CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade

Abstract

Immune regulatory CD4(+)CD25(+) cells play a vital role in the induction and maintenance of self-tolerance and are essential for T cell homeostasis and the prevention of autoimmunity. Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. To determine whether CD4(+)CD25(+) cells regulate T cell responses to alloantigen and are critical for tolerance induction, murine CD4(+) T cells were tolerized to alloantigen via ex vivo CD40 ligand (CD40L)/CD40 or CD28/cytotoxic T lymphocyte-associated antigen 4/B7 blockade resulting in secondary mixed leukocyte reaction hyporesponsiveness and tolerance to alloantigen in vivo. CD4(+)CD25(+) T cells were found to be potent regulators of alloresponses. Depletion of CD4(+)CD25(+) T cells from the CD4(+) responder population completely abrogated ex vivo tolerance induction to alloantigen as measured by intact responses to alloantigen restimulation in vitro and in vivo. Addback of CD4(+)CD25(+) T cells to CD4(+)CD25(-) cultures restored tolerance induction. These data are the first to indicate that CD4(+)CD25(+) cells are essential for the induction of tolerance to alloantigen and have important implications for tolerance-inducing strategies targeted at T cell costimulatory pathways.

Figure 1

CD4⁺CD25⁺ T cells are required for tolerance induction by CD40L/CD40 costimulatory blockade. (A) Primary MLR culture consisted of 10⁵ cells per well whole CD4⁺ or CD4⁺CD25⁻ T cell responders and 10⁵ cells per well bm12 splenic stimulators and/or anti-CD40L mAb. (B) Secondary MLR culture consisted of 10⁴ cells per well responder cells from 9-d bulk cultures as described in (A) and 10⁵ cells per well fresh bm12 stimulators. Tritiated thymidine was added 20 h before harvesting. On the y-axis are mean cpm ± 1 SEM. On the x-axis are days in culture. One of five representative experiments is shown.

Figure 1

CD4⁺CD25⁺ T cells are required for tolerance induction by CD40L/CD40 costimulatory blockade. (A) Primary MLR culture consisted of 10⁵ cells per well whole CD4⁺ or CD4⁺CD25⁻ T cell responders and 10⁵ cells per well bm12 splenic stimulators and/or anti-CD40L mAb. (B) Secondary MLR culture consisted of 10⁴ cells per well responder cells from 9-d bulk cultures as described in (A) and 10⁵ cells per well fresh bm12 stimulators. Tritiated thymidine was added 20 h before harvesting. On the y-axis are mean cpm ± 1 SEM. On the x-axis are days in culture. One of five representative experiments is shown.

Figure 2

CD4⁺CD25⁺ cells regulate alloresponses both in vitro and in vivo. (A and B) Primary MLR culture consisted of whole CD4⁺ or CD4⁺CD25⁻ T cell responders and bm12 splenic stimulators in the absence (A) or presence (B) of anti-CD40L mAb as in Fig. 1Purified CD4⁺CD25⁺ cells were added to CD4⁺CD25⁻ cells at the percentages indicated on the x-axis. One of three representative experiments is shown. *Significant (P < 0.05) reduction as compared with CD25⁻ culture. (C) 10⁵ naive whole CD4⁺, CD4⁺CD25⁺, whole CD4⁺ and CD4⁺CD25⁻, or whole CD4⁺ and CD4⁺CD25⁺ cells were adoptively transferred into bm12 recipients. On the x-axis are days after transfer of T cells. On the y-axis is the proportion of recipients surviving. CD4⁺CD25⁺ versus any other group, P < 0.001, n = 5–8 per group; CD4⁺ versus CD4⁺ + CD4⁺CD25⁺, P = 0.039; CD4⁺ versus CD4⁺ + CD4⁺CD25⁻, P = 0.007.

Figure 2

CD4⁺CD25⁺ cells regulate alloresponses both in vitro and in vivo. (A and B) Primary MLR culture consisted of whole CD4⁺ or CD4⁺CD25⁻ T cell responders and bm12 splenic stimulators in the absence (A) or presence (B) of anti-CD40L mAb as in Fig. 1Purified CD4⁺CD25⁺ cells were added to CD4⁺CD25⁻ cells at the percentages indicated on the x-axis. One of three representative experiments is shown. *Significant (P < 0.05) reduction as compared with CD25⁻ culture. (C) 10⁵ naive whole CD4⁺, CD4⁺CD25⁺, whole CD4⁺ and CD4⁺CD25⁻, or whole CD4⁺ and CD4⁺CD25⁺ cells were adoptively transferred into bm12 recipients. On the x-axis are days after transfer of T cells. On the y-axis is the proportion of recipients surviving. CD4⁺CD25⁺ versus any other group, P < 0.001, n = 5–8 per group; CD4⁺ versus CD4⁺ + CD4⁺CD25⁺, P = 0.039; CD4⁺ versus CD4⁺ + CD4⁺CD25⁻, P = 0.007.

Figure 2

CD4⁺CD25⁺ cells regulate alloresponses both in vitro and in vivo. (A and B) Primary MLR culture consisted of whole CD4⁺ or CD4⁺CD25⁻ T cell responders and bm12 splenic stimulators in the absence (A) or presence (B) of anti-CD40L mAb as in Fig. 1Purified CD4⁺CD25⁺ cells were added to CD4⁺CD25⁻ cells at the percentages indicated on the x-axis. One of three representative experiments is shown. *Significant (P < 0.05) reduction as compared with CD25⁻ culture. (C) 10⁵ naive whole CD4⁺, CD4⁺CD25⁺, whole CD4⁺ and CD4⁺CD25⁻, or whole CD4⁺ and CD4⁺CD25⁺ cells were adoptively transferred into bm12 recipients. On the x-axis are days after transfer of T cells. On the y-axis is the proportion of recipients surviving. CD4⁺CD25⁺ versus any other group, P < 0.001, n = 5–8 per group; CD4⁺ versus CD4⁺ + CD4⁺CD25⁺, P = 0.039; CD4⁺ versus CD4⁺ + CD4⁺CD25⁻, P = 0.007.

Figure 3

CD4⁺CD25⁺ cells upregulate CD40L on their cell surface during incubation with allostimulators. CD4⁺CD25⁻ (top) and CD4⁺ CD25⁺ (bottom) cells were phenotyped for constitutive and inducible expression of CD40L. Dotted line indicates negative control. The thin solid line indicates the constitutive expression of freshly purified naive cells. The bold line indicates CD40L expression after 4-d incubation with allostimulators.

Figure 5

Depletion of CD4⁺CD25⁺ cells abrogated protection from GVHD lethality induced by tolerization via costimulatory blockade. 10⁵ cells from washed, 9-d bulk primary MLR cultures as described in Fig. 1 and Fig. 4 were injected into bm12 recipients. (A) Anti-CD40L–tolerization. Two replicate experiments with similar results were pooled; n = 16. CD4⁺ Anti-CD40L versus any other group, P < 0.001; CD4⁺ Primed versus CD4⁺CD25⁻ Primed, P = 0.056. (B) Anti-B7–tolerization. n = 8 per group. CD4⁺ Anti-B7 versus any other group, P < 0.001.

Figure 5

Depletion of CD4⁺CD25⁺ cells abrogated protection from GVHD lethality induced by tolerization via costimulatory blockade. 10⁵ cells from washed, 9-d bulk primary MLR cultures as described in Fig. 1 and Fig. 4 were injected into bm12 recipients. (A) Anti-CD40L–tolerization. Two replicate experiments with similar results were pooled; n = 16. CD4⁺ Anti-CD40L versus any other group, P < 0.001; CD4⁺ Primed versus CD4⁺CD25⁻ Primed, P = 0.056. (B) Anti-B7–tolerization. n = 8 per group. CD4⁺ Anti-B7 versus any other group, P < 0.001.

Figure 4

CD4⁺CD25⁺ T cells are required for tolerance induction by CD28/CTLA-4/B7 costimulatory blockade. (A) Primary MLR culture consisted of whole CD4⁺ or CD4⁺CD25⁻ T cell responders and bm12 splenic stimulators and/or anti-B7 mAbs as in Fig. 1. (B) Secondary MLR culture consisted of 10⁴ cells per well responder cells from 9-d bulk cultures as described in (A) and fresh bm12 stimulators. One of four representative experiments is shown.

Figure 4

CD4⁺CD25⁺ T cells are required for tolerance induction by CD28/CTLA-4/B7 costimulatory blockade. (A) Primary MLR culture consisted of whole CD4⁺ or CD4⁺CD25⁻ T cell responders and bm12 splenic stimulators and/or anti-B7 mAbs as in Fig. 1. (B) Secondary MLR culture consisted of 10⁴ cells per well responder cells from 9-d bulk cultures as described in (A) and fresh bm12 stimulators. One of four representative experiments is shown.