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CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice.

Weigert O, von Spee C, Undeutsch R, Kloke L, Humrich JY, Riemekasten G - Arthritis Res. Ther. (2013)

Bottom Line: The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs.The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon).

Methods: Remission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.

Results: Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5×10⁶ purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.

Conclusions: Treg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.

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Cyclophosphamide (CTX) inhibits regulatory T cell (Treg) proliferation and causes a gap in Treg numbers. (A-E) Flow cytometry of CD4+Foxp3+ Treg and CD4+Foxp3- conventional T cells (Tcon) in the spleen (SN), lymph nodes (LN), thymus (TH) and peripheral blood (PB) of CTX-treated (CTX) (NZBxNZW) F1 mice with active disease compared to age-matched PBS-treated controls (Control). (A) Representative dot plots show 5-Bromo-2'-deoxyuridine (BrdU) incorporation of CD4+Foxp3+ Treg and of CD4+Foxp3- Tcon from CTX-treated and control mice in the respective compartments. The numbers in the quadrants indicate the percentage of the respective population among CD4+ cells. (B, C) Average percentage of BrdU+ cells among CD4+Foxp3- Tcon (B) and among CD4+Foxp3+ Treg (C). Data represent the means of four to five mice per group from two independent experiments. (D, E) Average percentage of CD4+Foxp3+ Treg among CD4+ T cells (D) and average absolute counts of CD4+Foxp3+ Treg in the respective organ (E). Data represent the means of five to ten mice per group from several independent experiments. (B-E) Error bars indicate standard error of the mean (SEM) (*P <0.05, **P <0.01, CTX vs Control).
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Figure 1: Cyclophosphamide (CTX) inhibits regulatory T cell (Treg) proliferation and causes a gap in Treg numbers. (A-E) Flow cytometry of CD4+Foxp3+ Treg and CD4+Foxp3- conventional T cells (Tcon) in the spleen (SN), lymph nodes (LN), thymus (TH) and peripheral blood (PB) of CTX-treated (CTX) (NZBxNZW) F1 mice with active disease compared to age-matched PBS-treated controls (Control). (A) Representative dot plots show 5-Bromo-2'-deoxyuridine (BrdU) incorporation of CD4+Foxp3+ Treg and of CD4+Foxp3- Tcon from CTX-treated and control mice in the respective compartments. The numbers in the quadrants indicate the percentage of the respective population among CD4+ cells. (B, C) Average percentage of BrdU+ cells among CD4+Foxp3- Tcon (B) and among CD4+Foxp3+ Treg (C). Data represent the means of four to five mice per group from two independent experiments. (D, E) Average percentage of CD4+Foxp3+ Treg among CD4+ T cells (D) and average absolute counts of CD4+Foxp3+ Treg in the respective organ (E). Data represent the means of five to ten mice per group from several independent experiments. (B-E) Error bars indicate standard error of the mean (SEM) (*P <0.05, **P <0.01, CTX vs Control).

Mentions: Donor cells were obtained from lymph nodes and spleens of (NZBxNZW) F1 mice between 6 to 10 weeks of age. CD4+ T cells were enriched with a CD4+ T cell isolation kit by negative selection (Miltenyi Biotec). Unlabelled CD4+ T cells were further stained with anti-CD4-FITC (BD Biosciences, clone RM4-5,) and anti-CD25-PE (Miltenyi Biotec, clone 7D4) and CD4+CD25+ Treg were purified by cell sorting. The purity of isolated CD4+CD25+ cells was greater than 95% and more than 95% of sorted CD4+CD25+ cells expressed Foxp3 (see also Figure 1B). Sorted Treg were incubated at 37° in cell culture medium (Roswell Park Memorial Institute (RPMI) 1640 with 10% FCS and penicillin/streptomycin) supplemented with 40 ng/ml of recombinant mouse IL-2 (rmIL-2, R&D Systems GmbH, Wiesbaden, Germany) for 4 h. Then cells were harvested, washed twice with PBS, and either 0.5 or 1.5 × 106 cells suspended in PBS were injected intravenously into (NZBxNZW) F1 mice five days after the last injection of CTX. Controls received an equal amount of PBS after treatment with GC/CTX.


CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice.

Weigert O, von Spee C, Undeutsch R, Kloke L, Humrich JY, Riemekasten G - Arthritis Res. Ther. (2013)

Cyclophosphamide (CTX) inhibits regulatory T cell (Treg) proliferation and causes a gap in Treg numbers. (A-E) Flow cytometry of CD4+Foxp3+ Treg and CD4+Foxp3- conventional T cells (Tcon) in the spleen (SN), lymph nodes (LN), thymus (TH) and peripheral blood (PB) of CTX-treated (CTX) (NZBxNZW) F1 mice with active disease compared to age-matched PBS-treated controls (Control). (A) Representative dot plots show 5-Bromo-2'-deoxyuridine (BrdU) incorporation of CD4+Foxp3+ Treg and of CD4+Foxp3- Tcon from CTX-treated and control mice in the respective compartments. The numbers in the quadrants indicate the percentage of the respective population among CD4+ cells. (B, C) Average percentage of BrdU+ cells among CD4+Foxp3- Tcon (B) and among CD4+Foxp3+ Treg (C). Data represent the means of four to five mice per group from two independent experiments. (D, E) Average percentage of CD4+Foxp3+ Treg among CD4+ T cells (D) and average absolute counts of CD4+Foxp3+ Treg in the respective organ (E). Data represent the means of five to ten mice per group from several independent experiments. (B-E) Error bars indicate standard error of the mean (SEM) (*P <0.05, **P <0.01, CTX vs Control).
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Figure 1: Cyclophosphamide (CTX) inhibits regulatory T cell (Treg) proliferation and causes a gap in Treg numbers. (A-E) Flow cytometry of CD4+Foxp3+ Treg and CD4+Foxp3- conventional T cells (Tcon) in the spleen (SN), lymph nodes (LN), thymus (TH) and peripheral blood (PB) of CTX-treated (CTX) (NZBxNZW) F1 mice with active disease compared to age-matched PBS-treated controls (Control). (A) Representative dot plots show 5-Bromo-2'-deoxyuridine (BrdU) incorporation of CD4+Foxp3+ Treg and of CD4+Foxp3- Tcon from CTX-treated and control mice in the respective compartments. The numbers in the quadrants indicate the percentage of the respective population among CD4+ cells. (B, C) Average percentage of BrdU+ cells among CD4+Foxp3- Tcon (B) and among CD4+Foxp3+ Treg (C). Data represent the means of four to five mice per group from two independent experiments. (D, E) Average percentage of CD4+Foxp3+ Treg among CD4+ T cells (D) and average absolute counts of CD4+Foxp3+ Treg in the respective organ (E). Data represent the means of five to ten mice per group from several independent experiments. (B-E) Error bars indicate standard error of the mean (SEM) (*P <0.05, **P <0.01, CTX vs Control).
Mentions: Donor cells were obtained from lymph nodes and spleens of (NZBxNZW) F1 mice between 6 to 10 weeks of age. CD4+ T cells were enriched with a CD4+ T cell isolation kit by negative selection (Miltenyi Biotec). Unlabelled CD4+ T cells were further stained with anti-CD4-FITC (BD Biosciences, clone RM4-5,) and anti-CD25-PE (Miltenyi Biotec, clone 7D4) and CD4+CD25+ Treg were purified by cell sorting. The purity of isolated CD4+CD25+ cells was greater than 95% and more than 95% of sorted CD4+CD25+ cells expressed Foxp3 (see also Figure 1B). Sorted Treg were incubated at 37° in cell culture medium (Roswell Park Memorial Institute (RPMI) 1640 with 10% FCS and penicillin/streptomycin) supplemented with 40 ng/ml of recombinant mouse IL-2 (rmIL-2, R&D Systems GmbH, Wiesbaden, Germany) for 4 h. Then cells were harvested, washed twice with PBS, and either 0.5 or 1.5 × 106 cells suspended in PBS were injected intravenously into (NZBxNZW) F1 mice five days after the last injection of CTX. Controls received an equal amount of PBS after treatment with GC/CTX.

Bottom Line: The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs.The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon).

Methods: Remission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.

Results: Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5×10⁶ purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.

Conclusions: Treg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.

Show MeSH
Related in: MedlinePlus