Rejection Of Bone Marrow Cells Research Articles

Bone Marrow Cell Rejection, MHC, NK Cells, and Missing Self Recognition: Ain't That Peculiar (with Apologies to Marvin Gaye).

Much modern research is quite focused, attempting to understand aspects of systems that are already revealed in broad outline. Researching a problem at its birth is an opportunity that many will never experience, a lonely exercise that many may avoid. In these situations, pre-existing findings from

A Critical Role for the TLR4/TRIF Pathway in Allogeneic Hematopoietic Cell Rejection by Innate Immune Cells

We show for the first time that signaling through the TLR4/TRIF pathway plays a critical role in allogeneic bone marrow cell (BMC) rejection. This appears to be unique to BMCs as organ allografts are rejected mainly via MyD88 signaling. Using T- or T-/B-cell-deficient mice, we found that BMC allorejection occurred early before T-cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. We further demonstrated the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF or TLR4 but not in MyD88 or TLR3. The restored cytotoxicity in TRIF-deficient recipients transferred with wild-type F4/80(+) or NK1.1(+) cells suggests TRIF signaling dependence on macrophages or NK cells in early BMC rejection. Production of the proinflammatory cytokine IL-6 and TRIF relevant chemokine MCP-1 was significantly increased early after bone marrow transplantation. In vivo specific depletion of macrophages or NK innate immune cells in combination with anti-CD154/rapamycin resulted in additive-enhanced allogeneic engraftment. The requirement for irradiation was completely eliminated when both macrophages and NK cells were depleted in combination with anti-CD154/rapamycin to target T- and B-cells, supporting the hypothesis that two barriers involving innate and adaptive immunity exist in mediating the rejection of allogeneic BMCs. In summary, our results clearly demonstrate a previously unappreciated role for innate immunity in BMC allorejection via signaling through a unique MyD88-independent TLR4/TRIF mechanism. These findings may have direct clinical impact on strategies for conditioning recipients for stem cell transplantation.

The Early Rejection of Allogeneic BMC by Macrophages or NK Cells Is TRIF Signaling Dependent

Abstract 1883The barrier for rejection of allogeneic bone marrow cells (BMC) has been attributed primarily to adaptive immunity, especially T cell immune responses. Significant progress has been made in developing immune-based nonmyeloablative conditioning strategies to achieve mixed chimerism in bone marrow transplantation (BMT) with targeting of T cells. The role of the innate immune system in BMC allorejection has not been adequately addressed. The fact that when T cells are targeted, alloengraftment requires additional conditioning from nonspecific reagents, such as irradiation and immunosuppressive drugs, suggesting the existence of another barrier. As humoral immunity is unlikely a barrier for BMC in unprimed naïve recipients, the innate immune system is most likely another barrier in BMT. The present study focused on a role for components of the innate immune response in allogeneic BMT. Using T cell-deficient (TCR-β/δ−/−) mice, we found that rejection of transplanted allogeneic BMC occurred very early, well before the time required for T cell activation and was T cell independent, suggesting an effector role for innate immune cells in BMC rejection.How the innate immune system recognizes or responds to allogeneic BMC remains unknown. Toll-like receptors (TLR) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors of MyD88 and/or TRIF. We have demonstrated that TRIF signaling is the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF but not MyD88. To further determine the cell populations of innate immunity in allogeneic BMC rejection mediated through TRIF signaling, TRIF deficient (TRIFLps2/Lps2) mice were used as recipients for in vivo cytotoxicity assays after adoptive transfer of wildtype innate immune cells: macrophages or NK cells. Wildtype F4/80+ macrophages were sorted from B6 spleens and peritoneal cavities and NK1.1+ NK cells from B6 spleens. The doses of transferred NK1.1+ and F4/80+ cells were 370,000 and 140,000 per recipient, respectively. One day after transfer, 20 × 106 CFSE-labeled BALB/c target (high intensity) and internal control B6 (low intensity) BMC were injected. TRIFLps2/Lps2 mice that did not receive transferred cells and wildtype B6 mice treated with saline served as controls.As expected, donor cells were rapidly eliminated in control wildtype B6 mice and rejection was complete by day 3. The rejection of donor cells was significantly less in TRIFLps2/Lps2 mice without receiving adoptively transferred cells compared with wildtype B6 controls, from marginal significance (P = 0.04) at 3 hr to the highest significance (P = 0.0001) at day 3 after cell infusion. At day 3, the killing rates were 91.4 ± 1.4% in TRIFLps2/Lps2 mice without transferred cells and 97.6 ± 1.5% in wildtype B6 controls. The eliminating rates of donor cells were increased in TRIFLps2/Lps2 recipients that received either F4/80+ or NK1.1+ cells, and the kinetics of elimination of donor cells was shifted to resemble B6 controls with no significant difference between them at these 3 time points (P values: 0.11 to 0.87). The cytotoxicity of donor cells was significantly increased in TRIFLps2/Lps2 recipients adoptively transferred with F4/80+ or NK1.1+ cells when compared with TRIFLps2/Lps2 controls at all time points (P values: 0.038 to 0.002), except the one at 3hr when compared between TRIFLps2/Lps2 recipients received NK1.1+ cells and TRIFLps2/Lps2 controls (P = 0.058). At day 1, the killing percentages of CFSE labeled BALB/c cells were 71.0 ± 5.1%, 69.1 ± 3.9%, or 61.1 ± 5.8% in TRIFLps2/Lps2 recipients that received either F4/80+ cells, NK1.1+ cells, or none, respectively. Taken together, the restored cytotoxicity in TRIF-deficient recipients transferred with wildtype F4/80+ or NK1.1+ cells suggests that TRIF signaling is essential for macrophage- and NK cell-mediated early rejection of allogeneic BMC, and that both cell types function as non-redundant effector cells in BMC rejection. Disclosures:Ildstad:Regenerex, LLC, a biotech start-up company: Equity Ownership.

Mouse NK cell–mediated rejection of bone marrow allografts exhibits patterns consistent with Ly49 subset licensing

AbstractNatural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49+ subsets in BMC rejection by using β2-microglobulin deficient (β2m−/−) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind “self-MHC” were found to be the dominant subset responsible for β2m−/− BMC rejection. This provides in vivo evidence for host MHC class I–dependent functional education. Interestingly, all H2d strain mice regardless of background were able to resist significantly greater amounts of β2m−/−, but not wild-type BMC than H2b mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.

A Critical Role for Innate Immunity In Allogeneic Hematopoietic Cell Rejection Via the TLR4/TRIF Pathway

AbstractAbstract 77Adaptive immunity, especially T cells, has long been believed to be the dominant immune barrier in allogeneic transplantation. Targeting host T cells significantly reduces conditioning for bone marrow cell (BMC) engraftment. Innate immunity has been recently shown to pose a significant barrier in solid organ transplantation, but has not been addressed in bone marrow transplantation (BMT). Using T cell deficient (TCR-β/δ−/−) or T and B cell deficient (Rag−/−) mice, we found that allogeneic BMC rejection occurred early before the time required for T cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. Therefore, we hypothesized that by controlling both innate and adaptive immunity, the donor BMC would have a window of advantage to engraft. Survival of BMC in vivo was significantly improved by depleting recipient macrophages and/or NK cells, but not neutrophils. Moreover, depletion of macrophages and NK cells in combination with co-stimulatory blockade with anti-CD154 and rapamycin as a novel form of conditioning resulted in 100% allogeneic engraftment without any irradiation and T cell depletion. Donor chimerism remained stable and durable up to 6 months. Moreover, specific Vβ5½ and Vβ11 clonal deletion was detected in host CD4+ T cells in chimeras, indicating central tolerance to donor alloantigens.Whether and how the innate immune system recognizes or responds to allogeneic BMCs remains unknown. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors. The adaptor MyD88 transmits signals emanating from all TLR, except TLR3 while TRIF specifically mediates TLR3 and TLR4 signaling via type 1 IFN. To further determine the innate signaling pathways in allogeneic BMC rejection, B6 background (H2b) MyD88−/− and TRIF−/− mice were conditioned with anti-CD154/rapamycin plus 100 cGy total body irradiation and transplanted with 15 × 106 BALB/c (H2d) BMC. Only 33.3% of MyD88−/− recipients engrafted at 1 month, resembling outcomes for wild-type B6 mice. In contrast, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month, significantly higher than in MyD88−/− and wild-type B6 controls (P < 0.005). To determine the mechanism of innate signaling in BMC rejection, we examined whether TRIF linked TLR3 or TLR4 is the key pattern recognition receptor involved in BMC recognition. To this end, TLR3−/− and TLR4−/− mice were transplanted with BALB/c BMC with same conditioning. None of the TLR3−/− mice engrafted. In contrast, engraftment was achieved in 100% of TLR4−/− mice up to 6 months follow up. Taken together, these results suggest that rejection of allogeneic BMC is uniquely dependent on the TLR4/TRIF signaling pathway. Thus, our results clearly demonstrate a previously unappreciated role for innate immunity in allogeneic BMC rejection. Our current findings are distinct from prior reports demonstrating a critical role of MyD88 in rejection of allogeneic skin grafts and lung, and may reflect unique features related to BMC. The findings of the role of innate immunity in BMC rejection would lead to revolutionary changes in our understanding and management of BMT. This would be informative in design of more specific innate immune targeted conditioning proposals in BMT to avoid the toxicity. Disclosures:Bozulic:Regenerex LLC: Employment. Ildstad:Regenerex LLC: Equity Ownership.

Innate Immunity Is Responsible for Early Allorejection of Donor Bone Marrow Cells.

Abstract 3523Poster Board III-460Adaptive immunity, especially T cells, has long been believed to be the dominant immune response in allogeneic transplantation. However, innate immunity has been recently shown to pose a significant barrier to the induction of tolerance to solid organ transplants. The role of the innate immune system in bone marrow cell alloreactivity has not been addressed. The innate immune system provides the first line of defense in the removal of pathogens because of the delay in generation of adaptive immune responses. Our present findings show that innate immunity is a significant first line barrier in bone marrow cell (BMC) rejection. To determine the effect of innate immune cell populations on rejection of donor BMCs, T cell deficient mice (TCR β/δ−/−) were used as BMC recipients in in vivo cytotoxicity assays (Figure A). TCRβ/δ−/− mice have normal innate immune cell populations but do not initiate adaptive cell-mediated cytotoxic or humoral responses. 20 × 106 CFSE labeled donor (high CFSE fluorescence intensity) and recipient control splenocytes (low CFSE fluorescence intensity) were injected into TCRβ/δ−/− and wildtype control B6 recipient mice. Donor cell survival was compared over time. Donor BALB/c splenocytes were eliminated in wildtype B6, with rejection complete by day 3. The kinetics of elimination of donor cells in TCRβ/δ−/− mice was similar to that for wildtype B6 controls, with donor cells eliminated by day 3. These results indicate that early rejection of the splenocytes in wildtype mice was T cell-independent. The acute rejection of BMC in wildtype B6 recipients occurred within 3 days, which is prior to the time required for T cell activation. Thus the effectors mediating BMC rejection would be the innate immune cells: macrophages, neutrophils, or NK cells. To rule out potential involvement of natural Abs in the cytotoxicity we observed in the TCRβ/δ−/− mice, Rag−/− mice were used as recipients (Figure B). Rag−/− mice do not produce mature T cells or B cells. 20 × 106 CFSE labeled donor (high CFSE intensity) and recipient control BM cells (low CFSE intensity) were injected into Rag−/−mice. Rag−/− and wildtype B6 control mice exhibited similar kinetics of donor cell cytolysis. The rapid elimination of allogeneic cells from immunocompetent mice is comparable with that observed in T cell- (TCRβ/δ−/−) or T and B cell- (Rag−/−) deficient mice indicates that allogeneic cells are subject to T cell-independent rejection at the early time period after cell infusion (≤ 3 days). As the kinetics of cytotoxicity were similar in experiments using either splenocytes or BMCs as target cells in our later experiments, our data suggest that the innate immune system is responsible for early allorejection of donor BMC at the early inductive period for adaptive immunity. These findings may have significant impact on the development of immune-based nonmyeloablative conditioning strategies and show for the first time that a dominant factor in BMC rejection is contributed by innate immune responses. [Display omitted] Disclosures:Bozulic:Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

NK Cell-Mediated Rejection of Bone Marrow Cells- In Vivo Evidence of NK Cell Subset Licensing.

Abstract 3537Poster Board III-474Natural killer (NK) cells have been shown to attack virally-infected and transformed cells as well allogeneic bone marrow cells (BMC) but not normal self-tissues. The mechanism of missing self recognition and self tolerance of NK cells is poorly understood. NK cells exist as subsets based on expression of inhibitory receptors (Ly49 in mouse, KIR in man) that bind MHC class molecules. In vitro data have shown that murine NK cell subsets bearing Ly49 receptors for self MHC class I molecules have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC class I-dependent functional education, allowing the NK cells bearing the appropriate Ly49 receptors to preferentially mediate effector function. Thus far, no in vivo evidence for this preferential licensing or arming has been shown. We assessed the intrinsic response potential of the different Ly49+ NK cell subsets in BMC rejection without having the complicating effects of binding MHC on the target cell (which delivers potent inhibitory signals to the NK cell) by using β2-microglobulin deficient (β2m−/−) mice as donors which totally lack class I MHC molecules. Using syngeneic, congenic, and allogeneic strains of mice as recipients and depleting the different Ly49 subsets, we found that NK cell subsets whose Ly49 molecules have been shown to bind “self-MHC Class I” (ie Ly49G2 in H2d and Ly49C in H2b haplotypes) were found to be the dominant subset responsible for mediating the rejection of the β2m−/− BMC. This provides the first in vivo evidence for host MHC class I-dependent functional education (licensing or arming). Importantly, we also demonstrated that prior activation of the NK cells in vivo could override this licensing effect and allow the “non-licensed subset” to mediate rejection. The pattern of NK mediated rejection ability by Ly49 subsets was observed in B10D2 allogeneic H2d strain mice but not observed in BALB/c allogeneic H2d strain mice indicating that licensing ability was not solely dependent on host MHC expression and supporting the role of MHC class I–independent system for NK-cell self-tolerance. Interestingly, all H2d strain mice (B10.D2, BALB/c, and B6D2F1) were able to resist significantly greater amounts of B2m-/- BMC than H2b strain mice indicating that the rheostat hypothesis regarding Ly49 affinities for MHC and NK cell function impacts BMC rejection capability. These results demonstrate that both MHC and non-MHC genes on the host has multiple effects on NK cell subset-mediated BMC rejection and that licensing or arming of Ly49 NK cell subsets can be observed in vivo. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Cell Rejection by Innate Immune Pathways Is Exclusively Dependent On the TRIF Pathway.

Abstract 3526Poster Board III-463Research on transplantation rejection, tolerance, and immunosuppressive agents has been directed toward the adaptive immune response as, until now, it has been believed to be the dominant mechanism for alloreactivity. The role of innate immunity in transplantation has not been fully defined. Despite the sentinel role of innate immunity in driving and shaping adaptive immunity, the contribution of innate immunity to bone narrow cells has only recently been identified. TLR4-driven MyD88-dependent immunity has been demonstrated to be critical for allogeneic innate immune responses to solid organ transplants. Whether innate immunity plays a role in BMC rejection has not been defined. In the present studies we tested whether the absence of MyD88 would enhance allogeneic engraftment in bone marrow transplantation (BMT). The core component of TLR signaling is activation of an IL-1-like pathway dependent upon the adapter MyD88. MyD88−/− B6 mice (n=6) were used to study the role of MyD88 signaling in allogeneic BMC rejection. As TRIF is another crucial adapter for TLR3 signaling and responsible for induction of signaling via type 1 IFNs, B6 TRIF−/− mice (n=6) were also investigated. Wild-type B6 (H2b, n=6) mice were used as controls. In the present study, MHC plus minor antigen-disparate BALB/c (H2d) mice served as allogeneic bone marrow donors. Recipients were nonmyeloablatively conditioned with anti-CD154 mAb (day0 and +3) to block CD40:CD154 co-stimulatory pathway and sirolimus (day0 to +4) to block late-stage T cell activation by inhibiting IL-2 responsiveness. Recipients were transplanted with 15 × 106 allogeneic (BALB/c; H2d) marrow cells 4-6 hours following conditioning with 100 cGy total body irradiation (TBI). Allogeneic engraftment was achieved in 33.3% of MyD88−/− recipients at 1 mo after BMT, a percentage similar to that for wild-type B6 mice. Surprisingly, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month after BMT, which was significantly higher than in MyD88−/− (1.8 ± 1.0%; P < 0.0001) or in wild-type B6 mice (1.3 ± 0.8%; P < 0.005) and thereafter through all time points tested (2, 4 and 5 months, P < 0.05). The levels of donor chimerism increased with time and the level of donor chimerism reached 14.7 ± 7.1% TRIF−/− at five months. These results suggest that allogeneic BMC rejection is uniquely independent of the MyD88 pathway but rather involves TRIF signaling. These data suggest that the innate immune response to BMC differs from that for solid organs and may reflect the specificity of BMT compared with other transplants. These findings demonstrate for the first time that BMC elicit an innate immune response via the TRIF pathway and may open the door to novel approaches for immune-based conditioning to promote engraftment. Disclosures:Ildstad:Regenerex: Equity Ownership.

Regulation of the NK Cell Alloreactivity to Bone Marrow Cells by the Combination of the Host NK Gene Complex and MHC Haplotypes

Host NK cells can reject MHC-incompatible (allogeneic) bone marrow cells (BMCs), suggesting their effective role for graft-vs leukemia effects in the clinical setting of bone marrow transplantation. NK cell-mediated rejection of allogeneic BMCs is dependent on donor and recipient MHC alleles and other factors that are not yet fully characterized. Whereas the molecular mechanisms of allogeneic MHC recognition by NK receptors have been well studied in vitro, guidelines to understand NK cell allogeneic reactivity under the control of multiple genetic components in vivo remain less well understood. In this study, we use congenic mice to show that BMC rejection is regulated by haplotypes of the NK gene complex (NKC) that encodes multiple NK cell receptors. Most importantly, host MHC differences modulated the NKC effect. Moreover, the NKC allelic differences also affected the outcome of hybrid resistance whereby F1 hybrid mice reject parental BMCs. Therefore, these data indicate that NK cell alloreactivity in vivo is dependent on the combination of the host NKC and MHC haplotypes. These data suggest that the NK cell self-tolerance process dynamically modulates the NK cell alloreactivity in vivo.

Lack of correlation between an assay used to determine early marrow allograft rejection and long-term chimerism after murine allogeneic bone marrow transplantation: Effects of marrow dose

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unit-granulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2b) mice were lethally irradiated and transplanted with various doses of BALB/c (H2d) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.

CD127-LOW MEMORY T CELLS AND IMMUNOGLOBULIN M ARE CRITICAL FACTORS FOR EARLY REJECTION OF ALLOGENIC BONE MARROW CELLS IN SENSITIZED RECIPIENT MICE.

O270 Aims: Allosensitization is major risk factor of graft failure in clinical bone marrow transplantation even in HLA-matched combination. It is still unclear what critical components of immunological memory act on graft rejection in allosensitized hosts. In this study, we investigated the impact of allosensitization on engraftment of donor bone marrow cells (BMC) and the key effectors in donor BMC-rejection using the conventional fully allogeneic murine bone marrow transplantation model. Methods: We used C57BL/6 recipient mice (B6; H-2Kb), which were intraperitoneally injected with 1×107 splenocytes of C3H (H-2Kk) mice on day -35 (5W-sensitized) or -7 days (1W-sensitized), as allosensitized recipients. The B6 recipient mice were lethally irradiated with 10 Gy whole-body irradiation (WBI) and were intravenously injected with 2×107 (low dose) or 10×107 (high dose) T-cell-depleted bone marrow cells (TCD-BMC). Results: The titer of IgG alloAb in sensitized B6 were peaked at 4-7 weeks after allosensitization. The titer of IgM alloAbs arrived at maximum level within the 5 days and rapidly decreased to undetectable level within 3 weeks. While all non-sensitized recipients transplated with TCD-BMC from C3H survived permanently {Mean survival time (MST) >100 days}, all 5W-sensitized recipients did not survived (MST=10.2 days). We confirmed the cause of death was not due to Graft-versus-Host disease. The rejection of BMC was donor-specific since all 5W-sensitised recipients reconstituted with third party, SJL(H-2s)-TCD-BMC survived permanently (MST >100 days). This rejection of BMC was not overcame by administration of high dose TCD-BMC or by antibody-mediated NK-cell-depletion. In spite of existence of high titer of IgG alloAb, antibody-mediated T-cell-depletion alone can improve the rejection (MST=53.9 days), but, in case using low dose TCD-BMC, late graft failure occurred in 5W-sensitized recipients mice. Antibody-mediated T-cell-depletion and using high-dose of TCD-BMC completely overcame the early and late graft rejection (MST>100 days) and the recipients had durable multilineage complete chimerism. We confirmed that radioresistant T cells were critical components to reject donor BMC using adoptive transfer model. The phenotype of surviving T cells after lethal irradiation was CD127-low CD44-high memory T cells. In contrast to 5W-sensitized B6, 1W-sensitized B6, rejected C3H-BMC in spite of the depletion of NK-cells or T-cells. To assess the effect of alloreactive IgM, naïve mice transferred with serum from B6 mice, which had been allosensitized five days before (5D-serum: high IgM and minimal IgG alloAb), or five weeks before (5W-serum: no IgM and high IgG alloAb), were lethally irradiated and transplanted with donor BMC. The recipients transferred with 5D-serum rejected donor BMCs (MST=8.6 days), but the recipients transferred with 5W-serum did not (MST>100 days). Conclusions: These findings suggested that alloreactive CD127-low memory T cells and IgM are the key effector for the rejection of donor BMCs in sensitized recipients.

The immunobiology of natural killer cells and bone marrow allograft rejection

Natural killer (NK) cells mediate the acute rejection of bone marrow cell (BMC) allografts, but not solid tissue grafts, in lethally irradiated mice. However, the mechanisms underlying this capability for rejecting BMC remain unclear. NK cells express (1) inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules and (2) activating receptors with diverse specificities. Inhibitory NK receptors confer to NK cells the ability to discriminate between MHC class I-positive and -negative target cells and are therefore involved in the control of NK cell tolerance to self, as well as in the elimination of cells that have downregulation of MHC class I molecules. Preclinical studies in mice have provided good evidence that subsets of NK cells that bear different combinations of both inhibitory and activating Ly49 receptors can interact with each other and target specific BMC rejection, as well as NK cell responses toward tumor cells. Recent clinical studies have also shown that the use of killer cell immunoglobulin-like receptor ligand incompatibility in patients with leukemia who received hematopoietic stem cell transplants correlated not only with the elimination of graft rejection, but also with eradication of tumor and prevention of graft-versus-host disease; this offers a significant advantage for survival. In this review, we attempt to bring together literature regarding the biology of NK cells and discuss the current issues in bone marrow transplantation and the potential clinical role of NK cell alloreactivity in the efficacy of this procedure for immunotherapy of cancer and infectious states.

Increased bone marrow allograft rejection by depletion of NK cells expressing inhibitory Ly49 NK receptors for donor class I antigens

Natural killer (NK) cells are the major effectors of acute rejection of incompatible bone marrow cell (BMC) grafts in lethally irradiated mice. The immunogenetics of BMC rejection are largely controlled by the coexpression (or not) of inhibitory and stimulatory Ly49 receptors whose ligands are class I major histocompatibility complex (MHC) molecules. The majority of the BMC rejection studies involved low numbers of BMCs that were resisted by host NK cells. In the present study, larger numbers of BMCs were given in which rejection was not detected and the role of different Ly49 NK subsets not presumably involved in the rejection of a particular BMC haplotype was examined. Surprisingly, the data show that the removal of NK cell subsets expressing Ly49 inhibitory receptors for donor class I antigens, which would be predicted to have no effect on the BMC rejection capability, resulted in the marked rejection of BMCs where no resistance was normally seen. These results extend the "missing self" hypothesis to suggest that NK Ly49 inhibitory receptors can both inhibit activation and killing by those cells, but also can in some way influence the function of NK cells that do not express that inhibitory receptor in a cell-cell interaction. This suggests that caution must be exercised before removal of host NK cell subset is applied clinically because enhanced BMC rejection may result. Altering the balance of Ly49 NK subsets may also affect other in vivo activities of these cells.

Immunobiology of natural killer cells and bone marrow transplantation: merging of basic and preclinical studies.

Natural killer (NK) cells mediate acute rejection of bone marrow, but not solid tissue, allografts in lethally irradiated mice. Precisely how and why this rejection occurs is still unclear. In allogeneic bone marrow transplantation (BMT), a spectrum of results is possible; one result can be marrow graft failure due to host rejection of the graft by NK and T cells and, at the opposite spectrum, the occurrence of graft-versus-host disease (GVHD). Donor NK cells, however, appear capable of improving donor engraftment without giving rise to GVHD and thus may be of use as an immunotherapy following BMT. As NK-cell inhibitory receptors play a role in bone marrow cell rejection, these same inhibitory receptors may also affect NK responses towards tumor cells. It has been demonstrated that blocking the interaction of inhibitory receptors with MHC determinants on tumor cells can result in greater antitumor effects. Thus, NK cells are capable of mediating both positive and negative effects during BMT depending on whether they are of host versus donor origin and their state of activation. Understanding their role in BMT provides insights as to their physiological roles and points the way to potential clinical uses.

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants.

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.

Role of classical (RT1.A) and nonclassical (RT1.C) MHC class I regions in natural killer cell-mediated bone marrow allograft rejection in rats.

We have studied the role of the different MHC (RT1) subregions in acute natural killer (NK) cell-mediated bone marrow allograft rejection in lethally irradiated, bone marrow cell (BMC) reconstituted rats. We employed a series of MHC congenic and intra-MHC recombinant rat strains so that effects of mismatches in defined RT1 subregions could be studied systematically. BMC allograft survival was measured as 125IUdR uptake in the spleen between day 5 and day 7 after irradiation and BMC reconstitution. We found that in certain RT1 haplotype combinations, nonclassical RT1.C disparities by themselves could determine graft rejection (i.e., in the u/av1 recombinant haplotypes), whereas in another combination (between the av1 and c haplotypes) a mismatch for an isolated classical RT1.A region was decisive for engraftment. Thus, PVG.R1 BMC failed to proliferate in PVG rats, differing in the RT1.A region only, whereas in PVG.1U rats rejection could be determined by isolated differences in the RT1.C region (LEW.1WR1). Also, RT1 homozygous rats (RT1.U) rejected semi-allogeneic F1 hybrid BMC. The acute rejection of BMC was mediated by NK cells, as athymic nude rats, lacking alloreactive T cells but with normal alloreactive NK cells, showed the same patterns of rejection as did normal rats. Nude rats also rejected allogeneic lymphocytes, a previously documented NK-mediated phenomenon, with identical requirements of MHC disparity. This investigation shows that rat effector NK cells are radioresistant, independent of the thymus, and capable of recognizing and rejecting MHC mismatched transplanted BMC on the basis of mismatches in both classical and nonclassical class I regions in vivo. The studies underline the importance also of NK cells in determining BMC allograft survival.

Differential Effects of the Rejection of Bone Marrow Allografts by the Depletion of Activating Versus Inhibiting Ly-49 Natural Killer Cell Subsets

Natural killer cells mediate the specific rejection of bone marrow cell (BMC) allografts in lethally irradiated mice. The Ly-49 family of molecules present on subsets of murine NK cells appears capable of binding class I MHC molecules, resulting in transmission of an inhibitory signal to the NK cell. These Ly-49 family members have been shown to have an immunoreceptor tyrosine-based inhibitory motif that is responsible for the inhibitory signal. However, a new Ly-49 family member was found that lacks this motif, Ly-49D, and evidence suggests that this may be an activating receptor. We therefore compared the role of the activating Ly-49 member with NK cells bearing inhibitory Ly-49 receptors in BMC rejection. Depletion of Ly-49D+ NK cells in H-2b mice abrogated their ability to reject H-2d BMC allografts. Similarly, Ly-49C+ NK cells also were shown to mediate the specific rejection of H-2d BMC. When both subsets were depleted, an additive enhancement of BMC engraftment was observed, indicating that both subsets play a role in the rejection of allogeneic H-2-homozygous H-2d BMC. However, rejection of H-2(b x d) or D8 (H-2b, Dd transgene) BMC allografts was unaffected by Ly-49C+ NK cell depletion in H-2b mice. In marked contrast, depletion of Ly-49D+ NK cells in H-2b mice totally abrogated the rejection of H-2(b x d) heterozygous BMC in support of in vitro data suggesting that Ly-49D+ NK cells receive activating signals. Therefore, NK subsets demonstrate a differential ability to reject H-2 homozygous and heterozygous BMC.

Natural killer cells recognize common antigenic motifs shared by H-2Dd, H-2Ld and possibly H-2Dr molecules expressed on bone marrow cells.

Murine natural killer (NK) cells can mediate specific rejection of bone marrow cell (BMC) allografts. Whereas positive recognition of allogeneic MHC antigens forms the basis for T cell alloreactivity, it has been postulated that NK cells are reactive against targets that do not express certain self-encoded MHC class I antigens. Here, we study the immunogenicity of BMC grafts from two class I transgenic mice, D8 (B6 mice with an H-2Dd transgene) and C3H.Ld (C3H mice with an H-2Ld transgene). D8 BMC grafts are acutely rejected by B6 but not D8 recipients. This suggests that antigenic motifs associated with the H-2Dd molecule are recognized. B6 mice depleted of their CD3+ but not NK1.1+ cells can still reject D8 BMC grafts. These data suggest that NK1.1+/CD3- cells recognize the H-2Dd derived antigenic motifs. Similarly, C3H.Ld BMC grafts are rejected by B6 x C3H F1 but not B6 x C3H.Ld F1 recipients. Thus, antigenic motifs associated with the H-2Ld molecule can also be recognized. Furthermore, expression of either H-2Dd or H-2Ld by the recipients renders them unable to reject D8 or C3H.Ld BMC grafts. Therefore, H-2Dd and H-2Ld molecules appear to express common antigenic motifs recognized by NK cells. Additional studies with B6.R4 (KbIbSbDr), an intra-H-2 recombinant mouse, indicated that a third class I molecule, possibly H-2Dr, also shared the common antigenic motifs with both H-2Dd and H-2Ld molecules. Thus, positive recognition of class I antigens by NK cells can occur. However, expression of some of these antigenic motifs appear to be negatively controlled by certain H-2r genes as suggested by rejection of D8 and B6.R4 BMC grafts by D8 x B10.RIII F1 and B6.R4 x B10.RIII F1 hybrids respectively.

Use of newborn liver cells as a murine model for cord blood cell transplantation.

There is current interest in using umbilical cord blood cells as a substitute for bone marrow cells (BMC) in human transplantation. However, T cell-depleted BMC are more susceptible to rejection. Because CBC lack mature T cells, mouse newborn liver cells (NLC) were used as a model to investigate the role of T cells in engraftment. BALB/c BMC, C.B-17 scid/scid (SCID) BMC, and BALB/c NLC were transplanted into lethally irradiated BALB/c and (B6 x DBA/2) F1 (B6D2F1) hosts. Splenic 125IUdR incorporation 5 days later assessed engraftment. BALB/c BMC, NLC, and SCID BMC grew well in BALB/c hosts, but only BALB/c BMC grew in B6D2F1 hosts. This suggests that T cells are necessary for engraftment of H-2d stem cells in the B6D2F1 host. Addition of BALB/c thymocytes to SCID BMC or NLC allowed engraftment in the F1 hosts. It appears that NK cells mediated the resistance because their depletion by mAb increased engraftment of SCID BMC or NLC. Anti-NK1.1 mAb and -asialo GM1 serum eliminate all NK cells, although anti-5E6 mAb eliminate a subpopulation of NK cells that responds to Hh-1d (determinant 2+) stem cells. Treatment of F1 hosts with any of these mAb prevented rejection of SCID BMC or BALB/c NLC. Also, activation of NK cells by poly I:C caused rejection of donor cells in the F1 that could not be overcome by the presence of thymocytes. This effect of the poly I:C could also be reversed by mAb depletion of host NK cells. Thus, engraftment of stem cells is influenced by the presence of donor T cells and the activation level of host NK cells.

Rejection of bone marrow cell allografts by natural killer cell subsets: 5E6+ cell specificity for Hh‐1 determinant 2 shared by H‐2d and H‐2f

The 5E6 antigen, defined by anti-5E6 mAb, is expressed on one-half of murine natural killer (NK) cells, and we have previously demonstrated (C. L. Sentman et al., J. Exp. Med. 1989. 170: 1991) that 5E6+ NK cells are necessary for the rejection of BALB/c (Hh-1d) but not C567BL/6 (Hh-1b) bone marrow cells (BMC). In experiments described here, we have characterized the specificity of 5E6+ and 5E6- NK cell subsets for hemopoietic histocompatibility-1 (Hh-1) antigens. Prospective recipient mice were treated with anti-5E6 mAb and challenged with BMC from a variety of donors. In addition, H-2d/Hh-1d C.B-17 scid 5E6+ or 5E6- NK cells were adoptively transferred into irradiated, NK cell-depleted hosts and challenged with H-2b/Hh-1b BMC. The data indicate that the 5E6+ NK cells are necessary for the rejection of only those BMC that express the Hh-1 determinant 2 shared by H-2d and H-2f haplotypes of strains BALB/c (d), A.Ca (f), and B10.M (f). No reactivity to other Hh-1 antigens resides in the 5E6+ population. In contrast, the ability of NK cells to lyse H-2d or H-2b tumor cells was independent of 5E6 expression. These results suggest that the 5E6 molecule is likely to be important in the specific recognition and rejection of BMC that express Hh-1 determinant 2, and is probably not involved in recognition of "tumor target cell structures".