Distinct roles for major and minor antigen barriers in chimerism‐based tolerance under irradiation‐free conditions

Eliminating cytoreductive conditioning from chimerism‐based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade [CB] and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell‐mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC‐mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell‐mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.

nephrotoxicity, and may not constitute a durable solution because long-term allograft survival is still limited by chronic rejection. 2,3 These and other caveats still fuel the dream for a state in which the patients indefinitely retain an allograft without requiring any immunosuppressive medication, namely transplantation tolerance.
Several approaches have been developed in the murine setting to achieve this desired state from which the induction of mixed chimerism emerged as a promising strategy. 4 Furthermore, it has been the only approach that has already been successfully translated in several independent clinical trials. [5][6][7] In the mid-1950s, it was discovered that transplanting allogeneic bone marrow (BM) into lethally irradiated mice can confer tolerance to solid tissues from the same donor. 8 Even back then, the irradiation required for successful BM engraftment in adult recipients raised serious concerns about clinical implementation. 9 It took 30 years until it was realized that replacing only a part of the recipient's BM is sufficient to achieve tolerance 10 -a state referred to as mixed chimerism. The low irradiation doses necessary to induce mixed chimerism provided a reasonable basis for clinical implementation. 6 Further progress could be achieved through the increased understanding of T cell activation and allorecognition, which led to the provision of new therapeutic possibilities. T cell-depleting antibodies broke the first ground 11 but it was the specific blockade of T cell costimulatory pathways that allowed the avoidance of global depletion of the recipient T cell repertoire. In particular, the concomitant use of CTLA4-Ig and α-CD40L proved to be highly effective 12 especially when combined with the mammalian target of rapamycin inhibitor rapamycin. 13 But even then, allogeneic BM was rejected unless low doses of total body irradiation (TBI) were applied 14 or unrealistically high BM doses were used. 15    Bio X Cell) either at the time of transplantation (d-1, d2, d5, d8, short-α-NK1.1) or regularly until the end of follow-up (d-1, d2, d5, d8, d28, d56, d84, 112, 140, 168) (long-α-NK1.1). Antibodies, fusion proteins, and rapamycin were administered intraperitoneally (i.p.).

| BMT and antibody treatment
Mixed chimerism was defined as having at least 2 lineages displaying >0.5% donor cells.

| Histology
Grafts were retrieved at the end of the observation period.
Samples were fixed in 7.5% formalin overnight. Paraffin blocks were subsequently sectioned and stained with hematoxylin and eosin. Slides were scanned with an Aperio ScanScope scanner (Aperio Technologies, Inc, Vista, CA). The Aperio ScanScope allowed scanning of the whole slide using a 920-objective lens with a numerical aperture of 0.75 coupled with a double objective to achieve a scan of whole slides at 920 magnification. Digitalized slides were viewed and annotated with an Aperio ImageScope.

| Statistics
Data were statistically analyzed with GraphPad Prism 5.0 (Graph Pad Inc, La Jolla, CA). A 2-sided Student's t test with equal variances was used to compare percentages of chimerism levels, Vβ subtypes, and Ki67 expression. Total chimerism levels were compared between groups by using analysis of variance (ANOVA). A P value below .05 was considered to denote statistical significance (*P < .05, **P < .01, ***P < .001, ****P < .0001, n.s. P > .05).  Figure 1I). To test the hypothesis that returning NK cells impede skin graft tolerance, we depleted NK cells long-term by α-NK1.1 (d-1, d2, d5, d8, d28, d56,…) ( Figure 1F). In the lasting absence of NK cells, chimerism was again achieved ( Figure 1G) albeit without any increase in chimerism levels ( Figure 1H). Unexpectedly, BALB/c skin graft survival was not extended in comparison to transient NK cell depletion ( Figure 1I). Therefore, we conclude that it is not the NK cells returning after temporal depletion that are

| Minor antigen disparities impede skin graft tolerance in the absence of NK cell alloreactivity
From previous results we knew that transplantation of F1 BM (triggering no NK alloreactivity) into parental recipients leads to chimerism and permanent acceptance of F1 skin grafts under CB and rapamycin. 18 Therefore, we hypothesized that tissue-specific  Figure 2B). Because F1.BALB/c mice have not yet been used for tolerance studies, we characterized their alloreactivity in comparison to F1 mice. Naïve C57BL/6 mice rejected F1 and F1.BALB/c skin allografts at the same time ( Figure 2C). In an in vitro proliferation assay, comparable numbers of recipient CD4 and CD8 T cells proliferated in response to F1, F1.BALB/c, and BALB/c splenocytes ( Figure 2D).
These observations indicate that F1 and F1.BALB/c trigger a similar degree of T cell alloreactivity.
In agreement with our previous results, transplanting 20 × 10 6 F1 BM cells into C57BL/6 recipients under the cover of CB and rapamycin ( Figure 3A) induced long-lasting mixed chimerism in multiple lineages ( Figure 3B). Moreover, F1 skin allografts survived indefinitely ( Figure 3F Figure 3H). Because of the limitations of the experimental system, however, no conclusion with regard to the deletion of mature CD8 cells can be drawn, which likely have a prominent role in the rejection of MiHA-disparate skin. In summary, these data suggest that MiHA disparities, which are present to a higher degree in F1.BALB/c than in F1 skin allografts, lead to lower levels of chimerism and skin graft rejection despite stable mixed chimerism.

| Minor antigens impede tolerance in the absence of MHC disparities
To assess the role of isolated MiHAs disparities in the absence of MHC disparities, we employed BALB.B mice as donors that express the same MHC haplotype as C57BL/6 recipients albeit on the background of BALB/c mice ( Figure 5A). To distinguish donor  This phenomenon, known as split tolerance, has not yet been fully clarified. Several factors have been proposed to favor the occurrence of split tolerance. Tissue-specific expression of MiHAs and minimal conditioning protocols that keep the endogenous T cell compartment largely intact are both currently considered as prime factors. 21 The marked difference between the survival of BALB/c and B10.D2 skin allografts that we observed in NK cell-depleted recipients strongly supports this hypothesis. In both cases, durable chimerism emerged after NK cell depletion but skin grafts only survived in the absence of MiHAs disparities (B10.D2 → C57BL/6).

| D ISCUSS I ON
Because durable chimerism levels were achieved with both donors, it seems reasonable to assume that MiHAs expressed by BALB/c but not B10.D2 resulted in different skin survival rates. The role of MiHAs for tolerance induction has often been neglected in early tolerance studies because most donors were MHC disparate but expressed the same MiHAs as the recipients. 12,22 We have recently addressed this issue and found that the absence of MiHA promotes chimerism and tolerance in mice receiving nonmyeloablative TBI and CB. In contrast to the current experimental setup, these mice did not receive rapamycin and showed significantly higher levels of mixed chimerism due to nonmyeloablative TBI. 23 We also recently found that adding regulatory T cells from the recipient to the donor BM transplant promotes MiHA tolerization under CB and rapamycin in nonirradiated mice. The role of MiHAs for the induction and maintenance of chimerism, however, was not directly addressed in this study. The exact mechanisms are not yet known, but regulatory mechanisms seem to prevail over deletional mechanisms with regard to MiHA tolerization. 24 One major difference to the present study is the use of a cellular therapy instead of cell-depleting antibodies. Antibody-dependent cellular cytotoxicity is accompanied by the release of inflammatory cytokines that might interfere with tolerization mechanisms. However, BALB.B skin grafts were also rejected in the absence of NK cell-depleting antibodies.
Therefore, we rather assume that indirectly alloreactive T cells are responsible for the late allograft loss. Therefore, we conclude that skin-specific MiHAs of F1.BALB/c skin grafts primarily elicit indirect T cell alloresponses that cause late graft rejection. However, we cannot rule out the alternative possibility that MiHAs inherited from C57BL/6 might offer a survival advantage to transplants from F1 mice by a-yet undefined-inhibitory mechanism.
Our recent data showed that NK cells play a major role in BM rejection in nonirradiated mice under CB and rapamycin. 18 15 Our study extended these observations to nonirradiated recipients in whom NK cell-mediated rejection is qualitatively and quantitatively different. 28 Another study also demonstrated that NK cell depletion can overcome CB resistant BM rejection. At this, a nonengrafting dose of donor BM (20 × 10 6 ) was given (d0) before the recipients received a nonmyeloablative dose of busulfan (d5) together with a second donor BM dose (d6) and an extended course of CB. NK cell-depleted recipients displayed multilineage chimerism and retained skin allografts long term. 29 In contrast to our study, NK cell-depleted mice exhibited high levels of donor chimerism (~50%) because of the nonmyeloablative pretreatment, which again underlines that rejection seems to be significantly different in the absence of cytotoxic treatment. It should also be noted that BALB/C skin grafts survived longer under CB and rapamycin if low doses of irradiation were used instead of NK cell depletion. 13 However, the differences between F1 and F1 BALB/c donors suggest that MiHAs may influence donor chimerism levels at later time points. Vice versa, the absence of MiHA disparities has been shown to increase chimerism levels in a nonmyeloablative BMT model. 23 The expression of NKG2D ligands on certain donor strains can reinforce NK cell-mediated BM rejection through missing self-recognition. 30 Because F1 donors are not subjected to NK cell alloreactivity we, however, suggest that rather indirect alloreactive T cells account for the differences between F1 and F1.BALB/c donor chimerism levels.
In summary, this study provides further insight into the mechanisms that induce and maintain tolerance through mixed chimerism and delineates the distinct roles of MHC and MiHA barriers in the noncytotoxic setting.

ACK N OWLED G M ENTS
BM and TW designed the experiments, analyzed the data, and wrote the manuscript. BM performed the experiments. NP, NG, MW, MM, LWU,