Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance - PubMed (original) (raw)

Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance

Adrian R Kendal et al. J Exp Med. 2011.

Abstract

A paradigm shift in immunology has been the recent discovery of regulatory T cells (T reg cells), of which CD4(+)Foxp3(+) cells are proven as essential to self-tolerance. Using transgenic B6.Foxp3(hCD2) mice to isolate and ablate Foxp3(+) T reg cells with an anti-hCD2 antibody, we show for the first time that CD4(+)Foxp3(+) cells are crucial for infectious tolerance induced by nonablative anti-T cell antibodies. In tolerant animals, Foxp3(+) T reg cells are constantly required to suppress effector T cells still capable of causing tissue damage. Tolerated tissue contains T cells that are capable of rejecting it, but are prevented from doing so by therapeutically induced Foxp3(+) T reg cells. Finally, Foxp3(+) cells have been confirmed as the critical missing link through which infectious tolerance operates in vivo. Peripherally induced Foxp3(+) cells sustain tolerance by converting naive T cells into the next generation of Foxp3(+) cells. Empowering Foxp3(+) regulatory T cells in vivo offers a tractable route to avoid and correct tissue immunopathology.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Ablation of FoxP3+ T cells with an anti–human CD2 antibody. (A) Ablation of Foxp3+ cells after B6.Foxp3hCD2 mice received 1 mg YTH655 (at anti-hCD2 IgG2b mAb) i.p. in indicated organs. Three mice/group/time point are shown. (B) Foxp3+ cells after seven daily i.p. injections of 250 µg YTH655. P < 0.001. Three mice/group/time point are shown. (C) Flow cytometry analysis of spleen, lymph, and blood cells 4 wk after 2–10 million B6.Foxp3hCD2 splenocytes were injected i.v. into lymphocyte-deficient B6.RAG−/− recipients with 1 mg anti-hCD2 (squares; n = 16 composite of four experiments). **, P < 0.01 for the ablative effect of antibody compared to controls. (D) Splenocytes from B6.Foxp3hCD2 mice pretreated (open triangles, n = 6) or not (open circles, n = 3) with 7 × 250 µg anti-hCD2 mAb (30–40% of Foxp3+ T cells survived) were harvested and transferred into B6 RAG−/− mice with 1 mg anti-hCD2. P < 0.0001 compared with control (circles, n = 3). In all cases, horizontal bars represent the arithmetic mean and error bars represent SEM.

Figure 2.

Figure 2.

Loss of transplant tolerance and suppression on adoptive transfer after ablation of FoxP3+ T cells. (A) (CBAxB6hCD2)F1 mice can be made tolerant to multiple minor mismatched Balb/K skin grafts with anti-CD4/CD8/CD40L blocking mAb (squares, n = 23; P < 0.0001 compared with control; circles, _n_ = 4). The skin grafts were accepted for >150 d and were not rejected despite challenge grafting at day 60 (arrow). (B) Tolerant spleen cells were harvested from another cohort of (CBAxB6hCD2)F1 mice that had accepted a Balb/K graft for at least 60 d and transferred into RAG−/− recipients in conjunction with a challenge Balb/K graft. 20 million tolerant splenocytes from male (CBAxB6hCD2)F1 mice suppressed the addition of five million naive splenocytes (triangles, n = 6) and one million primed splenocytes (circles, n = 5). This suppression was dependent on Foxp3+ T cells because anti-hCD2 mAb led to rapid rejection of the skin grafts. P = 0.0026 (n = 9) and P = 0.0168 (n = 5), respectively (one mouse in each group was euthanized). (C) 20 million tolerant splenocytes from male (CBAxB6hCD2)F1 mice that accepted the first Balb/K graft also accepted a Balb/K skin graft when adoptively transferred into lymphocyte deplete (RAG−/−) recipients. When 1 mg anti-hCD2 was co-administered, the grafts were rejected. P = 0.0008 (n = 11). (D) 20 million tolerant splenocytes from female (CBAxB6hCD2)F1 mice that accepted the first Balb/K graft also accepted a Balb/K skin graft when adoptively transferred into lymphocyte deplete (RAG−/−) recipients with (open triangles, n = 8) or without (closed triangles, n = 3) anti-hCD2 mAb. In the same experiment, 20 million tolerant splenocytes from male (CBAxB6hCD2)F1 mice rejected the second Balb/K graft when co-administered with anti-hCD2 mAb (circles, n = 4; P = 0.0047 one mouse was euthanized).

Figure 3.

Figure 3.

Loss of transplant tolerance after ablation of Foxp3+ T cells in tolerant hosts. (A) Male (CBAxB6hCD2)F1 mice treated with anti-CD4/CD8/CD40L blocking mAb (3 × 3 mg doses over 7 d) accepted an MHC mismatched Balb/C skin (n = 27; P < 0.0001). (B) After 100 d, a challenge Balb/C skin graft was transplanted. Mice that received ablative anti-hCD2 mAb to deplete Foxp3+ T cells (squares, n = 16) rapidly rejected the challenge graft. MST 12.0 versus 47.0 d of control group (circles, n = 11), P < 0.0001. (C) The original tolerizing Balb/C graft was also rapidly rejected when mice received anti-hCD2 mAb (squares, n = 16). MST (+100d) 17.5 versus 38.0 d of control group (circles, n = 11), P = 0.0012. (D) Even without a challenge graft, mice that received anti-hCD2 mAb to ablate Foxp3+ T cells (downward triangles, n = 4; squares, n = 9) rejected a Balb/C graft that had been well tolerated for 50 d, with (P = 0.045) or without (P < 0.0001) a co-transplanted syngeneic graft.

Figure 4.

Figure 4.

Foxp3+ iTreg cells are induced in vivo by co-receptor blockade, and FoxP3+ iTreg cells induced by TGF in vitro are suppressive in vivo. (A) Female transgenic RAG−/− Marilyn.Foxp3hCD2 mice can be tolerized to male B6 RAG−/− skin by anti-CD4 blocking mAb (squares, n = 10; P = 0.0001). (B) De novo hCD2+Foxp3+ T reg cells were found in the spleen, draining, and mesenteric lymph nodes, but not the thymus, of Marilyn.Foxp3hCD2 mice that accepted a male graft for at least 60 d but not in untreated rejecting RAG−/− Marilyn.Foxp3hCD2 mice (P < 0.0001). (C) 5 × 105 TGF-β–conditioned male H-Y antigen-specific hCD2+ CD4+ RAG−/− Marilyn.Foxp3hCD2 (DBYT) cells failed to reject a male CBA.Ca RAG−/− graft (circles, n = 7) when injected into B6 RAG−/− recipients compared with 5 × 105 hCD2− DBYT cells (open squares, n = 11; P = 0.0003) or 1 × 105 naive CD4+ RAG−/− Marilyn.Foxp3hCD2 cells (open triangles, n = 6; P = 0.0006). The hCD2− DBYT group consists of mice injected with MoFlow-sorted hCD2− DBYT cells. Alone, n = 3; plus anti-hCD2 mAb, n = 4; plus isotype control mAb, n = 4. (D) 5 × 105 unsorted (DBYT) cells (squares, n = 4) and 5 × 105 hCD2+ cells (diamond, n = 5) fail to reject a male CBA.Ca RAG−/− graft when injected into B6 RAG−/− recipients. Elimination of hCD2+Foxp3+ T cells, either by anti-hCD2 mAb (open squares, n = 5; P = 0.0095) or MoFlow cell sorting of hCD2− cells (open triangles, n = 5; P = 0.0039), results in graft rejection. A challenge graft co-transplanted with 1 × 105 naive RAG−/− MarilynWT CD4+ cells after 30 d (arrow) fails to break tolerance and is itself accepted long term. (E) A positive control group is included to demonstrate that 1 × 105 naive RAG−/− MarilynWT CD4+ cells normally reject male skin when injected into naive RAG−/− mice (open circles, n = 3; P = 0.0062). **, P < 0.01; ***, P < 0.001.

Figure 5.

Figure 5.

FoxP3+ T cells within grafted tissue contributed to the survival of tolerated skin grafts transferred onto lymphopenic recipients. Male B6 RAG−/− grafts that were tolerated by anti-CD4 mAb–treated female RAG−/− Marilyn.Foxp3hCD2 mice (Fig. 4 A) were then retransplanted onto lymphocyte-deficient female B6 RAG−/− recipients. Ablation with anti-hCD2 mAb (squares, n = 11) resulted in rapid graft rejection compared with isotype control mAb (circles, n = 10; P = 0.0040).

Figure 6.

Figure 6.

In vitro–generated Foxp3+ iTreg cells prevented graft rejection by naive CD4 T cells and allowed some naive T cells to become Foxp3+ themselves. Female B6.RAG−/− received a male CBA.RAG−/− skin graft in conjunction with either 5 × 105 in vitro–cultured female hCD2+ RAG−/− Marilyn.Foxp3hCD2 DBYT cells (squares, n = 6), 1 × 105 naive CD4+ cells from RAG−/− MarilynWT mice (circles, n = 6), or a mixture of both (diamonds, n = 8). (A) All of the mice that received 1 × 105 naive CD4+ cells rejected the male skin compared with none of those that received 5 × 105 hCD2+ cells (P = 0.0066) and half that received a mixture (P = 0.0247). After 100 d, the mice were killed and Foxp3+ cell composition was analyzed. (B) Total white cell spleen counts were similar between the three groups, but there were more CD4+ cells in mice that received a mixture of hCD2+ and naive CD4+ cells. (C) FACS analysis demonstrated conversion of naive CD4+ cells into Foxp3+ T cells. Over 98% of Foxp3+ cells in mice that received 5 × 105 isolated hCD2+ RAG−/− Marilyn.Foxp3hCD2 cells alone were hCD2+, whereas 18% of Foxp3+ cells in the draining lymph and 24% in the spleen of mice that received a 5:1 mixture (and had accepted the male graft) were hCD2 negative (P = 0.0009). Error bars represent SEM.

Similar articles

Cited by

References

    1. Alters S.E., Shizuru J.A., Ackerman J., Grossman D., Seydel K.B., Fathman C.G. 1991. Anti-CD4 mediates clonal anergy during transplantation tolerance induction. J. Exp. Med. 173:491–494 10.1084/jem.173.2.491 - DOI - PMC - PubMed
    1. Andersson J., Tran D.Q., Pesu M., Davidson T.S., Ramsey H., O’Shea J.J., Shevach E.M. 2008. CD4+ FoxP3+ regulatory T cells confer infectious tolerance in a TGF-β–dependent manner. J. Exp. Med. 205:1975–1981 10.1084/jem.20080308 - DOI - PMC - PubMed
    1. Atarashi K., Tanoue T., Shima T., Imaoka A., Kuwahara T., Momose Y., Cheng G., Yamasaki S., Saito T., Ohba Y., et al. 2011. Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 331:337–341 10.1126/science.1198469 - DOI - PMC - PubMed
    1. Battaglia M., Stabilini A., Migliavacca B., Horejs-Hoeck J., Kaupper T., Roncarolo M.G. 2006. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J. Immunol. 177:8338–8347 - PubMed
    1. Benghiat F.S., Graca L., Braun M.Y., Detienne S., Moore F., Buonocore S., Flamand V., Waldmann H., Goldman M., Le Moine A. 2005. Critical influence of natural regulatory CD25+ T cells on the fate of allografts in the absence of immunosuppression. Transplantation. 79:648–654 10.1097/01.TP.0000155179.61445.78 - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources