejection. However, the transcription factor FOXP3 encodes a protein that should be more extensively examined. Since one published study described the expression of FOXP3 protein in CD8⫹ T cells in the peripheral blood of allograft recipients. In that study Segundo et al8 did not detect any expression of FOXP3 protein. We suggest that the search for correlations between the FOXP3 marker and allograft function should focus on the FOXP3 protein. The aim of our study was to evaluate whether the FOXP3 protein was present in the CD8⫹ CD28⫺ peripheral blood population of renal allograft recipients and its level correlates with allograft function. MATERIALS AND METHODS The study was performed on 30 renal allograft recipients with a male/female ratio of 2/1. One cohort of subjects had uneventful stable courses (S, n ⫽ 18) and another biopsy-proven chronic rejection (CR, n ⫽ 12). The immunosuppressive protocol was based on cyclosporine (CsA; n ⫽ 21) or rapamycin (RAPA; n ⫽ 9). The median age of the patients in the CR group was 48.5 years (range, 31 to 65 years) and in the S group, 52 years (range, 25 to 64 years). The median time after transplantation was 47.5 months (range, 12 to 172 months) in the CR and 44.5 months (range, 20 to 187 months) in the S group. Control blood samples were obtained from 10 healthy volunteers with the median age of 32.5 years (range, 23 to 47 years). Peripheral blood mononuclear cells (PBMCs) were isolated from the recipient blood samples by density gradient centrifugation using Ficoll-Isopaque. After washing with CellWash (Becton Dickinson, New Jersey) and labeling with mouse anti-human MAbs, the cells were conjugated with the fluorochromes anti-CD8 MAb and anti-CD28 MAb (Pharmingen). The samples were incubated in the dark for 30 minutes, washed twice, and labeled using a PE anti-human FOXP3 Kit (eBioscence) according to the manufacturer’s protocol. The percentage of cells was determined by flow cytometry using a FACSCalibur instrument and Cell Quest software (BD Biosciences). The Mann-Whitney U test was used to assess differences between groups. Correlations were calculated with the Spearman rank order correlation test. A P ⱕ .05 was considered statistically significant.
RESULTS
The mean percentage of CD8⫹ CD28⫺ cells in the peripheral blood lymphocyte population was higher among the CR group than the S group: CR, 25.16 ⫾ 11.98% versus S, 17.93 ⫾ 9.79% (P ⬍ .05). The difference was also significant for the subgroup of patients who underwent immunosuppressive therapy based on CsA: CsA-CR, 26.43 ⫾ 12.83% (n ⫽ 11) versus CsA-S, 17.99 ⫾ 7.78% (n ⫽ 10, P ⬍ .05). There was no correlation between the percentage of CD8⫹ CD28⫺ cells in lymphocytes and the age of patients or their time after transplantation (data not shown). We did not observe FOXP3 protein expression either in the peripheral blood CD8⫹ CD28⫺ cells of renal allograft recipients (Fig 1, Fig 2C) or in the whole peripheral blood populations of
KORECKA-POLAK, DUSZOTA, WIERZBICKI ET AL
CD8⫹ (Fig 2B) or CD28⫺ cells (Fig 2A) from the either the CR or S groups. We noted the presence of FOXP3⫹ cells in the CD8⫺ and CD28⫹ populations of lymphocytes (Fig 2A,B). The Few CD8⫹ CD28⫺ cells in the peripheral blood of healthy volunteers showed a low expression of FOXP3 protein (Fig 1). DISCUSSION
Our data suggested that the increased percentage of CD8⫹ CD28⫺ cells in the peripheral blood of renal recipients was associated with graft rejection among subjects receiving immunosuppressive therapy with CsA but not with RAPA, possibly because of the small number of patients treated with the latter drug. Our results were consistent with the study by Baeten et al.3 which observed a higher percentage of CD8⫹ CD28⫺ cells in the peripheral blood of renal allograft recipients with CR who were treated with calcineurin inhibitors (cyclosporine or tacrolimus) than drugfree tolerant patients (DF-Tol) with stable graft function. We did not observe cells with FOXP3 protein either among the CD8⫹ CD28⫺ subpopulation nor the whole CD8⫹ or CD28⫺ populations in the peripheral blood of the renal allograft recipients. One study by Segundo et al.8 did not detect expression of FOXP3 protein in CD8⫹ T cells in the peripheral blood of cardiac transplant recipients receiving immunosuppressive therapy based either on rapamycin or on calcineurin inhibitors. The inhibition of CD8⫹ cell activation by immunosuppressive drugs is probably the reason for the lack of FOXP3 protein in these cells. Some studies have suggested that FOXP3 is not restricted to regulatory elements and can be a marker of cell activation.9,10 The question is why immunosuppressive drugs inhibit the expression of FOXP3 protein in CD8⫹ cells but not in CD8⫺ cells and why there is no difference in outcomes of immunosuppressive therapies based on rapamycin versus cyclosporine, as indicated in the case of CD4⫹ CD25high regulatory cells.8,11,12 The correlation between T cell activation and FOXP3 expression seems to be confirmed by the lack of this marker in CD28⫺ cells because as the CD28 molecule is a costimulator for the activation of T cells.13 This observation suggests a role for the CD28 signal in the induction of FOXP3 expression in human lymphocytes. Although it is generally believed that stable FOXP3 expression occurs in human CD4⫹ T cells after TCR, triggering it has also been shown that CD28 can induce FOXP3 expression in CD4⫹ T cells.14 This suggests questions about the role of the CD28 signal in FOXP3 expression in human T cells and whether the expression of FOXP3 protein is also possible in CD8⫹ CD28⫺ T cells. Most studies have considered the presence of FOXP3 mRNA in CD8⫹ CD28⫺ cells to correlate with stable graft function. For example, Manavalan et al.7 showed the presence of FOXP3 mRNA in CD8⫹ CD28⫺ cells in the peripheral blood of rejection-free heart transplant recipients. These cells triggered the transcription of ILT3 and
T CELLS AND ALLOGRAFT FUNCTION
2919
Fig 1. Lack of expression of FOXP3 protein in the CD8⫹CD28⫺ from the peripheral blood of renal allograft recipients. Abbreviations: CsA, immunosuppressive protocol based on cyclosporine A; RAPA, immunosuppressive protocol based on rapamycin; CR, biopsy-proven chronic rejection; S, uneventful stable course; healthy Ctrl, healthy volunteers control. Plots are representative of 10 healthy volunteers and all 30 patients with both CR and S graft function and CsA and RAPA immunosuppressive protocols.
ILT4 in ECS sharing HLA class I antigens with the donor. The authors considered this to be proof of the suppressive properties of these cells. However, CD8⫹ CD28⫺ FOXP3⫹ cells were in the peripheral blood of 12 of 15 rejection-free patients who were 10 to 12 months after post-transplantation and 7 of 10 rejection-free patients who were 3 years post-transplantation. All patients were treated with standard immunosuppressive therapy (cyclosporine, steroids, and azathioprine). There is no suggestion in the article about any possible differences between the rejection-
free patients with versus without FOXP3 mRNA in their CD8⫹ CD28⫺ cells. However, as mentioned above, the transcriptional factor FOXP3 acts as a protein, so it seems that such studies should focus on examining the level of FOXP3 protein, not its mRNA. In contrast, some researchers have suggested a lack of correlation between FOXP3 mRNA levels in peripheral blood T cells and immunological processes in the graft. Dijke et al15 examined FOXP3 mRNA expression patterns among PBMCs of heart transplant patients during quies-
2920
KORECKA-POLAK, DUSZOTA, WIERZBICKI ET AL
Fig 2. Lack of expression of FOXP3 protein in the CD28⫺ (A), CD8⫹(B), and CD8⫹CD28⫺ populations (C) in the peripheral blood of a renal allograft recipient. Plots are from the analysis of the CsA-CR patient’s blood sample and are representative of all 30 patients with both CR and S graft function and CsA and RAPA immunosuppressive protocols.
cence versus rejection compared with those in endomyocardial biopsy specimens. Cardiac allograft rejection was only associated with high FOXP3 mRNA levels in the graft, but not with those in the peripheral blood. Therefore, the
phenotype of peripheral blood cells may not be involved in allograft function. To sum up, we did not detect cells with FOXP3 protein among the CD8⫹ CD28⫺ T cell population in the periph-
T CELLS AND ALLOGRAFT FUNCTION
eral blood of renal allograft recipients displaying different graft function and under different immunosuppressive protocols. The expression of this marker in CD8⫹ CD28⫺ cells seems to be useless as a diagnostic tool; FOXP3 protein is probably not a marker of the Ts subset of CD8⫹ CD28⫺ cells. ACKNOWLEDGMENT We thank Lidia Malchar and Marcin Cichocki for technical assistance.
REFERENCES 1. Arosa FA: CD8⫹CD28⫺ T cells: certainties and uncertainties of a prevalent human T-cell subset. Immunol Cell Biol 80:1, 2002 2. Cortesini R, Renna-Molajoni E, Cinti P, at al: Tailoring of immunosuppression in renal and liver allograft recipients displaying donor specific T-suppressor cells. Hum Immunol 63:1010, 2002 3. Baeten D, Louis S, Braud C, et al: Phenotypically and functionally distinct CD8⫹ lymphocyte populations in long-term drug-free tolerance and chronic rejection in human kidney graft recipients. J Am Soc Nephrol 17:294, 2006 4. Suciu-Foca N, Manavalan JS, Scotto L, et al: Molecular characterization of allospecific T suppressor and tolerogenic dendritic cells: review. Int Immunopharmacol 5:7, 2005 5. Meloni F, Morosini M, Solari N, et al: Foxp3 expressing CD4⫹CD25⫹ and CD8⫹CD28⫺ T regulatory cells in the peripheral blood of patients with lung cancer and pleural mesothelioma. Hum Immunol 67:1, 2006 6. Zhou H, Wang ZD, Zhu X, et al: CD8⫹FOXP3⫹T cells from renal transplant recipients in quiescence induce immunoglobulin-
2921 like transcripts-3 and -4 on dendritic cells from their respective donors. Transplant Proc 39:3065, 2007 7. Manavalan JS, Kim-Schulze S, Scotto L, et al: Alloantigen specific CD8⫹CD28⫺FOXP3⫹ T suppressor cells induce ILT3⫹ ILT4⫹ tolerogenic endothelial cells, inhibiting alloreactivity. Int Immunol 16:1055, 2004 8. Segundo DS, Ruiz JC, Izquierdo M, et al: Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4⫹CD25⫹ FOXP3⫹ regulatory T cells in renal transplant recipients. Transplantation 82:550, 2006 9. Morgan ME, van Bilsen JH, Bakker AM, et al: Expression of FOXP3 mRNA is not confined to CD4⫹CD25⫹ T regulatory cells in humans. Hum Immunol 66:13, 2005 10. Pillai V, Ortega SB, Wang CK, et al: Transient regulatory T-cells: a state attained by all activated human T-cells. Clin Immunol 123:18, 2007 11. Coenen JJ, Koenen HJ, van Rijssen E, et al: Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27⫹ subset of human CD4⫹CD25⫹ regulatory T cells. Blood 107:1018, 2006 12. Korczak-Kowalska G, Wierzbicki P, Bocian K, et al: The influence of immuosuppressive therapy on the development of CD4⫹CD25⫹ T cells after renal transplantation. Transplant Proc 39:2721, 2007 13. Korecka A, Duszota A, Korczak-Kowalska G. [The role of the CD28 molecule in immunological tolerance]. Postepy Hig Med Dosw 61:74, 2007 14. Scotta C, Soligo M, Camperio C, et al: FOXP3 induced by CD28/B7 interaction regulates CD25 and anergic phenotype in human CD4⫹CD25⫺ T lymphocytes. J Immunol 181:1025, 2008 15. Dijke IE, Caliskan K, Korevaar SS, et al: FOXP3 mRNA expression analysis in the peripheral blood and allograft of heart transplant patients. Transpl Immunol 18:250, 2008