Xuan Zhang, Peking Union Medical College Hospital,Chinese Academy of Medical Science and Peking Union Medical College
9 February 2010
The Fluorescence intensity of CD25 is apparently influenced by the staining intensity of the conjugated antibody and the setup of flow cytometer parameters, therefore definition of CD25 expression as high or low, bright or dim, is relatively arbitrary and should be taken into consideration when comparing the results from different research groups.
Nevertheless, the expression of Foxp3 could not simply be substituted for by CD127-/low, but rather correlates with CD25 expression in CD4+CD127-/low subgroups[1]. The expression rate of Foxp3 is the highest (greater than 90%) in CD4+CD127-/lowCD25high subgroup, which is, without controversy, the classical regulatory T cell (Treg) that possessing immuno-regulatory capacity, and it is possible to use CD4+CD127-/lowCD25high as surrogate to sort out these classical Tregs, whereas this is not the case in CD4+CD127-/low subgroups that are CD25low or CD25-. For those CD25low, Foxp3 expression is not so high as in CD25 high subgroup (72%), and for those CD25- cells, Foxp3 expression dramatically drops to 10%. Similar finding was also reported by Miyara and colleagues[2]. They observed that when CD45RA was not taken into account, the expression of Foxp3 was downregulated when CD25 expression was low. Therefore it is not possible to sort out CD4+CD25-Foxp3+ subgroup using CD4+CD127-/lowCD25- as the surface surrogate markers. It is not surprising to see that CD4+CD127-CD25- cells sorted out by Bonelli M and colleagues[3] expressed Foxp3 at a rate of 53% and suppressed T-cell proliferation but not production of interferon-gamma, as these cells might be ‘contaminated’ by some CD4+CD127-CD25low cells. Indeed CD4+CD127-/lowCD25low cells can express considerable amount of Foxp3 and to some extent, also have anergic and suppressive capacity though not as potent as classical Tregs. Therefore it may not be feasible to pick out a homogeneous subgroup of cells that could represent CD4+CD25-Foxp3+ with combination surface markers of CD4,CD127 and CD25. Those abnormally increased CD4+CD25-Foxp3+cells in lupus [4] not only phenotypically resemble effector T cells (Teffc) on expressions of CCR4, GITR, CD152, but also can secret cytokines such as IL-2 and interferon-gamma[1]. In terms of CD4+CD127low/- cells, CD25low cells contamination in CD25- subgroup may affect the regulatory capacity detected.
It is possible that the intriguing CD4+CD25-/lowFoxp3+ cells is a mixture that contains CD45RA+CD25moderateFoxp3low Treg resting precursor cells as well as CD45RA-CD25-Foxp3low nonTreg cells as reported by Miyara and colleagues[2]. In their study, they divided FoxP3+CD4+ T cells into CD45RA+FoxP3low resting Treg cells, CD45RA-FoxP3high activated Treg cells, and cytokine-secreting CD45RA-FoxP3low nonsuppressive T cells, and found abnormal increase of CD45RA-FoxP3low in active SLE, which could also secrete more cytokines than the other two Foxp3+ cells. Therefore it is not surprising to see different results obtained by different research groups. Besides, Systemic lupus erythematosus (SLE) is a heterogeneous disease, of which ethnics, course of disease and immunotherapy may in some ways, have influence on its immunoregulatory network. We enrolled new-onset SLE patients as study subjects to eliminate the interference of therapy, though still we could not exclude the individual difference amplified by the disease per se.
Despite of the dispute discussed, research in this field have reached some consensus, in that in SLE patients do exist a group of abnormally increased CD4+CD25-Foxp3+ cells that decrease in most patients with active SLE after effective treatment, and may be an unique immunological feature of SLE and it does not occur in other autoimmune diseases such as rheumatoid arthritis or systemic sclerosis, though there is report that in non-obese diabetic (NOD) mice, intra-islet Treg cells express decrease levels of CD25[5]. These newly identified cells are not unanimous in property and function. They are neither traditional Treg with intact regulatory capacity nor pure Teffc. Recent studies have demonstrated that instead of being end-differentiated cells, Treg is plastic, and Foxp3+ cells could become IL-17 producers[6][7], expressing the Th1 transcription factor T-bet or produce interferon-gamma[8-10]. As we know, IL-2-induced STAT5 activation is required for Treg development and survival. The Treg-specific determining region in an intron of Foxp3 contains a STAT5 binding site and Tregs with low IL-2Ralpha (CD25) expression levels are unstable and lose FOXP3 expression when transferred to lymphopenic mice or in an IL-2-deficient autoimmune setting[11-15]. Given the plasticity of Foxp3+ cells, in human disease such as SLE, under the immunopathological condition, the upregulated CD4+CD25-Foxp3+ cells may likely be one of the mechanisms that contribute to the maintenance and expansion of autoimmune reponse. These cells may be abnormally differentiated Treg precursor or represent transitional cells during transformation from Treg to Teffc under pathological circumstances.
Advance research on the origin and development of this group of cells and their role in SLE will help us to better understand the immunopathogenesis of lupus. To do that, a specific surrogate marker for Foxp3 other than CD127low/- is needed.
Li-dan Zhao, Yang Li, Xuan Zhang Department of Rheumatology, Peking Union Medical College Hospital，Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, 100730 Corresponding author: Xuan Zhang, E-mail: zxpumch2003@yahoo.com.cn
1. Yang HX, Zhang W, Zhao LD, Li Y, Zhang FC, Tang FL, He W, Zhang X: Are CD4+CD25-Foxp3+ cells in untreated new-onset lupus patients regulatory T cells? Arthritis Res Ther 2009, 11(5):R153. 2. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T, Valeyre D et al: Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 2009, 30(6):899-911. 3. Bonelli M, Savitskaya A, Steiner CW, Rath E, Smolen JS, Scheinecker C: Phenotypic and functional analysis of CD4+ CD25- Foxp3+ T cells in patients with systemic lupus erythematosus. J Immunol 2009, 182(3):1689-1695. 4. Zhang B, Zhang X, Tang FL, Zhu LP, Liu Y, Lipsky PE: Clinical significance of increased CD4+CD25-Foxp3+ T cells in patients with new-onset systemic lupus erythematosus. Ann Rheum Dis 2008, 67(7):1037-1040. 5. Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, Sgouroudis E, Piccirillo CA, Salomon BL, Bluestone JA: Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 2008, 28(5):687-697. 6. Voo KS, Wang YH, Santori FR, Boggiano C, Wang YH, Arima K, Bover L, Hanabuchi S, Khalili J, Marinova E et al: Identification of IL-17-producing FOXP3+ regulatory T cells in humans. Proc Natl Acad Sci U S A 2009, 106(12):4793-4798. 7. Koenen HJ, Smeets RL, Vink PM, van Rijssen E, Boots AM, Joosten I: Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 2008, 112(6):2340-2352. 8. Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ: The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol 2009, 10(6):595-602. 9. Wei G, Wei L, Zhu J, Zang C, Hu-Li J, Yao Z, Cui K, Kanno Y, Roh TY, Watford WT et al: Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity 2009, 30(1):155-167. 10. Xu L, Kitani A, Fuss I, Strober W: Cutting edge: regulatory T cells induce CD4+CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta. J Immunol 2007, 178(11):6725-6729. 11. Burchill MA, Yang J, Vogtenhuber C, Blazar BR, Farrar MA: IL-2 receptor beta-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J Immunol 2007, 178(1):280-290. 12. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E et al: Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 2007, 5(2):e38. 13. Komatsu N, Mariotti-Ferrandiz ME, Wang Y, Malissen B, Waldmann H, Hori S: Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc Natl Acad Sci U S A 2009, 106(6):1903-1908. 14. Wei L, Laurence A, O'Shea JJ: New insights into the roles of Stat5a/b and Stat3 in T cell development and differentiation. Semin Cell Dev Biol 2008, 19(4):394-400. 15. Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, Ashby M, Nakayama M, Rosenthal W, Bluestone JA: Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol 2009, 10(9):1000-1007.
Competing interests
The authors declare they have no competing interests
The intriguing CD4+CD25-/lowFoxp3+ cells in SLE
9 February 2010
The Fluorescence intensity of CD25 is apparently influenced by the staining intensity of the conjugated antibody and the setup of flow cytometer parameters, therefore definition of CD25 expression as high or low, bright or dim, is relatively arbitrary and should be taken into consideration when comparing the results from different research groups.
Nevertheless, the expression of Foxp3 could not simply be substituted for by CD127-/low, but rather correlates with CD25 expression in CD4+CD127-/low subgroups[1]. The expression rate of Foxp3 is the highest (greater than 90%) in CD4+CD127-/lowCD25high subgroup, which is, without controversy, the classical regulatory T cell (Treg) that possessing immuno-regulatory capacity, and it is possible to use CD4+CD127-/lowCD25high as surrogate to sort out these classical Tregs, whereas this is not the case in CD4+CD127-/low subgroups that are CD25low or CD25-. For those CD25low, Foxp3 expression is not so high as in CD25 high subgroup (72%), and for those CD25- cells, Foxp3 expression dramatically drops to 10%. Similar finding was also reported by Miyara and colleagues[2]. They observed that when CD45RA was not taken into account, the expression of Foxp3 was downregulated when CD25 expression was low. Therefore it is not possible to sort out CD4+CD25-Foxp3+ subgroup using CD4+CD127-/lowCD25- as the surface surrogate markers. It is not surprising to see that CD4+CD127-CD25- cells sorted out by Bonelli M and colleagues[3] expressed Foxp3 at a rate of 53% and suppressed T-cell proliferation but not production of interferon-gamma, as these cells might be ‘contaminated’ by some CD4+CD127-CD25low cells. Indeed CD4+CD127-/lowCD25low cells can express considerable amount of Foxp3 and to some extent, also have anergic and suppressive capacity though not as potent as classical Tregs. Therefore it may not be feasible to pick out a homogeneous subgroup of cells that could represent CD4+CD25-Foxp3+ with combination surface markers of CD4,CD127 and CD25. Those abnormally increased CD4+CD25-Foxp3+cells in lupus [4] not only phenotypically resemble effector T cells (Teffc) on expressions of CCR4, GITR, CD152, but also can secret cytokines such as IL-2 and interferon-gamma[1]. In terms of CD4+CD127low/- cells, CD25low cells contamination in CD25- subgroup may affect the regulatory capacity detected.
It is possible that the intriguing CD4+CD25-/lowFoxp3+ cells is a mixture that contains CD45RA+CD25moderateFoxp3low Treg resting precursor cells as well as CD45RA-CD25-Foxp3low nonTreg cells as reported by Miyara and colleagues[2]. In their study, they divided FoxP3+CD4+ T cells into CD45RA+FoxP3low resting Treg cells, CD45RA-FoxP3high activated Treg cells, and cytokine-secreting CD45RA-FoxP3low nonsuppressive T cells, and found abnormal increase of CD45RA-FoxP3low in active SLE, which could also secrete more cytokines than the other two Foxp3+ cells. Therefore it is not surprising to see different results obtained by different research groups. Besides, Systemic lupus erythematosus (SLE) is a heterogeneous disease, of which ethnics, course of disease and immunotherapy may in some ways, have influence on its immunoregulatory network. We enrolled new-onset SLE patients as study subjects to eliminate the interference of therapy, though still we could not exclude the individual difference amplified by the disease per se.
Despite of the dispute discussed, research in this field have reached some consensus, in that in SLE patients do exist a group of abnormally increased CD4+CD25-Foxp3+ cells that decrease in most patients with active SLE after effective treatment, and may be an unique immunological feature of SLE and it does not occur in other autoimmune diseases such as rheumatoid arthritis or systemic sclerosis, though there is report that in non-obese diabetic (NOD) mice, intra-islet Treg cells express decrease levels of CD25[5]. These newly identified cells are not unanimous in property and function. They are neither traditional Treg with intact regulatory capacity nor pure Teffc. Recent studies have demonstrated that instead of being end-differentiated cells, Treg is plastic, and Foxp3+ cells could become IL-17 producers[6][7], expressing the Th1 transcription factor T-bet or produce interferon-gamma[8-10]. As we know, IL-2-induced STAT5 activation is required for Treg development and survival. The Treg-specific determining region in an intron of Foxp3 contains a STAT5 binding site and Tregs with low IL-2Ralpha (CD25) expression levels are unstable and lose FOXP3 expression when transferred to lymphopenic mice or in an IL-2-deficient autoimmune setting[11-15]. Given the plasticity of Foxp3+ cells, in human disease such as SLE, under the immunopathological condition, the upregulated CD4+CD25-Foxp3+ cells may likely be one of the mechanisms that contribute to the maintenance and expansion of autoimmune reponse. These cells may be abnormally differentiated Treg precursor or represent transitional cells during transformation from Treg to Teffc under pathological circumstances.
Advance research on the origin and development of this group of cells and their role in SLE will help us to better understand the immunopathogenesis of lupus. To do that, a specific surrogate marker for Foxp3 other than CD127low/- is needed.
Li-dan Zhao, Yang Li, Xuan Zhang
Department of Rheumatology, Peking Union Medical College Hospital，Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, 100730
Corresponding author: Xuan Zhang, E-mail: zxpumch2003@yahoo.com.cn
1. Yang HX, Zhang W, Zhao LD, Li Y, Zhang FC, Tang FL, He W, Zhang X: Are CD4+CD25-Foxp3+ cells in untreated new-onset lupus patients regulatory T cells? Arthritis Res Ther 2009, 11(5):R153.
2. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T, Valeyre D et al: Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 2009, 30(6):899-911.
3. Bonelli M, Savitskaya A, Steiner CW, Rath E, Smolen JS, Scheinecker C: Phenotypic and functional analysis of CD4+ CD25- Foxp3+ T cells in patients with systemic lupus erythematosus. J Immunol 2009, 182(3):1689-1695.
4. Zhang B, Zhang X, Tang FL, Zhu LP, Liu Y, Lipsky PE: Clinical significance of increased CD4+CD25-Foxp3+ T cells in patients with new-onset systemic lupus erythematosus. Ann Rheum Dis 2008, 67(7):1037-1040.
5. Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, Sgouroudis E, Piccirillo CA, Salomon BL, Bluestone JA: Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 2008, 28(5):687-697.
6. Voo KS, Wang YH, Santori FR, Boggiano C, Wang YH, Arima K, Bover L, Hanabuchi S, Khalili J, Marinova E et al: Identification of IL-17-producing FOXP3+ regulatory T cells in humans. Proc Natl Acad Sci U S A 2009, 106(12):4793-4798.
7. Koenen HJ, Smeets RL, Vink PM, van Rijssen E, Boots AM, Joosten I: Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 2008, 112(6):2340-2352.
8. Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ: The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol 2009, 10(6):595-602.
9. Wei G, Wei L, Zhu J, Zang C, Hu-Li J, Yao Z, Cui K, Kanno Y, Roh TY, Watford WT et al: Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity 2009, 30(1):155-167.
10. Xu L, Kitani A, Fuss I, Strober W: Cutting edge: regulatory T cells induce CD4+CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta. J Immunol 2007, 178(11):6725-6729.
11. Burchill MA, Yang J, Vogtenhuber C, Blazar BR, Farrar MA: IL-2 receptor beta-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J Immunol 2007, 178(1):280-290.
12. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E et al: Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 2007, 5(2):e38.
13. Komatsu N, Mariotti-Ferrandiz ME, Wang Y, Malissen B, Waldmann H, Hori S: Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc Natl Acad Sci U S A 2009, 106(6):1903-1908.
14. Wei L, Laurence A, O'Shea JJ: New insights into the roles of Stat5a/b and Stat3 in T cell development and differentiation. Semin Cell Dev Biol 2008, 19(4):394-400.
15. Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, Ashby M, Nakayama M, Rosenthal W, Bluestone JA: Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol 2009, 10(9):1000-1007.
Competing interests
The authors declare they have no competing interests