Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development This information is current as of September 23, 2021. Amir M. Alhajjat, Beverly S. Strong, Amanda E. Lee, Lucas E. Turner, Ram K. Wadhwani, John R. Ortaldo, Jonathan W. Heusel and Aimen F. Shaaban J Immunol 2015; 195:1506-1516; Prepublished online 1 July 2015; Downloaded from doi: 10.4049/jimmunol.1500463 http://www.jimmunol.org/content/195/4/1506 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2015/07/01/jimmunol.150046 Material 3.DCSupplemental References This article cites 45 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/195/4/1506.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development

Amir M. Alhajjat,* Beverly S. Strong,† Amanda E. Lee,† Lucas E. Turner,† Ram K. Wadhwani,† John R. Ortaldo,‡ Jonathan W. Heusel,x,{ and Aimen F. Shaaban†

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or . Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hema-

topoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that Downloaded from express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which http://www.jimmunol.org/ developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell–allospecific education. The Journal of Immu- nology, 2015, 195: 1506–1516.

he prenatal exposure to alloantigens is an important feature (11), significant changes occur within the NK cell compartment of immunologic development in eutherian mammals. Both that result in self-tolerance, but maintain otherwise normal T innate and adaptive components of the fetal immunity. Evidence also exists for the instructive influence of have evolved to temper the hazards of or NK cell–activating receptor interactions with environmental with the emergence of prenatal self-tolerance. Since the seminal ligands in altering the phenotype and function of the NK cell by guest on September 23, 2021 work of Owen (1), Burnet et al. (2), and Medawar and colleagues repertoire (12–14). However, animal models in which the target (3), much has been written about the origins of self-tolerance; ligand is ubiquitously expressed throughout development do not however, few studies have examined the mechanisms or signifi- adequately emulate the more complex setting of in utero he- cance of prenatal NK cell tolerance. matopoietic cellular transplantation (IUHCT) or perhaps an Current evidence suggests that NK cell self-tolerance results encounter between a developing fetal NK cell and a maternal from the interaction of inhibitory NK cell receptors with their cell during naturally occurring maternal–fetal cellular traffick- environment, resulting in a mature NK cell repertoire that is fine- ing (15). More specifically, these studies do not permit fine tuned to self-MHC class I expression (4–7). With the gain or loss modulation of the level of ligand exposure to multiple inhibitory of either cognate (8–10) or noncognate MHC class I self-Ags or activating receptors, which is logically the most significant parameter in determining prenatal tolerance or alternatively immunization. *Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, Indeed, we previously confirmed that a minimum level of cir- † IA 52242; Department of Surgery, Cincinnati Children’s Hospital Medical Center culating chimerism is necessary to induce durable NK cell toler- and University of Cincinnati College of Medicine, Cincinnati, OH 45229; ‡Experimental Therapeutics Section, National Cancer Institute, Frederick, MD 21702; xDepartment of ance to prenatally transplanted allogeneic hematopoietic cells (16). Pathology & Immunology, Washington University School of Medicine, St. Louis, MO Recipients with high chimerism levels established and maintained 63110; and {Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110 stable engraftment and exhibited donor-specific NK cell tolerance. Conversely, recipients with low chimerism levels displayed NK ORCID: 0000-0002-9459-107X (A.F.S.). cell–dependent graft rejection. The essence of this quantitative Received for publication February 24, 2015. Accepted for publication June 11, 2015. model for NK cell education is that allospecific tolerance requires This work was supported by National Institutes of Health Grant R01HL103745 and the Children’s Hospital Research Foundation (to A.F.S.). exposure to a critical level of ligand exposure during development— a chimerism threshold. In those experiments, host NK cells from Address correspondence and reprints to Dr. Aimen F. Shaaban, Center for Fetal Cellular and Molecular Therapy, Department of Surgery, Cincinnati Children’s Hos- chimeric mice naturally expressed both activating and inhibitory pital Medical Center, 3333 Burnet Avenue, MLC 11025, Cincinnati, OH 45229-3039. Ly49 receptors that were specific for the donor MHC class I ligands. E-mail address: [email protected] Following preimmune transplantation to an otherwise unmanip- The online version of this article contains supplemental material. ulated allogeneic fetal host, direct trans- recognition of donor cells Abbreviations used in this article: IUHCT, in utero hematopoietic cellular transplan- by activating and inhibitory receptors most likely played a dominant tation; LDMC, light-density mononuclear cell. role in the education of host NK cells, although indirect or even cis Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 recognition by inhibitory receptors resulting from MHC transfer www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500463 The Journal of Immunology 1507 may have had an important role in the education of host NK cells control) and PMA/ionomycin (positive control) wells. Following a 4-h (17–20). It may be speculated that a threshold level of circulating incubation at 37˚C in the presence of brefeldin A (BD Biosciences), the chimerism was critical to each of these mechanisms. In any case, splenocytes were harvested and extracellular staining was performed, as described above, followed by intracellular staining using a BD Cytofix/ current models of NK cell education do not explain how contra- Cytoperm kit (BD Biosciences). Because Abs against NK1.1 were used for dictory activating and inhibitory input signals are reconciled during stimulation, the percentages of NK cells that expressed IFN-g were de- 2 NK cell education to result in rejection or tolerance. In this study, termined by gating on CD3 DX5+ . prenatal allospecific NK cell tolerance was examined in prenatal In vivo BrdU assay chimeras. The present findings illustrate a leading role for the . instructive allorecognition by the activating receptor during Engrafter mice (peripheral blood chimerism 1.8%) and age-matched naive controls were selected at 2, 4, 7, and 10 wk of age. The animals development in determining the mature NK cell repertoire and the were injected with 200 mg BrdU (Sigma-Aldrich) twice daily for 3 d via i.p. functional competence of phenotypically distinct NK cell subsets in injection. On the fourth day, spleen was harvested from both groups and prenatal hematopoietic chimeras. LDMCs were prepared as above. Extracellular staining was first achieved, and the cells were then washed twice in preparation for intracellular staining. Atotalof106 cells was incubated in 1 ml 10% DMSO and 90% FBS (both Materials and Methods from Sigma-Aldrich) and frozen overnight at 280˚C. The cells were then Animals thawed, permeabilized, and incubated with DNase I at 37˚C for 1 h prior to washing and staining with BrdU FITC (PRB-1). Breeding stock of B6Ly5.2 (H2b, Ly5.2) and B6Ly5.1 (H2b, Ly5.1; The d Jackson Laboratory, Bar Harbor, ME) and BALB/c (H2 , Ly5.2; Charles Adoptive transfer River Laboratories, Wilmington, MA) mice were bred in our colony in the Cincinnati Children’s Research Foundation. All experimental protocols Splenic LDMCs were isolated from naive B6Ly5.1 mice at either 3 wk Downloaded from were approved by the Institutional Animal Care and Use Committee and in (immature) or 7 wk (mature), Ab labeled, and sorted on a FACSAria (BD compliance with the Department of Health Guide for the Care and Use of Biosciences) isolating CD32NK1.1+Ly49D+AFG2 NK cells (Ly49D+ NK Laboratory Animals. cells that do not coexpress Ly49A, Ly49F, or Ly49G; hostile NK cells). A total of 3–4 3 105 cells/mouse of purified hostile NK cells was transferred In utero transplantation to 7-wk-old engrafter B6Ly5.2 and age-matched naive B6Ly5.2 mice via retro-orbital injection. The transferred NK cells were followed serially in In utero transplants were performed as previously described (16) and il- + + the peripheral blood of the recipients, and the percentage of Ly49D AFG lustrated in Fig. 1A. Briefly, BALB/c fetal liver light-density mononuclear + http://www.jimmunol.org/ (Ly49D NK cells that coexpress any combination of Ly49A, Ly49F, or cells (LDMCs) were harvested from donor fetuses at embryonic day 14 + 2 Ly49G; friendly NK cells) and Ly49D AFG NK cells was calculated (day of plug = day 0) using Ficoll gradient separation (Histopaque 1077; among the transferred Ly5.1+ cells. Sigma-Aldrich, St. Louis, MO). Under isoflurane anesthesia, a midline laparotomy was made in the recipient, and the uterus was exposed. Re- Statistical analysis cipient B6 fetuses were injected through the translucent uterine wall with a5mL suspension of LDMCs using a 100 mm beveled glass micropipette. Statistical comparisons were performed using a two-tailed Student t test The uterus was returned and the abdomen closed with absorbable suture. assuming unequal variances. Engraftment rates for the in vivo deple- Pregnant dams were housed individually and given buprenorphine i.p. until tion experiments were compared using the Fisher’s exact test. All statis- delivery. Sixty-one percent of injected mice survived to weaning. Pups tical calculations were performed using Excel software (Microsoft, were weaned at 3 wk of age at which initial level of donor chimerism was Redmond, WA). determined, as outlined below. by guest on September 23, 2021 mAbs and flow cytometry Results Host NK cells expressing donor-specific activating receptors LDMCs were isolated from submandibular blood samples initially at weaning (3 wk) to determine early chimerism level and at subsequent time are responsible for graft rejection following IUHCT points according to experimental protocol. The following mAb were pur- B6 mice express high levels of the Ly49D-activating receptor on chased from eBioscience or BD Pharmingen (San Diego, CA), unless d ∼55% of their mature NK cells, although no self-ligand has been otherwise specified: CD45 anti-Ly5 (30-F11), H-2K (SF1-1.1), CD3e d (145-2C11), NK1.1 (PK136), DX5 (DX5), Ly49A (YE1/48; BioLegend, identified. This receptor binds to the H-2D MHC class I ligand San Diego, CA), Ly49C (4LO-3311; provided by W. Yokoyama, St. Louis, that is expressed on the surface of all BALB/c cells, and Ly49D+ b6 MO), Ly49D (4E5), Ly49I (YLI-90), Ly49G2 (CWY-3), Ly49H (3D10), cells have been shown to be responsible for rejection of BALB/c Ly49C/I (5E6), CD11b (M1/70), CD27 (LG.7F9), NKG2A (16A11), (H2d) marrow transplants by adult B6 (H2b) mice (21, 22). To Ly49F (HBF-719; Novus Biologicals, Littleton, CO), IFN-g (XMG1.2), and BrdU (PRB-1). Dead cells were excluded using Hoechst 33342. Where determine whether this mechanism is consistent in IUHCT, we applicable, when chimerism was determined, the percentage of non- employed a prenatal BALB/c→B6 allotransplantation model erythroid donor chimerism was calculated as the percentage of H-2Kd+ (Fig. 1A) in which tolerance or rejection is predicted by the level + cells within the total (donor + host) CD45 gate. Samples were analyzed on of early peripheral blood chimerism (16). As shown (Fig. 1B), a BD LSR II and acquired using BD FACSDiva software (both from BD . Biosciences, San Diego, CA). Data and figures were prepared using FlowJo a chimerism level 1.8% at 3 wk of age (chimerism threshold) (Tree Star, Ashland, OR). resulted in stable long-term engraftment and donor-specific tol- erance in 100% of the B6 recipients (engrafters). Conversely, In vivo NK cell subset depletion a chimerism level ,1.8% resulted in universal graft rejection in Rejecter animals with low-level chimerism (peripheral blood chimerism these recipients (rejecters). ranging from 0.9 to 1.7%) were subjected to in vivo depletion of Ly49D or To determine whether host NK cells expressing an allospecific Ly49C/I NK cells using monthly i.p. injections of purified 4E5 mAb or 5E6 activating receptor were responsible for the graft rejection, Ly49D+ mAb, respectively, at 100 mg/dose from 3 to 19 wk of age. Chimerism and the frequency of Ly49D or Ly49C/I NK cells were assessed weekly in the (4E5) NK cells were selectively depleted from the rejecter chi- + peripheral blood of treated recipients and littermate controls, including meras (0.9–1.7% chimerism), and chimerism and Ly49D NK cell immediately prior to the monthly redosing of the Ab to ensure .99% frequency were followed at serial time points (Fig. 1C). Intrave- depletion of the NK cell subset throughout the study period. nous injections with 4E5 mAb were initiated at 3 wk of age im- Intracellular assay mediately following baseline measurements. Effective elimination of Ly49D+ NK cells (.99%) was achieved by 4 wk of age and Twelve-well plates were coated overnight with 10 mg/ml anti-Ly49D and + anti-NK1.1 mAb. Plates were washed with sterile PBS. LDMCs were maintained through 20 wk, after which the Ly49D NK cells separated from freshly harvested splenocytes, washed twice, and cultured returned (Fig. 1C, Supplemental Fig. 1). As shown, the selective at a density of 106 cells into each of the coated wells or uncoated (negative in vivo depletion of Ly49D+ cells preserved engraftment in all of 1508 PRENATAL ALLOSPECIFIC NK CELL TOLERANCE Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Rejection of prenatally transplanted allografts is mediated by host Ly49D+ NK cells. (A) Prenatal allogeneic chimeras were generated by intrahepatic transplantation of light-density fetal liver cells between age-matched allogeneic fetuses at embryonic day 14 and subsequently allowed to progress toward delivery. At 3 wk of age, chimeras were sorted into engrafter or rejecter subgroups in relation to the chimerism threshold (1.8%). (B) Long- term engraftment rates for recipient mice according to initial chimerism level. (C) Rejecter mice were subjected to selective depletion of either donor- reactive (Ly49D) or nonreactive (Ly49C/I) NK cells by monthly injections of purified mAb from 3 to 19 wk of age. Representative histograms of gated CD3-NK1.1+ lymphocytes demonstrate the effective depletion of the Ly49D+ NK subset throughout the study period. Engraftment rate (D) and peripheral blood chimerism level (E) as a function of age for rejecter mice subject to selective phenotypic depletion and compared with chimerism-matched untreated controls. Graphs represent data from at least 12 separate mice in each group 6 SD. *p , 0.05 when compared with untreated control. (F) Serial chimerism levels in two Ly49D-depleted rejecter mice and an untreated rejecter control. Shaded area represents timing for return of host Ly49D+ NK cells following withdrawal of anti-Ly49D mAb. the rejecter mice when compared with the untreated or the Ly49C/ Ly49D+ NK cells. Interestingly, the chimerism level drifted above the I-depleted (5E6) rejecter controls (Fig. 1D, 1E). To further dem- threshold level of 1.8% in each of these mice during the Ly49D- onstrate that the rejection was specifically mediated by the depletion phase (Fig. 1F). Ly49D+ NK cells, the Ab injections were discontinued at 19 wk of age and the Ly49D+ NK cells were allowed to return. Graft rejection Host NK cells expressing donor-specific activating receptors was seen in 8 of the 12 previously depleted mice soon after the persist despite stable prenatal engraftment Ly49D+ NK cells reappeared. However, 4 of the 12 mice maintained Because Ly49D+ NK cells are responsible for rejection in BALB/ their engraftment despite the return of an almost normal frequency of c→B6 rejecter mice, we reasoned that they should be eliminated The Journal of Immunology 1509 in engrafter mice in which long-term engraftment is predictably lated as the frequency of friendly:hostile phenotypes. A ratio observed. Surprisingly, Ly49D+ NK cells persisted in the periph- ..1.4(averageratioinnaivecontrols) would indicate a selec- eral blood of engrafter mice at the earliest time point, but at a re- tive expansion of the friendly Ly49D+AFG+ subset and/or a selec- duced frequency when compared with naive controls (Fig. 2A). tive reduction of the hostile Ly49D+AFG2 subset had occurred. As Approximately 40% of NK cells in these tolerant hosts continue to summarized, a 4-fold higher selection ratio had already developed express Ly49D despite their potential for donor reactivity. in engrafter mice by 3 wk of age, reaching a plateau of ∼10-fold Similar changes were also present in bone marrow, raising the over the subsequent weeks as Ly49D receptor expression matured, possibility that the frequency of Ly49D+ NK cells had been al- whereas the average selection ratio in naive control mice remained tered during NK cell development (Fig. 2B) (23). No change was consistently equal to ∼1.4 (Fig. 3C). Furthermore, the changes in the observed in the frequency of NK cells expressing the irrelevant selection ratio required instructive input from the Ly49D-activating activating receptor Ly49H provided that these cells did not receptor as an analysis of inhibitory receptor coexpression in the coexpress Ly49D (Fig. 2C). Ly49D2 NK cells showed no differences between the engrafters and controls (Fig. 3C). NK cells expressing a donor-specific activating receptor without coexpressing a relevant inhibitory receptor are Lastly, a detailed analysis of multiple sites of NK cell lym- eliminated during NK cell maturation in engrafter mice phopoiesis reveals that engrafter mice exhibited an overall decrease The persistence of ostensibly alloreactive Ly49D+ NK cells in engrafter in total NK cell number in the developmentally immature envi- mice may result from the selection of developing Ly49D+ NK cells ronment of the bone marrow as well as among the more mature NK cells in the spleen and the peripheral blood (Fig. 3D). In each site, that coexpress donor-specific inhibitory receptors. In the context of Downloaded from BALB/c→B6 prenatal chimeras, each of the potentially donor-reactive the magnitude of this reduction was proportionate to the loss of host Ly49D+ NK cells would be expected to coexpress at least hostile NK cells. one inhibitory receptor that is specific for the BALB/c MHC class I ligands (e.g., Ly49A, Ly49F, or Ly49G) to remain toler- A low frequency of functionally anergic hostile NK cells ant. For simplification, these friendly phenotypes will be clus- persists in stable chimeras + +

tered and described as Ly49D AFG NK cells. Conversely, host Although their frequency was greatly diminished, phenotypically http://www.jimmunol.org/ NK cells that do not express a donor-specific inhibitory receptor hostile NK cells consistently accounted for ∼4% of all mature (Ly49D+AFG2) are considered hostile due to the unopposed engrafter NK cells (Fig. 3B). This may have resulted from the activating signals and should not propagate through the selec- coexpression of Ly49C, Ly49I, NKG2A, or other unknown in- tion process. hibitory receptors that exhibit weak binding with both donor and In support of a selection process, the frequency of friendly NK host ligands, thereby permitting these cells to remain tolerant (25, cells in peripheral blood was found to be slightly higher (40.3 6 26). However, the coexpression of any Ly49C, Ly49I, or NKG2A 5.9% versus 30.5 6 3.6%), whereas the frequency of hostile NK (CIN+) was similar between engrafter and control NK cells cells was dramatically lower (4.5 6 1.3% versus 26.8 6 2.1%) in (Supplemental Fig. 2). Furthermore, ∼20% of the residual Ly49D+ 2 engrafter mice when compared with naive controls (Fig. 3A, 3B). AFG hostile NK cells in engrafter mice did not express any by guest on September 23, 2021 2 To gauge the kinetics of NK cell selection relative to the critical Ly49C, Ly49I, or NKG2A (CIN ), which does not explain the phases of NK cell maturation (24), a selection ratio was calcu- persistence of these cells in engrafter mice (Supplemental Fig. 2).

FIGURE 2. Donor-reactive Ly49D+ NK cells persist at a reduced frequency in engrafter mice. (A) Ly49D+ NK cells persist at a lower frequency among the cir- culating pool of gated CD32NK1.1+ lymphocytes in blood over time. (B) Decreased frequency of Ly49D+ cells is consistent in blood, spleen, and bone marrow of engrafter mice. (C) The frequency of NK cells expressing the irrelevant activating receptor, Ly49H, is unchanged unless Ly49D is coexpressed. Data represent mean fre- quency 6 SD for at least five separate animals in either group at each time point. *p , 0.05. 1510 PRENATAL ALLOSPECIFIC NK CELL TOLERANCE Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Donor-specific tolerance in prenatal chimeras arises from the elimination of hostile phenotypes and the propagation of friendly phenotypes. (A and B) Comparison of donor-specific inhibitory receptor coexpression by Ly49D+ NK cells between engrafters and age-matched controls. (C) The selection ratio for donor-specific Ly49D+ NK cell phenotypes (%Ly49D+AFG+/%Ly49D+AFG2) in engrafter mice was several times that observed in control mice, whereas the selection ratio for nonspecific Ly49D2 NK cell phenotypes (%Ly49D2AFG+/%Ly49D2AFG2) was similar to controls. (D) The overall frequency of NK cell bone marrow spleen and peripheral blood is reduced in engrafter chimeras compared with age-matched controls. Subset analysis reveals that the reduction in frequency of NK cells is proportionate to the loss of hostile NK cells in the bone marrow, spleen, and peripheral blood. A CD32NK1.1 gate was used to define the total NK cell pool. Data represent mean frequency 6 SD for at least five separate animals in either group. *p , 0.05.

Alternatively, in the absence of a strong inhibitory signal, hostile Immature hostile NK cells accumulate in the hematopoietic NK cells may have persisted in the engrafter mice in a hypore- organs of prenatal chimeras and exhibit a low-avidity sponsive state, as has been shown for NK cells lacking self-specific phenotype inhibitory receptors in naive B6 mice (27–29). In support of this The selective hyporesponsiveness of hostile NK cells may have hypothesis, hostile NK cells from engrafter mice were markedly resulted from changes in their maturation or their avidity for the hyporesponsive relative to friendly NK cells following stimulation donor ligands similar to self-reactive T and B cells (31, 32). To with plate-bound Ly49D or NK1.1 mAb (Fig. 4A) using an estab- examine this possibility, we adopted the CD27/CD11b para- lished in vitro assay for NK cell responsiveness (30). This was in digm of NK cell maturation, which dissects NK cells into three sharp contrast to hostile NK cells in naive control mice, which were distinct subsets of maturation, as follows: CD27highCD11blow slightly more responsive than friendly NK cells in the same assays (R1); CD27highCD11bhigh (R2); CD27lowCD11bhigh (R3) (33, (Fig. 4A). Additionally, the response to calcium flux (PMA and 34). In examining the maturation of engrafter NK cells, we ionomycin) was maintained, confirming that the hostile NK cells noted an overall shift of hostile Ly49D+AFG2 NK cells toward + retained the capacity to produce IFN-g. Lastly, Ly49D NK cells a more immature phenotype. As shown (Fig. 5A), the frequency of coexpressing any combination of Ly49A, Ly49F, or Ly49G in the R1 subset of Ly49D+ NK cells in 3-mo-old engrafter mice engrafter mice exhibited normal responsiveness, indicating that ex- was higher than in age-matched control mice. The altered fre- pression of at least one inhibitory receptor for the donor prevented quency of R1 NK cells was most striking in the bone marrow. A the development of anergy (Fig. 4B). These observations demon- detailed comparison of Ly49D+ NK cells revealed significant strate that the selective hyporesponsiveness of hostile NK cells is at maturational differences between friendly and hostile NK cells least partially regulated by cell-intrinsic mechanisms that result in (Fig. 5B). The hostile NK cells displayed a notable shift toward donor-specific tolerance. a higher frequency of the R1 phenotype and lower frequencies The Journal of Immunology 1511 Downloaded from http://www.jimmunol.org/

FIGURE 4. Hostile NK cells from engrafter mice are selectively anergic to activating receptor-mediated signaling. Splenocytes from engrafters and controls were subjected to in vitro stimulation by plate-bound anti-Ly49D (4E5) mAb, anti-NK1.1 (PK136) mAb, or PMA and ionomycin, and the fre- quency of IFN-g+ cells was measured. (A) The resulting hostile:friendly IFN-g production ratio was calculated and compared between the groups: % Ly49D+AFG2 IFN-g+ NK cells/%Ly49D+AFG+IFN-g+ NK cells. (B) In separate experiments, a subset analysis of Ly49D+ NK cells coexpressing single or multiple donor-specific inhibitory receptors was performed. The IFN-g production ratio for each Ly49D+ NK cell subset relative to the friendly NK cell subset was calculated and compared: %subset+ IFN-g+ cells/%Ly49D+AFG+IFN-g+ NK cells. Values shown are derived from analysis of gated CD32 NK1.1+ lymphocytes with five separate animals in either group plus or minus SD. Values for Ly49D+F+AG2 NK cells are excluded, as the frequency of this subset is ,1% of all Ly49D+ NK cells and inadequate for statistical analysis. *p , 0.05. by guest on September 23, 2021 of the more mature R2 and R3 phenotypes in the bone marrow. possibility that the cell-intrinsic responses to activating receptor A similar pattern for R1 and R2 persisted in the spleen and signaling were fundamentally altered in hostile NK cells. Further blood, where the NK cell repertoire is progressively dominated study is needed to confirm whether the impairment in multiple by the more mature R2 and R3 subset. The differences were signaling mechanisms arises independently or through a common not significant in the lung, where relatively few immature NK defect in a downstream signaling pathway. cells are typically recovered. Conversely, friendly NK cells displayed no significant changes in maturation when compared Despite widely disparate frequencies, friendly and hostile NK with controls. cells from engrafter mice exhibit equivalent rates of To determine whether a diminished avidity for the donor cells homeostatic proliferation contributed to tolerance, the cell surface expression of Ly49D by Previous studies of NK cell maturation reveal a higher rate of hostile NK cells was examined in engrafter mice. Fig. 5C illustrates homeostatic proliferation by NK cells that express a self-specific that hostile Ly49D+AFG2 NK cells in engrafter mice expressed inhibitory receptor compared with NK cells that do not (23). significantly lower levels of Ly49D on their cell surface when Accepting this principle, we would expect that the friendly NK compared with controls, whereas a reduction in Ly49D expression cells in engrafter mice would display a higher homeostatic was not exhibited by friendly NK cells. The decreased expression proliferation rate compared with the hostile NK cells. To eval- appeared to be specific for Ly49D, as the expression level of uate this possibility, an in vivo BrdU assay was performed at Ly49H was unchanged when coexpressed on the same hostile NK different phases of murine NK cell development (24). A phe- cells (Fig. 5C). Furthermore, Ly49D downregulation was not ob- notypic analysis of friendly and hostile NK cells in the spleens served with coexpression of a donor-specific inhibitory receptor in of engrafter mice (Fig. 6A) revealed the skewed selection ratio the Ly49D+G+AF2 or Ly49D+A+FG2 NK cell subsets (Fig. 5C). that mirrored the peripheral blood findings (Fig. 3C) beginning A kinetic analysis revealed that the Ly49D downregulation was at the earliest stages of Ly49 receptor expression and reaching clearly evident by 3 wk of age and was maintained throughout the a plateau as receptor expression matured. Despite the markedly period of Ly49 receptor maturation (Fig. 5D). These selective diverging frequencies of these alternate phenotypes in the changes in the level of Ly49D expression offer an explanation for engrafter mice, the homeostatic proliferation of friendly and the anergic response of hostile NK cells to anti-Ly49D stimula- hostile NK cells was nearly identical, yielding a proliferation tion. However, the same hostile NK cells were also hypore- ratio that was approximately equal to 1 (Fig. 6B). As a result, the sponsive to anti-NK1.1 stimulation without changes in the cell preponderance of friendly NK cells could not be explained by surface expression of NK1.1 (Supplemental Fig. 3), raising the differences in homeostatic proliferation. 1512 PRENATAL ALLOSPECIFIC NK CELL TOLERANCE

FIGURE 5. Nondeleted hostile NK cells in stable chimeras exhibit an immature low-avidity phenotype. (A) Multiorgan analysis at 12 wk of age, demonstrating the relative maturation of Ly49D, hostile and friendly NK cells in an engrafter and a control mouse using the CD27/CD11b paradigm: CD27highCD11blow (R1); CD27high CD11bhigh (R2); CD27lowCD11bhigh (R3). (B) Analysis of hostile and friendly NK cell maturation at 5 mo of age re- vealing the selectively impaired mat- uration of hostile NK cells in the bone marrow, spleen, and blood of engrafter Downloaded from mice. (C) Measurement of the relative intensity of Ly49D expression by splenic NK cells from engrafter and control mice at 12 wk of age reveals selective downregulation by hostile phenotypes. The coexpression of a single donor-

specific inhibitory receptor is sufficient http://www.jimmunol.org/ to prevent Ly49D downregulation. The graphed data represent the mean fluo- rescence intensity of Ly49D receptor expression by friendly NK cells from at least five engrafter mice relative to the corresponding levels exhibited by con- trol mice (rMFI). (D) Ly49D expression was tracked on friendly and hostile pe- ripheral blood NK cells from engrafter and naive control mice from 3 to 12 wk by guest on September 23, 2021 of age. A CD3-NK1.1 lymphocyte gate was used to define the total NK cell pool. Values shown are derived from at least five separate animals in either group 6 SD. *p , 0.05.

Hostile NK cells in developmentally immature prenatal not display any signs of autoimmunity, and their chimerism level chimeras upregulate inhibitory receptors through activating was stable throughout the study period despite the transfer of receptor recognition of cognate donor ligand these cells (data not shown). Conversely, in the nonchimeric The higher selection ratio in engrafter mice might be alternatively control environment, the transferred hostile NK cells exhibited explained by the upregulation of inhibitory receptor expression by much less inhibitory receptor upregulation and only reached the hostile NK cells in engrafter mice, resulting in the peripheral level seen in naive mice. Surprisingly, hostile NK cells from crossover to a friendly phenotype. To examine this possibility, mature donor mice did not have the same capacity for inhibitory receptor upregulation as those from immature donors (Fig. 7C). hostile NK cells were sorted from naive B6Ly5.1 control mice and 2 2 adoptively transferred to BALB/c→B6Ly5.2 engrafter mice or Lastly, adoptively transferred Ly49D AFG NK cells exhibited B6Ly5.2 controls (Fig. 7A). The donor mice were either immature a stable phenotype without upregulation of Ly49D, Ly49A, Ly49F, (3 wk) or mature (7 wk) with regard to Ly49 receptor acquisition. or Ly49G in the chimeric secondary host, suggesting that signaling The upregulation of inhibitory receptor expression by the trans- input from the activating receptor is needed for inhibitory receptor ferred hostile Ly5.1+ NK cells was tracked in the new chimeric or upregulation (Supplemental Fig. 4). Therefore, the expression of nonchimeric (naive) secondary environment. As shown in Fig. 7B, donor-specific inhibitory receptors by hostile NK cells is favored by hostile NK cells that were transferred from an immature donor to activating receptor recognition of donor ligand in the peripheral a chimeric secondary recipient displayed a sharp upregulation in environment and most likely contributes to the preponderance of the expression of the donor-specific inhibitory receptors, eventu- phenotypically friendly NK cells in engrafter mice. The capacity for ally reaching the same relative frequency of friendly NK cells these adaptive changes appeared to be lost in developmentally seen in mature engrafter mice. The secondary recipient mice did mature mice. The Journal of Immunology 1513

FIGURE 6. Friendly and hostile NK cells demonstrate similar ho- meostatic proliferation despite their diverging frequencies. (A) The fre- quency of friendly and hostile NK cells among total splenic NK cell population in engrafter mice and age-matched controls. The resulting Ly49D+ selec- tion ratio is plotted at 2, 4, 7, and 10 wk of age and reveals a dramatic rise as maturation is achieved in engrafters. (B) The frequency of BrdU-positive NK cells among the friendly and hostile NK Downloaded from cell populations is shown. The Ly49D+ proliferation ratio (%friendly BrdU+ NK cells/% hostile BrdU+ NK cells) is plotted for the same data points and is thesameinengrafterandcontrolmice. A CD3-NK1.1 lymphocyte gate was used to define the total NK cell pool. http://www.jimmunol.org/ Data are derived from at least five experiments in each group 6 SD. *p , 0.05. by guest on September 23, 2021

Discussion necessary to convey tolerance to the Ly49D+ NK cells. This From the results of this study, a series of novel principles emerge conclusion is advanced by the findings in prenatal chimeras that that may fundamentally guide future investigations of develop- support phenotypic selection as the primary tolerance mechanism mental NK cell allospecific education. The first of these principles functioning at the earliest stages of Ly49 receptor development. is that host NK cells expressing a donor-reactive activating receptor Activating receptor recognition of the donor ligands appeared to 2 are key effectors of the prenatal allospecific response. This finding regulate the selection process because the Ly49D NK cells dis- establishes a mechanistic basis for an immune barrier to IUHCT played no differences in inhibitory receptor expression between that resides within the host. In prenatal BALB/c→B6 murine the engrafter and control mice. This stark contrast between 2 chimeras, this immune barrier exists as a subset of Ly49D+ NK Ly49D+ and Ly49D NK cells indicates that the checkpoint for cells. In the human fetus, it may exist in subsets of NK cells self-reactivity occurs after the expression of the Ly49D+ receptor expressing activating killer Ig-related receptors (35). If the prin- with the potential to subsequently coexpress Ly49A, L49F, or ciple is well conserved, then the potential for an allospecific im- Ly49G. mune response might include NK cells expressing killer Ig-related A third principle is that central NK cell selection is supported by receptors for ligands that do not normally exist within the host. the peripheral upregulation of inhibitory receptor expression by Surprisingly, the short-term depletion of the Ly49D+ NK cell hostile NK cells following recognition of the donor ligands through subset reliably preserved long-term engraftment in a subset of the the activating receptor. Whether this mechanism is a primary rejecter mice even after the treatment was discontinued. The po- feature of NK cell education or a secondary adaptive process was tential to rescue engraftment in this way suggests that the targeted addressed in the current report. The observation that the capacity depletion of donor-reactive NK cells may be a useful adjunct for for inhibitory receptor upregulation is greatly diminished in de- clinical IUHCT. velopmentally mature mice suggests that this is the primary process The second principle is that the appraisal for self-reactivity through which the final NK cell repertoire is attained. However, during NK cell selection in prenatal chimeras is based on the although a mechanism involving inhibitory receptor upregulation composite receptor phenotype and is directed by activating receptor might explain the increased frequency of friendly NK cells, it does recognition of the donor ligands. This concept was first examined not account for the overall decrease in the total number of NK cells by George et al. (36), who found that 97.8% of Ly49D+ NK cells in seen in the engrafter mice. Remarkably, we found very few hostile b/d B6 3 Balb F1 hybrid mice (H2D ) coexpressed at least one H- NK cells in the engrafter mice at any time point even as early as 2Dd–specific inhibitory receptor and concluded that this was 2 wk of age (Fig. 6A), suggesting that the elimination of this 1514 PRENATAL ALLOSPECIFIC NK CELL TOLERANCE Downloaded from

FIGURE 7. Adoptive transfer of hostile NK cells into a chimeric environment results in upregulation of inhibitory receptor coexpression. (A) Schematic http://www.jimmunol.org/ representation of the adoptive transfer experiments. Hostile NK cells from either immature (3 wk) or mature (7 wk) naive B6Ly5.1 mice were harvested and sorted to isolate hostile NK cells. The hostile NK cells were adoptively transferred to mature engrafters and age-matched B6Ly5.2 controls. The adoptively transferred cells were followed for their expression of the donor-specific inhibitory receptors. (B) The upregulation of inhibitory receptors in the adoptively transferred hostile NK cells from both the immature and mature donor is plotted. This is reported as the fraction of Ly5.1+ NK cells that are Ly49D+AFG+ and is followed serially in peripheral blood. (C) The upregulation of inhibitory receptor is compared between mature and immature donor NK cells transferred into the chimeric environment. A CD32NK1.1 lymphocyte gate was used to define the total NK cell pool. Data are derived from at least three separate experiments in each group 6 SD. *p , 0.05. phenotype occurred at a very early stage in NK cell maturation. toward the donor. This could be brought about by the unop- by guest on September 23, 2021 The similarities in homeostatic proliferation between hostile and posed chronic Ly49D stimulation of hostile NK cells resulting friendly NK cells at early and late time points (Fig. 6B) further in the exhaustion of DAP10 and DAP12 signaling adaptors support that tolerance was established at a very early age. Hence, leading to the functional hyporesponsiveness of other DAP10/ a model of NK cell education that begins with phenotypic selec- 12-dependent activating receptors and potentially DAP10/12- tion in the bone marrow and is subsequently supported by a de- independent receptors through an additional downstream sig- velopmentally limited capacity for fine-tuning of the peripheral naling defect (38–40). In this way, disarming would explain the repertoire provides a more comprehensive explanation for these pattern of anergy exhibited by the hostile NK cells. However, findings. Instructive recognition of ligand through the activating disarming does not provide a stand-alone explanation for the receptor appears to be an essential feature of each step. Conclusive emergence of only a small percentage of Ly49D+AFG2 NK support for such a process awaits the future characterization of cells at the earliest developmental time points. Instead, dis- stage-specific markers for NK cell education. arming may serve as the prevailing adaptive process in devel- A fourth principle is that the terminal development of hostile NK opmentally mature mice rather than during NK cell education cells is fundamentally altered in engrafter mice and results in a state in immature mice, where the capacity for inhibitory receptor of selective anergy. The pattern of altered activating receptor ex- upregulation is preserved (Fig. 7C). If the capacity to induce the pression and maturation displayed by NK cells from the bone coexpression of ligand-specific inhibitory receptors during devel- marrow, spleen, and peripheral blood further suggests that the opment is not possible, broad disarming of NK cells expressing appraisal for self-responsiveness began during the final stages of the activating receptor may occur. Relevant to this postulate, NK cell maturation in the bone marrow. Low-avidity, immature Tripathy et al. (12–14) examined the effect of developmental hostile NK cells may have exited the selection process and relo- exposure of the m157 viral ligand to its exclusive DAP10/12- cated to peripheral sites such as the spleen, where they remained dependent Ly49H-activating receptor in transgenic mice in relatively hyporesponsive to receptor-mediated signaling. There- which no known m157-specific inhibitory receptor has been after, defective signaling through the activating receptor may have defined. The authors observed hyporesponsiveness in Ly49H+ been insufficient to trigger upregulation of the inhibitory receptors NK cells to DAP10/12-dependent and independent activation in these cells. These alterations clearly result following develop- without alteration in the expression of inhibitory Ly49 receptors. ment in the chimeric environment. In addition to the lack of a known m157-specific inhibitory re- A number of previous reports have advanced a disarming model ceptor, differences in ligand-binding affinity, stromal interaction, for developmental regulation of potentially self-reactive NK cells cis versus trans recognition, membrane confinement, and synapse and are germane to the analysis of the current findings (37). Ac- formation are all possible determinants for the manner in which the cordingly, hostile Ly49D+AFG2 NK cells in BALB→B6 prenatal activating receptor input was managed differently during NK cell chimeras must be disarmed due to their unrestrained reactivity education between the m157 transgenic mice and the prenatal The Journal of Immunology 1515 chimera models (41–44). As such, the developmental regulation of References hostile NK cells in prenatal chimeras was more complex than could 1. Owen, R. D. 1945. Immunogenetic consequences of vascular anatomoses be- be predicted by current models. 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