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Suppression of natural killer cell-mediated 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 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 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 responses both in vitro and in vivo (12), cells represent an important component of innate 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 and can mediate MHC unrestricted Enhanced BM Rejection in Full MHC-Mismatched and Hybrid Resistance 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 (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 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 maturation (9) and the generation of cytotoxic T surface expression was down-modulated in a percentage of cells 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 -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 ␣ (CD25) ʈTo whom correspondence should be addressed. E-mail: [email protected]. (17), cytotoxic T -associated 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. 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, - 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 colony-forming unit–granulocyte͞monocytes (CFU-GMs) in receptors on NK cells whose ligands are MHC class I molecules 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. To confirm the role of of polyinosinic:polycytidylic acid [poly(I:C)] also increased BMC host CD4ϩCD25ϩ Treg cells in the suppression of the NK-mediated rejection (P Ͻ 0.001), as demonstrated by us and others (3, 23). The rejection, recipient mice were pretreated with anti-CD8 or anti- increased level of resistance observed by anti-CD25 administration CD4 and͞or anti-CD25-depleting mAbs. Antibody treatment re- was comparable with that seen with poly(I:C). Interestingly, coad- sulted in the depletion of Ϸ99% of the relevant CD4ϩ or CD8ϩ cell ministration of anti-CD25 mAb with poly(I:C) significantly en- populations (data not shown). As seen in Fig. 3, no change in donor hanced graft resistance compared with recipients receiving either BMC resistance was observed in mice treated with anti-CD8 mAbs treatment alone (B6, P Ͻ 0.05; CB6F1, P Ͻ 0.01), suggesting that compared with rat IgG-treated mice. In contrast, mice treated with the mechanisms by which the NK activity was increased were anti-CD4 demonstrated significant increases in BMC rejection (Fig. ϩ ϩ separate and distinct. Thus, in vivo removal of host CD4 CD25 3A, B6, P Ͻ 0.01; and Fig. 3B, CB6F1, P Ͻ 0.01) comparable with Treg cells strongly enhances NK cell-mediated allogeneic and mice depleted of CD25ϩ cells. Coadministration of anti-CD4 and parental BM rejection. anti-CD25 mAbs did not result in a significant increase of BM To verify that the removal of host CD4ϩCD25ϩ Treg cells was rejection when compared with the administration of each of the not deleterious to normal hematopoietic recovery, syngeneic BMT mAbs alone. These data suggest that CD4ϩCD25ϩ Treg cells are studies were performed. Anti-CD25 was administered to BALB͞c the T cell population that mainly regulates NK cell-mediated H2d mice before receiving a myeloablative dose of TBI and followed by BMC rejection. infusion of escalating doses of BALB͞c BMCs (1 ϫ 106 to 10 ϫ 106). Surprisingly, analysis of splenic CFU-GMs demonstrated that early In Vivo Administration of Anti-CD25 mAb Results in Decreased Long- hematopoietic recovery was significantly enhanced (P Ͻ 0.05) in Term Survival͞Donor Chimerism After Allogeneic BMT. We have mice depleted of CD25ϩ cells, signifying that removal of these cells recently shown that rejection of hematopoietic progenitors shortly was not impairing hematopoietic engraftment (Fig. 2C). Thus, the after allogeneic BMT does not always correlate with long-term increased NK cell-mediated rejection of the donor BMCs observed reconstitution and donor chimerism (26). To determine whether in mice after anti-CD25 treatment occurred despite potential depletion of CD25ϩ cells impairs long-term reconstitution of the

increases in myelopoiesis, which was observed in the syngeneic donor graft, we transplanted B6 (H2b) recipients with increasing IMMUNOLOGY models. doses of allogeneic (BALB͞c; H2d) BMCs after the infusion of

Barao et al. PNAS ͉ April 4, 2006 ͉ vol. 103 ͉ no. 14 ͉ 5461 Downloaded by guest on October 1, 2021 ϩ b Fig. 2. In vivo depletion of host CD25 cells enhances allogeneic and parental BM rejection and syngeneic BM engraftment. B6 (H2 ) mice (A and D) and CB6F1 hybrid (H2bxd) mice (B and E) received various treatments before irradiation and transplantation: rat IgG or anti-CD25, anti-Ly49C͞I (5E6), or anti-Ly49G2 (4D11) 6 at day Ϫ4 and day Ϫ2, anti-NK1.1 at day Ϫ2, and͞or poly(I:C) at day Ϫ2. Lethally irradiated B6 mice (9.0 Gy) and CB6F1 hybrid mice (11.0 Gy) received 20 ϫ 10 and 10 ϫ 106 H2d BMCs, respectively (three to four mice per group). On day 6 after BMT, splenic CFU-GMs were assessed. Enhanced allogeneic (A) and parental (B) BM graft rejection occurred after removal of host CD4ϩCD25ϩ Treg cells. A representative experiment from a total of four independent experiments is presented. (C) BALB͞c (H2d) mice were treated with rat IgG and anti-CD25 mAb as described above and received lethal TBI (7.0 Gy) and escalating doses of syngeneic BMCs (three to four mice per group). Enhanced syngeneic BM engraftment occurred after the depletion of host CD4ϩCD25ϩ Treg cells. One of three b bxd experiments is presented. The effect of coadministration of anti-Ly49 and anti-CD25 mAbs was determined in B6 (H2 ) mice (D) and F1 hybrid (H2 ) mice (E). Enhanced allogeneic and parental BM graft engraftment occurred after removal of Ly49C͞Iϩ NK cells with 5E6 and CD4ϩCD25ϩ Treg cells. In contrast, depletion of Ly49G2ϩ NK cells with 4D11 and CD4ϩCD25ϩ Treg cells increases BM rejection. Three independent experiments were performed. Values are represented as mean Ϯ SD. One-way ANOVA (A, B, D, and E) and unpaired Student’s t tests (C) were used. NRS, normal rat serum.

anti-CD25 or rat IgG mAbs. The results mirror those seen with the recipients that were depleted of CD25ϩ cells (Fig. 4) compared with CFU assays. At lower cell doses (2.5 ϫ 106 and 5 ϫ 106 BMCs), recipients that received control antibody infusions. In surviving death caused by BM failure was increased in allogeneic graft mice that received 5 ϫ 106 or 10 ϫ 106 allogeneic BMCs, lower

Fig. 3. Enhanced allogeneic and parental BM rejec- tion after anti-CD4 and anti-CD25 treatment. B6 (H2b) bxd and F1 (H2 ) mice received various mAb treatments (rat IgG or anti-CD25 mAb at day Ϫ4 and day Ϫ2 and anti-CD4 or anti-CD8 mAb at day Ϫ2) before irradia- tion and transplantation. Lethally irradiated B6 (H2b) bxd (9.0 Gy) (A) and F1 (H2 ) (11.0 Gy) (B) mice were injected with 20 ϫ 106 and 10 ϫ 106 H2d BMCs, respec- tively (three to four mice per group). Splenic CFU-GMs were assessed on day 6 after BMT. Three separate experiments were performed. Enhanced allogeneic and parental BM rejection occurred after treatment with anti-CD4 and anti-CD25 but not with anti-CD8 mAbs. Values are represented as mean Ϯ SD (one-way ANOVA).

5462 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509249103 Barao et al. Downloaded by guest on October 1, 2021 multipotential donor progenitors in recipient 1 week after transplantation and significantly increased long-term multilineage donor chimerism in sublethally irradiated recipients (34). We have now assessed the capacity of transferred CD4ϩCD25ϩ Treg cells to affect donor BMC rejection in the hybrid resistance model, which bxd is solely mediated by host NK cells. As seen in Fig. 6A,F1 (H2 ) hybrid mice were conditioned with TBI (7.0 Gy) and received a BMT 1 day later with donor H2b T cell-depleted BM from B6-GFP mice with and without coadministration of enriched CD4ϩCD25ϩ Treg cells. The sublethal conditioning dose of 7.0 Gy was selected to leave intact a significant myeloid compartment and increased ϩ ϩ host resistance capacity. CB6F1 hybrid mice receiving CD4 CD25 Treg cells at the time of BMT demonstrated significantly higher Fig. 4. Decreased survival and reduced donor chimerism in CD25-treated numbers of donor CFU-GMs compared with control recipients mice. B6 (H2b) mice received rat IgG or anti-CD25 mAbs at day Ϫ4 and day Ϫ2 (Fig. 6B, averaged 13.3-fold increases, P Ͻ 0.001) with donor before irradiation and transplantation. Mice were then irradiated (9.0 Gy) and chimerism confirmed by flow cytometry (data not shown). These transplanted with 2.5 ϫ 106, 5.0 ϫ 106,or10ϫ 106 BALB͞c (H2d) BMCs (six mice results support the depletion data and confirm that CD4ϩCD25ϩ per group). Mice were monitored for survival. On day 35 after BMT, surviving Treg cells strongly suppress NK cell-mediated BM graft rejection. mice were assessed for donor chimerism. Spleen cells were labeled with d b d anti-H2D and anti-H2K . The frequency of donor H2D expression in surviving Discussion mice is shown. A representative experiment from two independent experi- ϩ ϩ ments is presented. Values are shown as mean Ϯ SD. In this article, we provide evidence that CD4 CD25 Treg cells suppress NK cell-mediated BMC rejection in vivo. Removal of these immunosuppressive cells augmented NK activity, whereas their levels of donor (H2d) cells were observed in the spleen on day 35 addition inhibited activity as reflected by BMC engraftment. These after BMT. Survival of the recipients correlated with the chimerism data reconcile early observations of heightened NK activity in T results in which greater survival was observed in mice not treated cell-deficient mice, including heightened ability to reject BMC with anti-CD25. These results demonstrate that the removal of allografts and represent an additional link between adaptive and CD4ϩCD25ϩ Treg cells results in decreased long-term survival͞ innate immunity. NK cells have been previously shown by us and donor chimerism in allogeneic BMT. others to be capable of both up- and down-modulating T cell function (10, 11, 35, 36). The results presented here demonstrate In Vivo Administration of Neutralizing to TGF-␤ Enhances that a reciprocal arrangement exists in which T cells can also d ϩ ϩ H2 BMC Rejection in B6 and CB6F1 Hybrid Mice. CD4 CD25 T cells down-regulate NK cell function. Although NK cell activity appears have been shown to produce the immunosuppressive to be the predominant parameter affected, it remains to be deter- TGF-␤ (27, 28) and inhibit NK cell activity in vitro (29) and in vivo mined whether prolonged Treg depletion also alters NK cell (30–33). To assess the effect of the neutralization of endogenously number and͞or phenotype. It will also be of particular interest to produced TGF-␤ on BMC graft rejection, B6 and F1 mice were assess models in which increased Treg cells (e.g., models where injected with a neutralizing anti-TGF-␤ mAb (1D11) before BMT. heavy or prolonged tumor burden is present) may exist. The effects Anti-TGF-␤ mAb treatment significantly increased BM graft re- of Treg depletion on the NK cell populations may be even more jection in both mouse strains (Fig. 5A, B6, P Ͻ 0.01; and Fig. 5B, striking. ϩ ϩ F1, P Ͻ 0.01). These results suggest that TGF-␤ may be a contrib- CD4 CD25 Treg cells have been shown to suppress T cell (12, uting mechanism by which Treg cells suppress NK cell-mediated 37), NK T cell (38), and monocyte͞macrophage function (39). BM rejection in vivo. However, studies demonstrating direct effects of T cells in regu- lating NK cell-mediated BMC rejection have been lacking. NK and Adoptive Transfer of Donor CD4؉CD25؉ Treg Cells Suppresses NK T cells can resist engraftment of fully MHC-mismatched allogeneic Cell-Mediated BMC Rejection in the Hybrid Resistance BMT Model. marrow (5), but in hybrid resistance, NK cells have been identified Recently, we reported that the addition of donor CD4ϩCD25ϩ Treg as the only host immune elements capable of rejecting parental- cells affected the outcome in a complete MHC-mismatched BMT strain hematopoietic cell grafts (5, 40). We found that depletion of model (B6 3 BALB͞c) in which both host T and NK cells can host CD4ϩCD25ϩ Treg cells enhances NK cell-mediated BM mediate resistance. In this model, the addition of donor rejection in the full MHC-mismatched and hybrid resistance BMT CD4ϩCD25ϩ Treg cells resulted in greater lineage-committed and models. Moreover, adoptive transfer of donor-type CD4ϩCD25ϩ

Fig. 5. Enhanced allogeneic and parental BM rejec- b bxd tion after anti-TGF-␤ treatment. B6 (H2 ) and F1 (H2 ) mice received rat IgG or anti-TGF-␤ mAb at days Ϫ4, Ϫ2, and 0 before irradiation and transplantation. Le- b bxd thally irradiated B6 (H2 ) (9.0 Gy) (A) and F1 (H2 ) (11.0 Gy) (B) mice were injected with 20 ϫ 106 and 10 ϫ 106 H2d BMCs, respectively (three to four mice per group). Splenic CFU-GMs were assessed on day 6 after BMT. Three separate experiments were performed. Enhanced allogeneic and parental BM rejection oc- curred after treatment with anti-TGF-␤ mAb. Values are represented as mean Ϯ SD (unpaired Student’s IMMUNOLOGY t test).

Barao et al. PNAS ͉ April 4, 2006 ͉ vol. 103 ͉ no. 14 ͉ 5463 Downloaded by guest on October 1, 2021 Fig. 6. Donor CD4ϩCD25ϩ Treg cells enhance progenitor cell recovery after transplant into recipients that can mediate NK- but not T cell-dependent marrow b ϩ ϩ 6 b bxd allograft resistance. (A) Enriched H2 CD4 CD25 T cells (1 ϫ 10 ) were cotransplanted with H2 -GFP T cell-depleted BMCs into 7.0-Gy conditioned F1 (H2 ) (10 ϫ 106 BM T cell-depleted) recipients. (B) Spleen cells were removed from recipients (n ϭ 3 per group), pooled, and plated for CFU-GMs. Data are presented as the mean Ϯ SD from triplicate cultures. The results of two individual H2b 3 H2bxd experiments averaged 13.3-fold increases (unpaired Student’s t test).

bxd Treg cells into sublethally conditioned F1 (H2 ) hybrid mice myeloid reconstitution after autologous BMT. However, the use of resulted in decreased rejection of H2d BM grafts mediated by NK Treg cell depletion in allogeneic BMT may result in increased donor cells. With the advent of reduced intensity conditioning in clinical BMC rejection. The observations that increased NK cell-mediated BMT, there will likely be an increased incidence of graft rejection rejection occurred after Treg cell removal despite concurrent by host NK cells. The data presented here suggest that transfer of promotion of hematopoietic recovery suggests that the increased Treg cells can indeed be used to promote engraftment. NK activity is even more prominent. It will be also of interest to The mechanisms by which CD4ϩCD25ϩ T cells mediate their ascertain the role of Treg cells in other models (e.g., viral) where suppressive effects on NK cells are not yet determined and may NK cells play an important role. involve multiple events, particularly in vivo. The immunosuppres- In summary, we demonstrate that CD4ϩCD25ϩ Treg cells can sive properties of Treg cells appear to be mediated mainly by the suppress in vivo responses mediated by NK cells. This unique immunosuppressive cytokine TGF-␤. The inhibitory effect of immunoregulatory link between adaptive and innate lymphocytes TGF-␤ on NK cell activity in vitro is well known (29), and in vivo provides evidence for reciprocal interactions between the two arms models strongly imply that TGF-␤ is essential for suppression of NK of the immune system. The present findings enhance overall cells by CD4ϩCD25ϩ T cells (30). The cellular source of the TGF-␤ understanding of the regulation of immune responses and are was not addressed in our studies but can be derived from the particularly important for the development of therapeutic strategies immunosuppressive regulatory T cells. Recent studies have dem- to improve both BMT and cancer treatment involving NK cells. onstrated that Treg cells can suppress NK cell-mediated tumor killing by membrane-bound TGF-␤ (30). Our data showing that Materials and Methods anti-TGF-␤ mAb treatment increases NK cell-mediated BM graft Animals. BALB͞c (H2d), C57BL͞6 (B6; H2b), and BALB͞c ϫ bxd rejection raises the possibility that Treg-mediated NK suppression C57BL͞6F1 (CB6F1 or F1 H2 ) mice were obtained from the is exerted through TGF-␤ in our BMT models. However, it cannot Animal Production Area at the National Cancer Institute (Fred- be excluded that other mechanisms may be involved. For instance, erick, MD), and B6 CD8Ϫ/Ϫ (H2b) mice were from The Jackson the impaired production of IL-2 by CD4ϩ T cells (41) or IL-12 by Laboratory. C57BL͞6 TgN(act-EGFP)OsbC15-001-FJ001 GFP dendritic cells (42), potent NK stimulators, may be an indirect way transgenic breeder mice (B6-GFP) were originally provided by by which Treg cells modulate NK cell activity. Moreover, Timothy Ley (Washington University, St. Louis). All mice were CD4ϩCD25ϩ Treg cells may indirectly suppress NK cell activity maintained in microisolators under specific -free condi- through the modulation of function by inhibiting the tions. Mice were 8–12 weeks of age at the start of experiments. production of IL-15 and IL-18. NKG2D has been demonstrated to d be critical in the rejection of H2 BMCs (43). Recent studies also Myeloablative BMT Studies. B6, BALB͞c, and CB6F1 hybrid mice suggest that modulation of NKG2D may be a mechanism by which were given sulfomethoxazole-trimethoprim (Schein Pharmaceuti- Treg cells suppress NK cell function (30, 43–45). However, our cal, Corona, CA) in the drinking water 7 days before BMT. Before adoptive transfer data demonstrating that B6 (H2b) BMC rejection BMT, mice were injected i.p. with the following antibodies: 1 mg of is abrogated with Treg cell transfer into CB6F1 hybrid recipients rat IgG (Jackson ImmunoResearch); 1 mg of anti-CD25 (PC61; would suggest that NKG2D is not the only pathway that is affected. National Cell Culture Center, Minneapolis) at days Ϫ4 and Ϫ2 In the BMT scenario, Treg cells may function to inhibit donor cell before BMT (CD4ϩCD25ϩ cells were undetectable on lymph nodes engraftment and immune reconstitution either by suppressing for 8 days from day 0); 200 ␮g of anti-NK1.1 (PK136), a gift from direct NK cell killing of BM precursors and͞or the production of J. R. Ortaldo (National Cancer Institute); 300 ␮g of anti-CD4 inhibitors of hematopoiesis by these effectors (e.g., IFN-␥ and (GK1.5) or 300 ␮g of anti-CD8 (YTS169.4), generously provided by TNF-␣) (6). The increased engraftment of syngeneic BMCs ob- G. B. Huffnagle (University of Michigan, Ann Arbor) at day Ϫ2 served after Treg depletion is of interest. In this instance, before BMT; 5% of normal rat serum (NRS; Jackson ImmunoRe- CD4ϩCD25ϩ Treg cells may suppress hematopoietic progenitors search); 500 ␮g of anti-Ly49C͞I (5E6) and 200 ␮g of anti-Ly49G2 through the production of cytokines that impede hematopoiesis (4D11) provided by M. Bennett (University of Texas Southwestern directly (e.g., TGF-␤ and IL-10) or suppress cells that promote Medical Center, Dallas) at days Ϫ4 and Ϫ2 before BMT or 120 ␮g hematopoiesis (e.g., T cell production of IL-3͞GM-colony- of poly(I:C) (Sigma) at day Ϫ2; and 200 ␮g of anti-TGF-␤ (1D11), stimulating factor or macrophage production of colony-stimulating a gift from F. W. Ruscetti (National Cancer Institute), at days Ϫ4, factors). Although the mechanism remains to be elucidated, the Ϫ2, and 0. On the day of transplant (day 0), mice were lethally 137 data suggest that the removal of Treg cells increases immune and irradiated with a Cs source (B6 at 9.0 Gy, CB6F1 at 11.0 Gy, and

5464 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509249103 Barao et al. Downloaded by guest on October 1, 2021 BALB͞c at 7.0 Gy) and later injected i.v. with 20 ϫ 106 and 10 ϫ Adoptive Transfer of CD4؉CD25؉ Treg Cells and Sublethal BMT. One 6 10 BMCs from BALB͞c mice. BALB͞c recipients were trans- day before transplantation, CB6F1 recipients were conditioned with planted with 1 ϫ 106,5ϫ 106,or10ϫ 106 BALB͞c BMCs. The level a sublethal 7.0-Gy dose of TBI (60Co, 44.5 cGy͞min). BMCs of BMC engraftment was determined by measuring CFU-GM obtained from flushing of femurs and tibias were resuspended at a content in the spleen 6 days after BMT as described below. For the concentration of 2.5 ϫ 107 cells per ml, incubated for 30 min with long-term donor chimerism studies, mice were previously treated anti-Th1.2 mAb (H0-13-4) at 4°C (for 30 min), washed, and with 1 mg of rat IgG or anti-CD25 mAb at days Ϫ4 and Ϫ2 before incubated at 37°C for 30 min in the presence of complement (Rabbit they were irradiated and transplanted with different doses of Low Tox-M; Cedarlane Laboratories). Cells were washed before BALB͞c(H2d) BMCs (2.5 ϫ 106,5ϫ 106,or10ϫ 106). Mice were i.v. injection alone or together with B6 CD4ϩCD25ϩ T cells. monitored for survival and analyzed for levels of H2d 35 days after CD4ϩCD25ϩ Treg cells from spleen and lymph nodes of B6 BMT. Six mice per group were used. CD8Ϫ/Ϫ mice were obtained as described above. B6-GFP T cell- depleted BMCs were injected i.v. alone or together with B6 CFU-GM Assay. CFU-GM assays were performed as described (26, CD4ϩCD25ϩ T cells, and, 6 days after BMT, the level of BMC 34). Briefly, recipient spleens were collected on day 6 after BMT, engraftment was determined by measuring CFU-GM content in the and a single-cell suspension of each spleen was prepared. The spleen and by flow cytometry (FACScan) as described (26, 34). The ϫ 5 ϫ 5 spleen cells (1.5 10 to 5 10 ) were cultured in colony assay results are presented as the average total CFU-GMs per spleen Ϯ medium supplemented with colony stimulatory factors in 35-mm SD after multiplying CFU frequency by the total number of Petri dishes. Cultures were established in triplicate for each con- nucleated splenocytes. centration and maintained for 5–7 days at 37°C in humidified air Ͼ with 5% CO2. Colonies consisting of 50 cells were enumerated on Statistical Analysis. ANOVAs (one- and two-way) were used to a stereo microscope (Nikon). The results are presented as the total determine statistically significant differences between more than CFU-GMs per spleen Ϯ SD after multiplying CFU frequency by two experimental groups. Unpaired Student t tests were used to the total number of nucleated splenocytes. determine statistically significant differences between two experi- mental groups. P Ͻ 0.05 was considered significant. Flow Cytometric Analysis. Three-color flow cytometric analysis of cell suspensions was performed by using the following mAbs: We thank Dr. Ruth Gault for assistance with the manuscript; PE-anti-CD25 mAb (7D4) purchased from Miltenyi Biotec (Au- Kory Alderson for help with flow cytometry; and Weihong Ma, burn, CA); FITC-anti-Foxp3 (eBioscience, San Diego); and FITC- Myra Godfrey, and Danice Wilkins for excellent technical assistance. CD3, PE-CD8, PE-DX5, and PerCP-anti-CD4 (Pharmingen). This work was supported by National Institutes of Health Grants Nonspecific binding was corrected with isotype-matched controls. R01-CA93527, R01-HL63452, R01-AI34495, 2R37-HL56067, RR11576, Cell staining was prepared as described (24) and analyzed on a AI-46689, T32-CA09563, and P20-RR016464 and American Cancer FACScan with CELLQUEST software (Becton Dickinson). Society Grant RSG-020169.

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