Durable remissions of hematological malignancies regularly observed following allogeneic hematopoietic stem cell transplantation (aHSCT) are due to the conditioning regimen, as well as an immunological phenomenon called graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effect. The development of GVL is closely linked to graft-versus-host disease (GVHD), the main side effect associated with aHSCT. Both, GVHD and GVL are mediated by donor T cells that are initially activated by antigen-presenting cells that present recipient-derived alloantigens in the context of either matched or mismatched MHC class I molecules. Using murine models of aHSCT we show that ubiquitously expressed minor histocompatibility alloantigens (mHAg) are no relevant target for GVT effects. Interestingly, certain ubiquitously expressed MHC alloantigens augmented GVT effects early after transplantation, while others did not. The magnitude of GVT effects correlated with tumor infiltration by CD8+ cytotoxic T cells and tumor cell apoptosis. Furthermore, the immune response underlying GVHD and GVT was oligoclonal, highlighting that immunodominance is an important factor during alloimmune responses. These results emphasize that alloantigen expression on non-hematopoietic tissues can influence GVT effects in a previously unrecognized fashion. These findings bear significance for harnessing optimal GVL effects in patients receiving aHSCT.
Allogeneic hematopoietic stem cell transplantation (aHSCT) is an established treatment option for hematological malignancies, including leukemias [1,2,3,4]. The graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effect observed after aHSCT is closely linked to graft-versus-host disease (GVHD) a major contributor to the morbidity and mortality associated with this therapeutic approach [5,6,7]. While it has been demonstrated experimentally that GVT reactions can occur without significant GVHD [8,9,10,11], there is controversy whether the GVT reaction observed clinically is actually a beneficial aspect of otherwise detrimental GVHD. T cell-mediated GVT effects are heterogeneous with respect to effector cell populations, target antigens and their interrelation with GVHD. In general, the expression of alloantigens in three immunologically distinct locations—antigen-presenting cells (APC), host tissues and cancer cells—is relevant for the development GVHD and GVT effects [11, 12]. Several studies have demonstrated that alloantigen expression by host APC is critical to initiate both processes [13,14,15,16]. While alloantigen-reactive donor-derived T cells-mediate GVHD and GVT [17, 18], inflammatory cytokines can facilitate GVHD even in recipient tissues that lack expression of alloantigens . Relevant alloantigens are major histocompatibility antigens (MHC) [14, 15, 17] after MHC-mismatched or haploidentical transplantation, minor histocompatibility antigens (mHAg) [11, 12, 20,21,22] derived from normal polymorphic proteins, and tumor-associated antigens (TAA) [23, 24], which originate from selectively and aberrantly expressed non-mutated or mutated proteins. TAAs represent potential targets for T cell-mediated GVT effects [25, 26] that are in principle separable from the generalized GVHD reaction that targets widely expressed mHAg and MHC alloantigens. Expanding our understanding of how major and minor histocompatibility antigens on APC, host tissues and cancer cells influence the pathophysiology of GVHD and GVT effects, will be instrumental in developing and refining more effective therapies for hematological malignancies. Therefore, we addressed how the differential expression of mHAg and MHC alloantigens on tumor and normal tissue influence the development of GVT and GVHD.
Tumors and mouse models
C1498 [27, 28] and MPC-11  were obtained from the American Type Culture Collection (Manassas, VA, USA) and stably transfected using the gateway cloning system and pcDNA3.1+ (Invitrogen, Life technologies, Carlsbad, CA, USA), respectively. C1498-Ova was provided by M. Sauer (Klinik für Pädiatrische Hämatologie und Onkologie Hannover, Germany). [CBA/J × BALB/c]F1 (CCBAF1) and [Tg(CAG-OVA)916Jen × Balb/c]F1 (CB6F1-OVA) were bred at our facility. C57BL/6-Tg(CAG-OVA)916Jen/J  (B6-OVA) and OT-I  mice were from the Jackson Laboratory (Bar Harbor, MA), all other mice were from Charles River (Sulzfeld, Germany). Ages of 12–18-week-old mice were randomly assigned to experimental groups. Animals for which transplantation failed were excluded from the experiments. All procedures in accordance with European regulations were approved by the regional governmental authorities.
Cell transplantation and assessment of GVHD and GVT
Transplantation and monitoring of GVHD and tumor growth was performed as previously described [10, 25, 32]. Briefly, recipient mice received 9 Gy total body irradiation (TBI) from a 60Co source at a dose rate of 128 cGy/min 1 day before transplantation (day 1). Bone marrow cells obtained by flushing tibias and femurs of donor mice were administered via the tail vein at 1.0 × 106/g body weight, either alone or mixed with splenic lymphocytes (0.5 × 106/g body weight) as indicated. To generate B6-OVA bone marrow chimeras mice received 2x 4.5 Gy TBI at intervals of 6 hours. Four weeks later, chimerism was verified by PCR and mice received again 2x 4.5 Gy before second transplant. For UTY-experiments, C57BL/6 donors were immunized with 1.0 × 107 male or female splenocytes 4 and 2 weeks before transplantation. Recipients were inoculated with 1.0 × 106 MPC-11 or 1.0 × 105 C1498 cells at indicated time points.
Fluorescence reflectance imaging (FRI)
The IZKF Core Unit PIX—OPTI (Institute of Clinical Radiology, University Hospital Muenster, Germany) performed fluorescent measurements using an in vivo Multispectral FX PRO system (Bruker BioSpin MRI GmbH, Ettlingen, Germany).
Histopathology and immunostaining
Histopathology was performed in a blinded fashion. For immunostaining anti-CD8a (53–6.7) (Biolegend, San Diego, USA) and goat anti-Rat IgG (ThermoFisher, Waltham, USA) were used.
Antibodies against CD3 (145.2C11), CD8 (KT15), H-2Kb (AF6-88.5), H-2Kd (SF1-1.1), and H-2Kk (H100-5-28) were from Biolegend (San Diego, CA, USA). IFN-γ of co-culture (E:T 10:1; 72 h; 37 °C at 5% CO2) supernatants was measured using the BD CBA Mouse IFN-γ Flex Set (558296, BD, East Rutherford, NJ, USA). Samples were acquired using a BD FACSCanto. CBA-data were analyzed using FCAP Array v3.0.1 software.
T cell-specific activity assay
T cells were isolated using Pan T cell isolation Kit II (130-095-130, Miltenyi Biotec, Bergisch Gladbach, Germany). Mouse IFN-γ ELISpotPLUS kits (Mabtech, Nacka Strand, Sweden) were used for ELISpots and data were analyzed using a CTL ImmunoSpot S5 UV Analyzer (Ohio, USA). Cytotoxicity was measured using a GloMax Discover system (CytoTox-ONE, Promega, Madison, USA).
CDR3 spectratyping analysis of the TCR repertoire
T cells were stimulated for 5 days using irradiated (120 Gy) tumors (10:1), isolated by single-cell dilution, and restimulated using α-CD3e, α-CD28 (145-2C11, 37.51, BD), and 50 IU/ml IL-2. TCRβ sequences were amplified, extended, and resolved as described [33, 34]. Peak sizes of CDR3 lengths were analyzed using Peak Scanner 2 Software (Applied Biosystems, Foster City, USA).
The data are presented as means ± standard deviation (SD) or ± standard error of the mean (SEM) as indicated. The two-tailed Mann–Whitney U-test was used for statistical analysis. Experiments have been repeated at least once with at least six mice per group.
The MHC class I alloantigen H-2Kb does not contribute to GVT effects when simultaneously expressed on tumor and host non-hematopoietic tissues
To determine how TAA expressed by non-hematopoietic tissues influence GVT effects, we modified a BALB/c (H-2d) myeloma cell line (MPC-11 ) to also express the MHC class I antigen H-2Kb (MPC-11-Kb). To evaluate the immunogenicity of H-2Kb alloantigens in GVT reactions, we first examined tumor growth in a parent-into-F1 model mimicking clinical haploidentical aHSCT (Fig. 1a–i). BALB/c donor cells were transferred into lethally irradiated (C57BL/6 × BALB/c)F1 (CB6F1) recipients. One day after transplantation mice where inoculated with either unmodified tumors (MPC-11-wt) or tumors additionally expressing the alloantigen H-2Kb (MPC-11-Kb). As expected, weight loss (Fig. 1a–c) and clinical signs of GVHD  (Fig. 1d–f) where dependent on lymphocyte numbers contained within the graft. While GVT effects increased with more severe GVHD, growth kinetics of MPC-11-wt and MPC-11-Kb did not differ significantly indicating that H-2Kb alloantigens are not relevant GVT targets in a setting of ubiquitous alloantigen expression (Fig. 1g–i). While wild-type MPC-11 tumors (H-2d) grew progressively in syngenic hosts, tumors expressing the H-2Kb alloantigen where rejected by naive BALB/c hosts (Fig. 1j), indicating that H-2Kb is immunogeneic in immunocompetent allogeneic hosts. In centrally tolerant CB6F1 mice, on the other hand, both, MPC-11, as well as the MPC-11-Kb tumors grew progressively and with similar kinetics (Fig. 1j). The expression of H-2Kb in MPC-11-Kb tumors was stable throughout experiments since tumors expressed the H-2Kb-antigen 26 days after inoculation in naive and transplanted CB6F1 mice (Fig. 1k). Complete donor cell chimerism was confirmed by flow cytometry (Fig. 1l).
The MHC class I alloantigen H-2Kk contributes to GVT effects early after transplantation when simultaneously expressed on tumor and host non-hematopoietic tissues
To examine the contribution of the alloantigen H-2Kk to GVT effects when expressed either selectively as a TAA or ubiquitously we generated MPC-11 tumors (H-2d) that also expressed the MHC class I antigen H-2kk (MPC-11-Kk). To determine the immunogenicity of the H-2Kk alloantigen in GVT reactions (Fig. 2a–f) bone marrow with or without additional lymphocytes from BALB/c donors was transferred into lethally irradiated (CBA/J × BALB/c)F1 (CCBAF1) recipients which were inoculated at days 3, 6, or 13 with either unmodified tumors (MPC-11-wt) or tumors that expressed the additional alloantigen H-2Kk (MPC-11-Kk). CCBAF1 recipients developed more severe GVHD (Fig. 2a, d) when transplanted with grafts containing additional lymphocytes. As observed before using MPC-11-Kb tumors (Fig. 1g), no significant GVT effects against MPC-11-Kk developed in CCBAF1 mice that had received bone marrow alone (Fig. 2b). In recipients transplanted with additional donor lymphocytes, GVT effects were more pronounced when tumors additionally expressed the H-2Kk alloantigen compared to unmodified controls (Fig. 2c). GVT effect were less pronounced (Fig. 2e) or absent (Fig. 2f) when tumors were inoculated on day 6 or day 13, respectively, following aHSCT. As observed with MPC-11-Kb tumors (Fig. 1j) MPC-11-Kk tumors were rejected by naive BALB/c mice (Fig. 2g) but grew progressively in CCBAF1 hosts (Fig. 2h) indicating that GVT effects were mediated exclusively by the donor immune system. Flow cytometry confirmed that expression of the H-2Kk-antigen was stable in transplanted CCBAF1 mice (Fig. 2i).
MHC class I alloantigens are GVT targets, when selectively expressed on tumors but not on non-hematopoietic recipient tissue
Lethally irradiated CCBAF1 recipients were transplanted from BALB/c donors and inoculated with either unmodified (MPC-11-wt) or additionally H-2Kb (MPC-11-Kb) expressing tumors to evaluate selectively expressed tumor-antigens as targets of GVT effects. Again, recipients developed more severe GVHD when transplanted with lymphocyte-containing grafts (Fig. 2j). In mice transplanted without additional splenocytes, wild-type MPC-11 tumors cells grew progressively while growth of the MPC-11-Kb tumor was significantly reduced (Fig. 2k), indicating that H-2Kb was a relevant GVT target even in the absence of significant GVHD. In recipients developing GVHD, growth of wild-type MPC-11 tumor volumes were reduced to <10% compared to recipients without GVHD (Fig. 2k, l) and MPC-11-Kb tumors were rejected completely in most recipients (Fig. 2l). These results indicate that the tumor-restricted MHC class I antigen H-2Kb serves as a potent rejection antigen. Stable expression of H-2Kb in MPC-11-Kb tumors (Fig. 2m) and complete donor cell chimerism (Fig. 2n) were confirmed by flow cytometry. Finally, during GVHD the release of IFN-γ by T cells was antigen-specific toward MPC-11-Kb targets as measured by ELISpot (Fig. 2o).
The mHAg UTY only serves as a target of GVT reactions when expressed selectively by the tumor
In MHC-matched transplantation minor histocompatibility antigens (mHAgs) facilitate both GVT activity and GVHD [20, 21]. Therefore, we examined how the expression of mHAgs on non-hematopoietic host tissues influenced GVT effects [35, 36]. We employed a transplant model that allowed us to differentially express the enzyme histone demethylase (UTY) on leukemic cells, non-hematopoietic tissues, or both. UTY is an mHAg encoded by a Y chromosome gene  and is known to induce rejection of male tissues by the female immune system . For this purpose, the female C57BL/6 [H-2b] derived myeloid leukemia cell line C1498 [27, 28] was transduced with UTY to generate C1498-UTY (Fig. 3a). Naive C57BL/6 (B6) female mice failed to reject C1498 and C1498-UTY tumors (Fig. 3b), but once they had been immunized with male B6 splenocytes female donors could mount a relevant immune response against C1498-UTY (Fig. 3c). Female (Fig. 3d–f) or male (Fig. 3g–i) CB6F1 recipients were transplanted from female B6 donors that had been primed with female (HX) or male (HY) splenocytes. All recipients lost weight (Fig. 3d, g) and developed GVHD (Fig. 3e, h) following aHSCT. A significant GVT effect against C1498-UTY was only observed in recipients of HY-primed female splenocytes (Fig. 3f). In male CB6F1 mice, tumors grew progressively and identical to controls (Fig. 3i). As a control female (data not shown) or male CB6F1 mice (Fig. 3j–l) received an aHSCT from HY- or HX-primed male B6 donors. In this setting no GVT effects could be observed (Fig. 3l), indicating that male donors fail to elicit immune responses to the UTY-self antigen. In summary, these results indicate that the mHAg HY was a relevant GVT target only when expressed exclusively by tumors but not when expressed ubiquitously.
UTY-specific GVT effects are characterized by clonal T cell responses
To determine whether the observed GVT effects were UTY-specific, lymphocytes from C1498-UTY tumor-bearing mice that had undergone aHSCT from HY-primed female B6 donors were analyzed by ELISpot. The release of IFN-γ by T cells was UTY-specific (Fig. 4a, b) and when we compared tumor sections (Fig. 4c, d) there was more infiltration by cytotoxic CD8+ T cells in recipients transplanted from HY-immunized donors (Fig. 4c). To further characterize the post-transplant immune response toward the mHAg UTY, we compared the T cell receptor (TCR) repertoire of mice that were transplanted from HX- or HY-primed B6 donors by CDR length spectratyping (Fig. 4e–g). Compared to recipients from HX-primed B6 donors, T cells from mice transplanted from HY-primed donors showed two prominent TCRs of the TBV3/7 and TBV21/22 families, indicating that these TCRs were candidates for mediating UTY-specific GVT effects. Since, the TBV3/7 family TCR was absent in male CB6F1 recipients (Fig. 4g) with widespread TAA expression, that also lacked UTY-specific GVT effects (Fig. 3i), the T-cell clone mediating antigen-specific GVT effects in this setting most likely expressed TCRs of the TBV21/22 family. Furthermore, when we used C1498-UTY to clonally expand T cells of recipients transplanted from HY-primed female donors that had shown C1498-UTY specific GVT activity (Figs. 3f, 4c, e) the most prominent detectable TCR belonged to the differentially expressed TBV21/22 family (Fig. 4h). Finally, these T cells (Fig. 4h) exhibited up to 73% specific cytotoxicity toward C1498-UTY that was absent in recipients of naive B6 grafts (Fig. 4i) or when exposed toward C1498 or C1498-GFP that lacked UTY expression (Fig. 4j). These results suggest that a clonal T cell population with TCRs of the TBV21/22 family was the effector of the observed UTY-specific GVT effects.
Hematopoietic cell-restricted expression of the mHAg Ova augments GVT reaction
To further evaluate the impact of differential antigen expression on GVT effects we used Ovalbumin (OVA) as an antigen and OT-1 mice with a transgenic TCR that recognizes ovalbumin residues 257–264 in the context of H-2Kb as donors. The C57BL/6 [H-2b] derived acute myeloid leukemia cell line C1498 stably expressing ovalbumin (C1498-Ova) was used to assess GVT effects. First, we generated mice with expression of OVA limited to the hematopoietic system by using female B6-OVA mice (B6-Ova->B6) that express ovalbumin ubiquitously in all tissues as donors. The mice transplanted from wild-type donors (B6->B6) served as controls. Three weeks after aHSCT chimerism was confirmed by PCR (Fig. 5a). These chimeras then received a second transplant with B6 bone marrow with either B6 or OT-1 splenocytes. Engraftment of OT-1 leukocytes was confirmed by PCR for the transgenic TCRα-V2 and TCRβ-V5 genes (Fig. 5b). One day after transplantation all recipients were inoculated with C1498-Ova tumors. None of the recipients developed GVHD (Fig. 5c, e, left panels). Following transplant with grafts containing T cells from OT-1 mice, all recipients rejected C1498-Ova tumors regardless of whether the target antigen was expressed solely by the tumor (positive control, Fig. 5c right panel, recipient (B6->B6)) or by, both, the tumor and hematopoietic tissue (Fig. 5c, right panel, recipient (B6-Ova->B6)). Fluorescence reflectance imaging of the whole tumor showed similar signal intensities for GFP (Fig. 5d). Next we transplanted B6-Ova->B6 bone marrow chimeras with either B6 bone marrow and splenocytes (negative control) or B6 bone marrow and B6-Ova splenocytes (Fig. 5e). Only mice receiving leukocytes from OT-1 mice rejected the tumor, demonstrating that the OVA-specific T cells were required to elicit GVT effects (Fig. 5e, right panel). Fluorescence of GFP was significantly reduced and widespread tumor cell necrosis was evident only in recipients of OT-1 cell containing grafts (Fig. 5f). To examine the effects of ubiquitous mHag expression in the setting of aHSCT, CB6F1-Ova recipients ([Balb/c×C57BL/6-Ova]F1, H-2d×b) received a transplant from B6 bone marrow with either B6 or B6-OT-1 splenocytes. When ovalbumin was expressed in all tissues OT-1 T cells neither influenced the development of GVHD (Fig. 5g, h) nor facilitated rejection of C1498-Ova tumors (Fig. 5i). Similarly, no significant influence on GVT effects was observed when recipients with ubiquitous expression of ovalbumin were used (not shown). In line with our previous findings these results support the observation that ubiquitously expressed mHAgs do not regularly contribute to GVT effects.
To study the role of MHC alloantigens, as well as mHAgs in GVT reactions, we employed parent-into-F1 murine transplant models with donor derived tumors that naturally expressed no MHC alloantigens. Therefore, GVT reactions could only be mediated via recognition of molecularly defined MHC or mHAg alloantigens expressed by either the tumor alone, or ubiquitously on all host tissues. Previous studies suggested that expression of mHAgs on non-hematopoietic tissues results in T-cell exhaustion and impaired GVT effects [11, 12]. Although it is possible that differences in tumor biology rather than alloantigens alone influenced the outcome of individual experiments the results of our study are consistent with previous reports, adding the observation that certain MHC alloantigens might serve as relevant GVT targets, even when expressed ubiquitously. Findings made in the murine model of GVHD used in this study bear relevance to human GVHD, and some have even been translated into the clinical setting [39, 40].
It has been suggested that the primary targets of GVT are immunodominant allogeneic minor histocompatibility antigens rather than tumor-associated antigens  and that for optimal GVT responses to occur alloantigens need to be expressed on both APCs and tumor tissue . In addition to experimental data, the observation that GVT effects are severely attenuated after syngeneic transplant [41, 42] supports the notion that mHAgs rather than TAAs are the main targets of GVT effects routinely observed in clinical HSCT. Relevant GVT effects have, however, also been observed in transplant models where identical genetic backgrounds of effector T cells and tumor precluded allorecognition of tumor cells [25, 26, 43], and tumor-specific T cells have been identified in patients undergoing HSCT . Therefore, while APCs and alloantigen expression on tumors are important determinants for GVL, experimental evidence, as well as clinical observations suggest that TAAs can contribute to GVT effects, particularly during inflammatory conditions like GVHD.
Tumors downregulate the expression of MHC molecules to evade detection by the immune system [44, 45]. When we compared tumor growth in a setting where the MHC alloantigen H-2Kb was either expressed ubiquitously on all host tissues including tumors or expressed on non-malignant host tissue only, there was no appreciable difference in growth kinetics. These findings suggest that GVT effects in this setting where mediated predominantly by TAA with little contribution of MHC alloantigens expressed by MPC-11-Kb tumors. Furthermore, these TAA-directed GVT effects were only evident in the presence of GVHD since tumors grew progressively in recipients without GVHD, as well as naive CB6F1 mice. This observation was not due to a lack of expression or immunogenicity since H-2Kb protein was detectable throughout the experiments and naive BALB/c mice readily rejected H-2Kb-positive tumors.
On the other hand, MPC-11-Kk tumors expressing H-2Kk alloantigens elicited significantly stronger GVT effects compared to wild-type tumors. While GVT effects directed against TAA were also evident, immunologic tumor control was significantly enhanced when, in addition to other tissues, tumors also expressed H-2Kk. These GVT effects seem to be initiated early after aHSCT since they vanished when tumors were inoculated at later time points. Alternatively, tolerance mechanisms similar to those preventing progressive GVHD might limit GVT effects targeting ubiquitously expressed MHC alloantigens. The finding that ubiquitously expressed MHC-class I alloantigens represent relevant GVT targets experimentally are in line with clinical observations showing that following haploidentical aHSCT cancer immunoediting can select leukemic blasts to sometimes lose the entire recipient’s MHC haplotype not shared by the donor [46,47,48,49,50]. Nonetheless, GVT effects were stronger against tumor-restricted MHC alloantigens (MPC-11-Kb) than ubiquitously expressed MHC alloantigens (MPC-11-Kk). Since high levels and re-exposure to antigen can cause energy, as well as activation-induced cell death, a possible explanation for the differences in the observed GVT effects might be the levels of H-2Kb and H-2Kk protein expression [51,52,53].
The extent to which different MHC alloantigens contribute to immunological processes, such as autoimmune disease or GVHD has long been established and knowledge of the immunogenetic basis of GVHD is used routinely during donor selection for allogeneic HSCT [54,55,56]. Our unexpected finding that ubiquitously expressed H-2Kb and H-2Kk produced different GVT effects during the early phase following transplantation might therefore be a reflection of intrinsic molecular determinants that affect the magnitude of tumor-specific alloimmune responses against MHC molecules.
Using the HY-derived male UTY, as well as OVA as molecularly defined and immunogenic mHAg targets for GVH and GVT immune responses, we were able to corroborate observations that ubiquitously expressed mHAg serve as poor GVT targets compared to antigens with expression limited to the tumor [11, 12]. In keeping with previous work analyzing immunodominance among mHAgs in aHSCT, adaptive immune responses targeting tumor-specific UTY were clonal and consisted of T cells that lysed tumor cells in an antigen-specific manner .
Our data lend credence to therapeutic approaches that selectively target antigens with limited expression within the hematopoietic system. Upon engraftment and clearance of predominantly hematopoietic antigens the alloimmune reaction abates without causing further detrimental GVHD. Ubiquitously expressed mHAgs, on the other hand, promote GVHD while contributing little to GVT effects [11, 12]. The exception to this rule appears to be MHC alloantigens, which can be potent GVT targets even in the context of widespread expression. In summary, our study adds to the understanding of how differentially expressed alloantigens influence the development of GVHD and GVT effects and highlights the need to identify suitable tissue-restricted antigens to allow for novel targeted immunotherapies in the context of clinical aHSCT [20, 21, 35, 36].
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This work was supported by grants from the German José Carreras Leukemia-Foundation (DJCLS R 05/35, R 08/31 f)
S.R., K.F., C.O., J.U., C.W., and C.H., helped to design the experiments and performed experiments; M.S. developed the overall concept; S.R., J.C.A., K.F., W.E.B., and M.S. analyzed and discussed the data and wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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Robert, S., Albring, J.C., Frebel, K. et al. Alloantigen expression on malignant cells and healthy host tissue influences graft-versus-tumor reactions after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 53, 807–819 (2018). https://doi.org/10.1038/s41409-017-0071-7