Specific immunosuppression with inducible Foxp3-transduced polyclonal T cells

Abstract

Forkhead box p3 (Foxp3)-expressing regulatory T cells are key mediators of peripheral tolerance suppressing undesirable immune responses. Ectopic expression of Foxp3 confers regulatory T cell phenotype to conventional T cells, lending itself to therapeutic use in the prevention of autoimmunity and transplant rejection. Here, we show that adoptive transfer of polyclonal, wild-type T cells transduced with an inducible form of Foxp3 (iFoxp3) can be used to suppress immune responses on demand. In contrast to Foxp3-transduced cells, iFoxp3-transduced cells home "correctly" into secondary lymphoid organs, where they expand and participate in immune responses. Upon induction of iFoxp3, the cells assume regulatory T cell phenotype and start to suppress the response they initially partook in without causing systemic immunosuppression. We used this approach to suppress collagen-induced arthritis, in which conventional Foxp3-transduced cells failed to show any effect. This provides us with a generally applicable strategy to specifically halt immune responses on demand without prior knowledge of the antigens involved.

Figure 1. Polyclonal T_H::Foxp3 Cells Fail to Suppress CIA and Exhibit Altered Homing Behavior

(A) Arthritis was induced on day 0 by immunization with cII in CFA. Mice that did not receive any further treatment (black, n = 27) and mice that received 1 × 10⁶ T_H::Foxp3 cells 1 d prior to immunization (red, n = 7) are shown. The average arthritis scores of all mice in the two groups are shown. (B and C) Comparison of the homing behavior of (B) CFSE-labeled T_H (black) and T_R (red) cells and (C) GFP-expressing T_H::control (black) and T_H::Foxp3 (red) cells. A total of 1 × 10⁶ cells were transferred into each mouse (T_H, n = 3; T_R, n = 3; control, n = 4; and Foxp3, n = 6), and the tissues were analyzed 48 h later by flow cytometry. The diagrams represent the percentage of cells in each tissue, calculated from the total number of cells recovered in all tissues together (1.2 × 10⁵ ± 0.1 × 10⁵ T_H cells and 1.1 × 10⁵ ± 0.2 × 10⁵ T_R cells; 8.3 × 10⁴ ± 2.7 × 10⁴ T_H::control cells and 5.1 × 10⁴ ± 0.9 × 10⁴ T_H::Foxp3: cells; values ± SEM). Error-bars represent the SEM.

Figure 2. Foxp3-Mediated Regulation of CD62L

(A–D) CD62L expression on CD4⁺Foxp3⁻ T_H cells (black) and CD4⁺Foxp3⁺ T_R cells (red). (A) Representative FACS profiles for CD62L expression on T_H and T_R cells prepared from spleen (n = 3 in each case) with unstained T_H cells (grey) shown as control. (B) Mean fluorescence intensity (MFI) of CD62L on T_H and T_R cells from indicated tissues (n = 2 in each case). (C) Representative FACS profiles of CD4⁺CD25⁻ T_H (black) and CD4⁺CD25⁺ T_R (red) cells activated for 72 h (n = 3 in each case). (D) Total splenocytes were incubated in the absence of any treatment (solid line) or activated by addition of 100 ng/ml PMA in the presence (dashed line) or absence (dotted line) of 50 μM TAPI-2 (n = 3 in each case). (E–I) CD62L expression in T_H::control (black) and T_H::Foxp3 cells (red). CD4⁺CD25⁻ cells were activated for 36 h and transduced (0 h) with either m6p8[control] (black line; n = 3) or m6p8[Foxp3] (red line; n = 3). (E and F) Representative FACS profiles of CD62L expression on transduced cells at (E) 0 h and (F) 24 h after transduction. (G) Percentage of CD62L^hi cells within the transduced populations in the presence (dashed line) or absence (solid line) of 50 μM TAPI-2. (H) Amount of soluble CD62L in the supernatant measured by ELISA (representative of two independent experiments). (I) Relative CD62L expression in CD4⁺CD25⁻ T_H and CD4⁺CD25⁺ T_R cells (n = 3 in each case), as well as T_H::control and T_H::Foxp3 cells 48 h after transduction (n = 2 in each case) determined by qPCR and normalized to HPRT. Error bars represent the SEM.

Figure 3. Inducible Foxp3

(A) Diagram of iFoxp3 containing retroviral vectors m6pg[iFoxp3] either coexpressing GFP or a GPI-linked ratCD8 α-chain m6p8[iFoxp3], and m6p8[GFP-iFoxp3], which contains a fusion of GFP and iFoxp3. (B) MFI of intracellular stain for Foxp3 in T_H::Foxp3 and T_H::iFoxp3 cells compared to CD4⁺ T_R and T_H cells (n = 2 in each case). (C and D) Subcellular localization of GFP-iFoxp3 in T_H::GFP-iFoxp3 cells (C) in vitro after 48 h in the presence or absence of 50 nM 4-OHT or (D) in vivo after three injections of tamoxifen or carrier. (E–G) Gain of T_R cell function upon induction of iFoxp3. (E) Proliferation of T_H::control, T_H::Foxp3, and T_H::iFoxp3 cells upon anti-CD3ɛ (0.6 μg/ml) stimulation measured by ³H-thymidine incorporation in the absence (white bars; n = 3 in each case) or presence of 50 nM 4-OHT (grey bars; n = 3 in each case). (F) A total of 1 × 10⁵ CFSE-labeled CD4⁺CD25⁻ target T cells were cocultured with 1 × 10⁵ T_H::control, T_H::Foxp3 or T_H::iFoxp3 cells and activated with anti-CD3ɛ (0.6μg/ml) (n = 2 in each case). The proliferation of target cells was measured based on CFSE dilution after 72 h, and the percentage of cells that had undergone at least one cell cycle is shown. The assay was performed in the absence (white bars) or the presence (grey bars) of 50 nM 4-OHT added to the transduced cells 24 h prior to setup. (G) MFI of CD25 48 h after transduction on T_H::control, T_H::Foxp3, and T_H::iFoxp3 in the absence (white bars; n = 2 in each case) or presence of 50 nM 4-OHT (grey bars; n = 2 in each case). (H and I) Comparison of CD62L expression on T_H::control, T_H::Foxp3, and T_H::iFoxp3 48 h after transduction with m6p8. (H) Representative FACS profiles of CD62L expression (n = 2 in each case). (I) Percentage of CD62L^hi cells within the transduced populations. (J) Comparison of the homing behavior of T_H::control (black) and T_H::iFoxp3 (red) cells. A total of 1 × 10⁶ cells were transferred into each mouse (T_H::control, n = 2; T_H::iFoxp3, n = 3), and the tissues were analyzed 48 h later by flow cytometry. The diagrams represent the percentage of cells in each tissue calculated from the total number of cells recovered in all tissues together (5.4 × 10⁵ ± 0.7 × 10⁵ T_H::control cells and 3.1 × 10⁵ ± 0.4 × 10⁵ T_H::iFoxp3 cells; values ± SEM).

Figure 4. T_H::iFoxp3 Cells Partake in the Immune Response and Suppress It upon Induction

(A–C) Balb/c mice received 2 × 10⁴ T_H::Foxp3 or T_H::iFoxp3 cells prepared from DO11.10xSCID mice before being immunized s.c. with either ova in CFA (+ova) or CFA alone (−ova) (n = 3 in each case). (A) The frequency of GFP⁺ cells was measured eight days after immunization and the relative expansion was calculated as %GFP⁺ (+ova) / %GFP⁺ (-ova). (B) Total ova-specific antibodies in prebleeds (d0, white bars; n = 2 in each case) and 8 d after immunization (d8, grey bars; n = 3 in each case) in immunized and naive mice. (C) Total splenocytes were isolated from mice that had received T_H::iFoxp3 cells and were challenged with the indicated amounts of ova for 72 h in the absence (white bars) or presence (grey bars) of 50 nM 4-OHT. The total proliferation was measured by ³H-thymidine incorporation, and the relative proliferation was calculated as (+ova / (−ova). (D and E) Mice received 1 × 10⁶ polyclonal T_H::iFoxp3 cells and were immunized s.c. with ova in CFA. A week later, various tissues were analyzed. (D) The total number of recovered T_H::iFoxp3 cells from immunized mice (red, n = 3) or nonimmunized mice (black, n = 3) was calculated. (E) The relative number of endogenous and T_H::iFoxp3 cells was calculated as a ratio between immunized and nonimmunized mice. All error bars represent SEM, and p-values were determined using an unpaired t-test.

Figure 5. T_H::iFoxp3 Cells Suppress Collagen-Induced Arthritis upon iFoxp3 Induction

(A and B) Arthritis was induced on day 0 by immunization with cII in CFA. (A) Mice that received 1 × 10⁶ T_H::iFoxp3 cells (dark grey, n = 17), mice that did not receive any further treatment (black, n = 27), mice that received tamoxifen injections (light grey, n = 14), and mice that received 1 × 10⁶ T_H::iFoxp3 cells and tamoxifen injections to induce iFoxp3 (red, n = 25) are shown. The average arthritis scores of all mice in the groups are shown for each day. (B) Maximum arthritis score reached by individual animals that had received no transfer of cells, T_H::Foxp3 cells (see Figure 1A), and T_H::iFoxp3 cells ± tam. tam, tamoxifen. (C and D) Arthritis was induced by immunization with cII in CFA. (C) Mice that had received 1 × 10⁶ T_H::iFoxp3 cells the day before cII immunization and tamoxifen injections (red, n = 4) when the mice reached a score of 3 (day 0) and mice that did not receive any further treatment (black, n = 9) are shown. (D) Maximum arthritis score reached by individual animals. Error bars represent the SEM, and p-values were determined by Fisher's Exact Test.

Figure 6. T_H::iFoxp3 Cell-Mediated Suppression Is Specific

(A and B) Mice were immunized with cII in CFA on day 0. tam, tamoxifen. (A) On day 35, ex vivo recall reactions to cII were performed on cells purified from mice that did not receive any further treatment (control, n = 10), mice that had received 1 × 10⁶ T_H::iFoxp3 cells and tamoxifen injections (T_H::iFoxp3 + tam, n = 10), and naive mice (naive, n = 10). (B) Some of the mice described in (A) were immunized on day 28 with ova, and ex vivo recall reactions to ova were performed in parallel (control, −ova: n = 3, +ova: n = 7; T_H::iFoxp3 + tam, −ova: n = 3, +ova: n = 7; and naive, −ova: n = 5, +ova: n = 5). (C) Mice were immunized simultaneously with cII and ova in CFA on day 0. Ex vivo antigen-specific recall reactions to ova (closed) and cII (half-closed) were performed on day 28. Mice that did not receive any further treatment (naive, n = 4), mice that received 1 × 10⁶ T_H::iFoxp3 cells and tamoxifen injections (T_H::iFoxp3 + tam, n = 4), and mice that received 1 × 10⁶ T_H::iFoxp3 cells (T_H::iFoxp3, n = 4) are shown. p-Values were determined using an unpaired t-test.

Figure 7. T_H::iFoxp3 Cell Longevity

(A) Representative FACS profiles of splenocytes purified from the indicated mice 52 d after transfer of 1 × 10⁶ T_H::iFoxp3 cells. tam, tamoxifen. (B) Summary of the frequency of GFP⁺ cells in the spleen 52 d after transfer (n = 3 in each case). (C) Representative FACS profiles of specified tissues 52 d after transfer of 2 × 10⁶ T_H::iFoxp3 cells (n = 4 in each case; for auxiliary lymph node [aux. LN], a pooled sample was analyzed). (D) Summary of the frequency of T_H::iFoxp3 cells in the various tissues 17 and 52 d after transfer. (E–H) T_H::iFoxp3 cell survival upon 4-OHT withdrawal (E). T_H::control and T_H::iFoxp3 were cultured in the continuous presence [+ > +] or absence [− >−] of 50 nM 4-OHT. In the case of [+ >−], 4-OHT was withdrawn for 72 h after an initial induction for 48 h, before their suppressive activity was measured. A total of 1 × 10⁵ cells of the indicated populations were cocultured at a 1:1 ratio with 1 × 10⁵ CD4⁺CD25⁻ target cells in 96-well plates coated with anti-CD3ɛ [0.6 μg/ml]. The proliferation of the cells was measured after 72 h based on ³H-thymidine incorporation (n = 3 in each case). (F–H) T_H::control and T_H::iFoxp3 were cultured in the presence or absence of 4-OHT (50 nM) and anti-CD3ɛ (0.6 μg/ml). After 48 h, 4-OHT and anti-CD3ɛ was withdrawn. The viability of the cells was assessed by flow cytometry at 0 h, 24 h, and 48 h by measuring the coexpression of GFP. (F) Ratio of cells after 4-OHT withdrawal and cells that were cultured in the absence of 4-OHT from the start. (G and H) Representative FACS profiles of T_H::control and T_H::iFoxp3 cells. All error bars represent the SEM.