Suppression of natural killer cell-mediated bone marrow cell rejection by CD4؉CD25؉ regulatory T cells Isabel Barao*, Alan M. Hanash†, William Hallett*, Lisbeth A. Welniak*, Kai Sun*, Doug Redelman‡, Bruce R. Blazar§, Robert B. Levy†¶, and William J. Murphy*¶ʈ Departments of *Microbiology and Immunology and ‡Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557; †Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33101; and §Department of Pediatrics, Division of Bone Marrow Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455 Edited by Shimon Sakaguchi, Kyoto University, Kyoto, Japan, and accepted by the Editorial Board February 10, 2006 (received for review October 23, 2005) Naturally occurring CD4؉CD25؉ T regulatory (Treg) cells have been transcription factor Foxp3 (19). CD4ϩCD25ϩ Treg cells have been shown to inhibit adaptive responses by T cells. Natural killer (NK) shown to suppress T cell responses both in vitro and in vivo (12), cells represent an important component of innate immunity in although the precise mechanisms underlying the Treg-mediated both cancer and infectious disease states. We investigated whether inhibition of immune responses remain to be defined. CD4؉CD25؉ Treg cells could affect NK cell function in vivo by using There have been numerous reports that the functional activity of allogeneic (full H2-disparate) bone marrow (BM) transplantation NK cells may be under the influence of T cell control. For example, and the model of hybrid resistance, in which parental marrow T cell-deficient athymic mice were found to have augmented NK -grafts are rejected solely by the NK cells of irradiated (BALB͞c ؋ cell function in tumor resistance models (20, 21). We have previ ͞ C57BL 6) F1 recipients. We demonstrate that the prior removal of ously reported that mice with severe combined immune deficiency ؉ host Treg cells, but not CD8 T cells, significantly enhanced NK and that lack T and B cells not only could reject allogeneic BMCs cell-mediated BM rejection in both models. The inhibitory role of but actually displayed markedly heightened BMC rejection capa- Treg cells on NK cells was confirmed in vivo with adoptive transfer bility and could resist allogeneic BMC (3). These studies would ؉ ؉ studies in which transferred CD4 CD25 cells could abrogate NK suggest that T cells can possibly down-regulate NK cell-mediated cell-mediated hybrid resistance. Anti-TGF-␤ mAb treatment also BM rejection in vivo. increased NK cell-mediated BM graft rejection, suggesting that the In this article, we provide direct evidence that CD4ϩ ϩ ؉ ؉ CD25 Treg NK cell suppression is exerted through TGF-␤. Thus, CD4 CD25 cells can modulate NK cell function in vivo. NK cell-mediated BMC Treg cells can potently inhibit NK cell function in vivo, and their rejection was significantly augmented with prior Treg depletion of depletion may have therapeutic ramifications for NK cell function the recipient mice. Further, transfer of CD4ϩCD25ϩ Treg cells in BM transplantation and cancer therapy. could suppress this rejection in vivo. These results demonstrate a potentially important regulatory link between adaptive and innate ͉ ͉ ͉ anti-CD25 bone marrow transplantation hybrid resistance Foxp3 immune responses. atural killer (NK) cells represent a key component of the Results Ninnate immune system and can mediate MHC unrestricted Enhanced BM Rejection in Full MHC-Mismatched and Hybrid Resistance cytotoxicity against neoplastic and virally infected cells; they also BMT Models by CD25؉ Cell Depletion. To determine the role of host are capable of secreting numerous effector cytokines (1, 2). In CD4ϩCD25ϩ Treg cells on allogeneic and parental BM graft addition, NK cells can mediate rejection of bone marrow (BM) but rejection, we used anti-CD25 mAbs to deplete this population in not solid-tissue allografts (3, 4). Studies by Cudkowicz and Bennett vivo. Recipient mice were treated with anti-CD25 or control (5) have shown that NK cells are responsible for the phenomenon antibody for 2 days before BMT. Treatment with anti-CD25 of ‘‘hybrid resistance,’’ in which parental BM cells (BMCs) are antibody (PC61) essentially eliminated CD25ϩ T cells (as detected rejected by lethally irradiated F1 hybrid recipients. Aside from their with the 7D4 clone) and caused a marked, but not complete, ability to kill target cells directly, NK cells have also been shown to reduction in the percentage of Foxp3ϩ cells in the lymph nodes of modulate adaptive immune responses, presumably in part through recipients (Fig. 1). The fact that we have found that PC61 anti-CD25 the release of numerous cytokines (6). NK cells have been shown does not block the binding of 7D4 anti-CD25 (data not shown) to promote T helper 1-type responses (7, 8) and participate in indicates that either all CD25ϩ cells were eliminated or CD25 cell dendritic cell maturation (9) and the generation of cytotoxic T surface expression was down-modulated in a percentage of cells lymphocytes and tumor-specific memory T cells against various after in vivo treatment with anti-CD25. This observation for the tumors (10, 11). Thus, although the influence of NK cells on presence of CD4ϩCD25ϪFoxp3ϩ cells after anti-CD25 treatment is adaptive immunity has been well documented, little has been described in ref. 22. After CD25ϩ cell depletion, lethally irradiated elucidated regarding the influence of components of the adaptive b bxd B6 (H2 ) and F1 hybrid (H2 ) recipients (9.0 and 11.0 Gy, immune system on NK cells. respectively) were transplanted with BALB͞c (H2d) BMCs at BMC Naturally occurring CD4ϩCD25ϩ T regulatory (Treg) cells have been shown to be critical immunomodulatory cells capable of ϩ ϩ suppressing immune responses. CD4 CD25 Treg cells have been Conflict of interest statement: No conflicts declared. shown to be important for maintaining self-tolerance (12), regu- This paper was submitted directly (Track II) to the PNAS office. S.S. is a guest editor invited lating the homeostasis of the peripheral T cell pool (13), contrib- by the Editorial Board. uting to tolerance induction after solid organ transplantation (14), Abbreviations: BM, bone marrow; BMC, BM cell; BMT, BM transplantation; CFU-GM, and providing protection from graft-versus-host disease lethality in colony-forming unit–granulocyte͞monocyte; Treg, T regulatory; NK, natural killer; BM transplantation (BMT) models (15, 16). These immunosup- poly(I:C), polyinosinic:polycytidylic acid; TBI, total body irradiation. pressive thymus-derived cells represent a small fraction (5–10%) of ¶R.B.L. and W.J.M. contributed equally to this work. ϩ CD4 T cells that constitutively express IL-2 receptor ␣ (CD25) ʈTo whom correspondence should be addressed. E-mail: [email protected]. (17), cytotoxic T lymphocyte-associated antigen 4 (18), and the © 2006 by The National Academy of Sciences of the USA 5460–5465 ͉ PNAS ͉ April 4, 2006 ͉ vol. 103 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509249103 Downloaded by guest on October 1, 2021 Fig. 1. Decrease in Foxp3 level in CD4ϩCD25ϩ Treg cells in anti-CD25- treated mice. Lymph node cells from rat IgG- and anti-CD25-treated mice (1 mg at days Ϫ4 and Ϫ2) were stained for CD4 and CD25, followed by anti-Foxp3 intracellular staining and analysis by flow cytometry. In comparison with untreated mice (A), the CD4ϩ cells of lymph nodes from anti-CD25-treated mice (B) exhibited few Foxp3ϩ Treg cells. In C, isotype- matched controls were used. These results are representative of three different experiments. doses in which resistance was only partial. Six days after BMT, the Allogeneic and parental BMC graft rejection is largely controlled level of BMC engraftment was determined by measuring the by the coexpression of various inhibitory and stimulatory Ly49 colony-forming unit–granulocyte͞monocytes (CFU-GMs) in receptors on NK cells whose ligands are MHC class I molecules spleen as an indicator of early post-BMT donor-derived hemato- (24). It has been previously shown that removal of the NK cell poiesis that occurs after lethal total body irradiation (TBI). The subset expressing Ly49C͞I from homozygous H2b or heterozygous data (Fig. 2 A and B) demonstrate that lethally irradiated B6 and H2bxd recipient abrogates rejection to donor H2d BM grafts, d ϩ d CB6F1 hybrid mice were not capable of significantly resisting H2 whereas the removal of Ly49G2 NK cells increases H2 BMC BMC at these doses (20 ϫ 106 and 10 ϫ 106, respectively). In these rejection (24, 25). We investigated the impact of the depletion of anti-CD25-treated recipients, the rejection of the donor BMCs was CD4ϩCD25ϩ Treg cells in combination with the removal of significantly increased after the depletion of host CD4ϩCD25ϩ T Ly49C͞Iϩ or Ly49G2ϩ NK cells on H2d marrow engraftment in B6 b bxd cells in comparison with the control group (B6, P Ͻ 0.01; CB6F1, (H2 ) and F1 (H2 ) mice (Fig. 2 D and E). The codepletion of P Ͻ 0.001). Rejection depended on host NK cells, as demonstrated Ly49C͞Iϩ NK cells and CD25ϩ cells from recipient mice signifi- by the increased engraftment of BMCs in recipient mice treated cantly promoted H2d BMC engraftment compared with control first with anti-NK1.1 (P Ͻ 0.001 compared with rat IgG treatment). mice (P Ͻ 0.01). These results indicate that CD4ϩCD25ϩ Treg cells The combined depletion of both host NK cells and CD25ϩ cells suppress a defined NK cell subset that mediates BMC rejection resulted in similar levels of BMC engraftment in comparison with based on Ly49͞H2-specific recognition. mice treated with anti-NK1.1 mAbs alone, indicating that the anti-CD25-mediated effects on engraftment were contingent on In Vivo Depletion of Host CD4؉, but Not CD8؉, T Cells Enhances H2d host NK cells. Prior in vivo activation of NK cells by administration BMC Rejection in B6 and CB6F1 Hybrid Mice.