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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.

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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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Changes in regulatory T cell (Treg) and conventional T cell (Tcon) subsets during induction of remission and sequential Treg transfer. Treg and Tcon subsets were analyzed by flow cytometry in the peripheral blood of (NZBxNZW) F1 mice during induction of remission and after an additional adoptive transfer of 1.5 × 106 Treg/mouse (Treg) and compared to age-matched mice that received only glucocorticosteroid (GC)/cyclophosphamide (CTX) and PBS (Control). (A) Representative dot plots show expression of CD25 and CD44 among CD4+ gated Foxp3+ Treg and Foxp3- Tcon. (B) Average percentage of Foxp3+ Treg among CD4+ T cells at the indicated time points during the course of the study (*P <0.05; Treg vs Control). Bar diagrams compare the average percentage of Foxp3+ Treg among CD4+ T cells before (at day -4) and after the Treg transfer (at day 14) within the respective group (*P <0.05; before vs after). (C) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3+ Treg at the indicated time points during the study. (D) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3- Tcon throughout the study. (B-D) Data are the summary of two independent experiments with six to eight mice per group in total. Error bars indicate standard error of the mean (SEM).
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Figure 5: Changes in regulatory T cell (Treg) and conventional T cell (Tcon) subsets during induction of remission and sequential Treg transfer. Treg and Tcon subsets were analyzed by flow cytometry in the peripheral blood of (NZBxNZW) F1 mice during induction of remission and after an additional adoptive transfer of 1.5 × 106 Treg/mouse (Treg) and compared to age-matched mice that received only glucocorticosteroid (GC)/cyclophosphamide (CTX) and PBS (Control). (A) Representative dot plots show expression of CD25 and CD44 among CD4+ gated Foxp3+ Treg and Foxp3- Tcon. (B) Average percentage of Foxp3+ Treg among CD4+ T cells at the indicated time points during the course of the study (*P <0.05; Treg vs Control). Bar diagrams compare the average percentage of Foxp3+ Treg among CD4+ T cells before (at day -4) and after the Treg transfer (at day 14) within the respective group (*P <0.05; before vs after). (C) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3+ Treg at the indicated time points during the study. (D) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3- Tcon throughout the study. (B-D) Data are the summary of two independent experiments with six to eight mice per group in total. Error bars indicate standard error of the mean (SEM).

Mentions: The percentage and phenotype of CD4+Foxp3+ Treg and of CD4+Foxp3- Tcon was analyzed as outlined in Figure 5A. Consistent with our previous observations, treatment with GC/CTX led to a significant decrease in the percentage of Foxp3+ Treg among CD4+ T cells in the peripheral blood (day -10 vs day -4, P <0.01) (Figure 5B). However, mice that received Treg had a significantly higher frequency of CD4+Foxp3+ Treg at day 28 compared to control mice (P <0.05) (Figure 5B). In addition, by separately comparing the frequencies of CD4+Foxp3+ Treg before (day -4) and after (day 14) transfer in each treatment group, we observed a 2-fold increase in Treg frequency in mice that received Treg (P <0.05), whereas in mice that were treated with GC/CTX alone Treg frequencies remained unaffected (Figure 5B). Thus, adoptive transfer of Treg led to an increase in Treg in the recipients and compensated the CTX-induced gap in Treg numbers.


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)

Changes in regulatory T cell (Treg) and conventional T cell (Tcon) subsets during induction of remission and sequential Treg transfer. Treg and Tcon subsets were analyzed by flow cytometry in the peripheral blood of (NZBxNZW) F1 mice during induction of remission and after an additional adoptive transfer of 1.5 × 106 Treg/mouse (Treg) and compared to age-matched mice that received only glucocorticosteroid (GC)/cyclophosphamide (CTX) and PBS (Control). (A) Representative dot plots show expression of CD25 and CD44 among CD4+ gated Foxp3+ Treg and Foxp3- Tcon. (B) Average percentage of Foxp3+ Treg among CD4+ T cells at the indicated time points during the course of the study (*P <0.05; Treg vs Control). Bar diagrams compare the average percentage of Foxp3+ Treg among CD4+ T cells before (at day -4) and after the Treg transfer (at day 14) within the respective group (*P <0.05; before vs after). (C) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3+ Treg at the indicated time points during the study. (D) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3- Tcon throughout the study. (B-D) Data are the summary of two independent experiments with six to eight mice per group in total. Error bars indicate standard error of the mean (SEM).
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Figure 5: Changes in regulatory T cell (Treg) and conventional T cell (Tcon) subsets during induction of remission and sequential Treg transfer. Treg and Tcon subsets were analyzed by flow cytometry in the peripheral blood of (NZBxNZW) F1 mice during induction of remission and after an additional adoptive transfer of 1.5 × 106 Treg/mouse (Treg) and compared to age-matched mice that received only glucocorticosteroid (GC)/cyclophosphamide (CTX) and PBS (Control). (A) Representative dot plots show expression of CD25 and CD44 among CD4+ gated Foxp3+ Treg and Foxp3- Tcon. (B) Average percentage of Foxp3+ Treg among CD4+ T cells at the indicated time points during the course of the study (*P <0.05; Treg vs Control). Bar diagrams compare the average percentage of Foxp3+ Treg among CD4+ T cells before (at day -4) and after the Treg transfer (at day 14) within the respective group (*P <0.05; before vs after). (C) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3+ Treg at the indicated time points during the study. (D) Average percentage of CD25+ cells and CD44hi cells among CD4+Foxp3- Tcon throughout the study. (B-D) Data are the summary of two independent experiments with six to eight mice per group in total. Error bars indicate standard error of the mean (SEM).
Mentions: The percentage and phenotype of CD4+Foxp3+ Treg and of CD4+Foxp3- Tcon was analyzed as outlined in Figure 5A. Consistent with our previous observations, treatment with GC/CTX led to a significant decrease in the percentage of Foxp3+ Treg among CD4+ T cells in the peripheral blood (day -10 vs day -4, P <0.01) (Figure 5B). However, mice that received Treg had a significantly higher frequency of CD4+Foxp3+ Treg at day 28 compared to control mice (P <0.05) (Figure 5B). In addition, by separately comparing the frequencies of CD4+Foxp3+ Treg before (day -4) and after (day 14) transfer in each treatment group, we observed a 2-fold increase in Treg frequency in mice that received Treg (P <0.05), whereas in mice that were treated with GC/CTX alone Treg frequencies remained unaffected (Figure 5B). Thus, adoptive transfer of Treg led to an increase in Treg in the recipients and compensated the CTX-induced gap in Treg numbers.

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