Allogeneic Strain Research Articles

OC-009 SYNGENEIC VERSUS ALLOGENEIC NON-VASCULARIZED RECTUS FASCIA TRANSPLANTATION IN A RABBIT MODEL WITHOUT IMMUNOSUPPRESSION: SHORT TERM OUTCOME

Abstract Background and aim Transplantation (Tx) of non-vascularized rectus fascia (NVRF) is a valid option in solid organ transplant recipients to avoid open abdomen after Tx. To extrapolate this technique for non-Tx patients, we investigated in a preclinical rabbit model the feasibility of transplanting NVRF in a syngeneic (Syn) versus allogeneic (Allo) strain without using immunosuppression (IS). Short-term outcome was evaluated after 4 weeks. Material and methods A validated rabbit model of NVRF Tx was used comparing 6 Syn New Zealand White (NZW) rabbits versus 6 Allo Mixed breed to NZW Tx without IS. Animals were macroscopically analyzed at harvesting after 4 weeks for graft integration, herniation, adhesions, seroma, hematoma, and surgical site infections. Histological and immunohistochemical analysis was performed to assess inflammatory cell reaction and neovascularization. Mechanical testing was performed to assess the thickness, stiffness and strength. Results Results showed similar sufficient macroscopic ingrowth of the NVRF in both groups. At the histological level, cell infiltration suggested a clearing reaction more than a rejection-based inflammation, which was more pronounced in the Allo group. No significant differences were seen concerning mechanical properties. Conclusions In a validated rabbit model of NVRF Tx, we showed that Tx was possible in an Allo strain without the need for IS, resulting in satisfying short term inflammatory and mechanical outcomes. Longer-term experiments are needed to evaluate the effect of graft integration and possible hernia development.

The importance of MHC class II in allogeneic bone marrow transplantation and chimerism-based solid organ tolerance in a rat model.

Mixed hematopoietic chimerism enables donor-specific tolerance for solid organ grafts. This study evaluated the influence of different serological major histocompatibility complex disparities on chimerism development, graft-versus-host disease incidence and subsequently on solid organ tolerance in a rat model. For bone marrow transplantation conditioning total body irradiation was titrated using 10, 8 or 6 Gray. Bone marrow transplantation was performed across following major histocompatibility complex mismatched barriers: complete disparity, MHC class II, MHC class I or non-MHC mismatch. Recipients were clinically monitored for graft-versus-host disease and analyzed for chimerism using flow cytometry. After a reconstitution of 100 days, composition of peripheral leukocytes was determined. Mixed chimeras were challenged with heart grafts from allogeneic donor strains to define the impact of donor MHC class disparities on solid organ tolerance on the basis of stable chimerism. After myeloablation with 10 Gray of total body irradiation, chimerism after bone marrow transplantation was induced independent of MHC disparity. MHC class II disparity increased the incidence of graft-versus-host disease and reduced induction of stable chimerism upon myelosuppressive total body irradiation with 8 and 6 Gray, respectively. Stable mixed chimeras showed tolerance towards heart grafts from donors with MHC matched to either bone marrow donors or recipients. Isolated matching of MHC class II with bone marrow donors likewise led to stable tolerance as opposed to matching of MHC class I. In summary, MHC class II disparity was critically associated with the onset of graft-versus host disease and was identified as obstacle for successful development of chimerism after bone marrow transplantation and subsequent donor-specific solid organ tolerance.

Novel Targeting to XCR1+ Dendritic Cells Using Allogeneic T Cells for Polytopical Antibody Responses in the Lymph Nodes.

Vaccination strategy that induce efficient antibody responses polytopically in most lymph nodes (LNs) against infections has not been established yet. Because donor-specific blood transfusion induces anti-donor class I MHC antibody production in splenectomized rats, we examined the mechanism and significance of this response. Among the donor blood components, T cells were the most efficient immunogens, inducing recipient T cell and B cell proliferative responses not only in the spleen, but also in the peripheral and gut LNs. Donor T cells soon migrated to the splenic T cell area and the LNs, with a temporary significant increase in recipient NK cells. XCR1+ resident dendritic cells (DCs), but not XCR1− DCs, selectively phagocytosed donor class I MHC+ fragments after 1 day. After 1.5 days, both DC subsets formed clusters with recipient CD4+ T cells, which proliferated within these clusters. Inhibition of donor T cell migration or depletion of NK cells by pretreatment with pertussis toxin or anti-asialoGM1 antibody, respectively, significantly suppressed DC phagocytosis and subsequent immune responses. Three allogeneic strains with different NK activities had the same response but with different intensity. Donor T cell proliferation was not required, indicating that the graft vs. host reaction is dispensable. Intravenous transfer of antigen-labeled and mitotic inhibitor-treated allogeneic, but not syngeneic, T cells induced a polytopical antibody response to labeled antigens in the LNs of splenectomized rats. These results demonstrate a novel mechanism of alloresponses polytopically in the secondary lymphoid organs (SLOs) induced by allogeneic T cells. Donor T cells behave as self-migratory antigen ferries to be delivered to resident XCR1+ DCs with negligible commitment of migratory DCs. Allogeneic T cells may be clinically applicable as vaccine vectors for polytopical prophylactic antibody production even in asplenic or hyposplenic individuals.

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29H(sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29H(sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice.

Multiple allogeneic progenitors in combination function as a unit to support early transient hematopoiesis in transplantation.

Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34(+) cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34(+) cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft.

Mechanisms of B Cell Tolerance after in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive transplant approach that results in donor cell engraftment across immune barriers. Although a significant amount of work has investigated the fate of T cells following IUHCT, little attention has been paid to B cell tolerance and the fate of donor derived host reactive or host derived donor reactive B cells following IUHCT. B cell tolerance is broadly believed to occur by a combination of 3 mechanisms: deletion, receptor editing, and functional inactivation (anergy). In the current study we attempt to elucidate the mechanism(s) by which B cell tolerance occurs following IUHCT.Methods: 10x106 donor bone marrow (BM) cells were injected intravenously via the vitelline vein into gestational day 14 murine fetuses. IUHCT was performed in the congenic (C57Bl/6-GFP [H2Kb ] into C57Bl/6 [H2Kb ]) and allogeneic (C57Bl/6-GFP into Balb/c [H2Kd ]) strain combinations. Naive Balb/c and C57Bl/6 mice served as controls. Mice were sacrificed at day of life 3 (P3), 1 month and 4 months of age at which time their BM, spleen, and serum were harvested. To assess B cell deletion, flow cytometry was used to determine the absolute # and % of host and donor immature and pre B cells in the BM. Additionally, apoptosis of host and donor BM derived B cells was determined by annexin staining. Central receptor editing was evaluated using RT qPCR to measure the amount of Vκ-RS rearrangments in BM pre-B cells. Peripheral receptor editing was studied by calculating the % of λ light chains in mature splenocytes identified by flow cytometry. Finally, functional inactivation of donor reactive host B cells was assessed by measuring anti-H2Kb serum antibodies (ab) of allogeneic chimeras, naive, and immunized mice at 1 month of age.Results: The absolute number of BM immature B cells was decreased in allogeneic recipients of IUHCT compared to noninjected Balb/c controls at 1 month of age (fig 1). This effect was lost by 4 months of age. The decrease in B cells resulted primarily from a decrease in immature donor as opposed to host B cells compared to controls (% immature donor B cells in allogeneic recipients vs. controls: 16.2% vs. 39.9%; p<0.0005). Donor B cells in allogeneic chimeras also demonstrated a trend toward increased apoptosis compared to controls (24.8 vs. 18.7%; p=0.2) which was not seen in immature host B cells (18.3 vs. 18.6%; p=0.9). There was no significant decrease in the absolute number of immature B cells or increased apoptosis in congenic recipients compared to uninjected controls. These findings suggest deletion of autoreactivedonor B cells.Light chain receptor editing involves rearrangements within the κ and λ gene loci and may occur in BM pre-B cells or mature B cells in the spleen. We found no difference in the Vκ-RS rearrangements of pre B cells in allogeneic chimeras and controls at 1 month. In contrast, the quantity of total λ+ mature splenic B cells was increased in allogeneic chimeras at P3 (10.8 vs. 8.4%; p=0.02) and resulted from an increased host λ+ % compared to controls (10.8 vs. 8.4% p=0.03) suggesting peripheral receptor editing of host cells (fig.2). The λ+ % increase in allogeneic chimeras was lost by 1 month.Autoreactive B cells that escape deletion and receptor editing can be functionally inactivated. Neither allogeneic nor naive mice developed ab to H2Kb splenocytes, however, Balb/c mice immunized to H2kb antigen showed high ablevels (MFI fold change: allo-0.89 naive-1.37 imm-2.77; p<0.05).Conclusion:B cell tolerance after IUHCT is achieved by distinct mechanisms for host and donor cell populations. Donor derived host reactive B cells undergo deletion and apoptosis while receptor editing and functional inactivation are the primary mechanisms of B cell tolerance of host derived donor reactive B cells. We hope use this and future studies of antigen specific B cell tolerance to harness the immunologic potential of IUHCT for many hematopoietic and immunologic congenital diseases. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antibody Responses in Mice to Particles Formed from Adsorption of a Murine Monoclonal Antibody onto Glass Microparticles

Immunogenicity of therapeutic monoclonal antibodies (mAbs) is a concern because of the effects of anti-drug antibodies (ADAs) on therapeutic efficacy. Particulate matter has been suggested as a potential contributing factor to immunogenicity. In this study, we investigated ADA levels in mice in response to administration of a murine immunoglobulin G (IgG)2c/κ mAb (mAb1) that was generated in C57BL/6J mice. Particles of mAb1 were formed by adsorbing the protein to glass microparticles. Formulations containing microparticles were administered subcutaneously to mice of either the syngeneic strain, C57Bl/6J, or the allogeneic strain, BALB/c. ADA levels were measured using an isotype-specific enzyme-linked immunosorbent assay method. Whereas BALB/c mice showed strong IgG1 and IgG2b responses against both the particulate and native mAb1 samples, adsorption of mAb1 to particles rendered it slightly more immunogenic than its native, soluble form. In BALB/c mice, immunoglobulin M (IgM) was produced after the first week of injections and then faded gradually. In contrast, C57BL/6J mice showed moderate IgM, IgG1, IgG2b, and IgG3 responses to injections of glass particle-adsorbed mAb1. ADA responses were higher in the allogeneic BALB/c mice, which do not produce mAbs of the IgG2c/κ isotype. Thus, the presence of both foreign epitopes and particles may be important in inducing ADA responses.

Altered Thymocyte Development in Allogeneic in Utero Hematopoietic Cell Transplantation in the Mouse Model

AbstractAbstract 4668 IntroductionIn Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been analyzed. In this study, we utilized the murine model of allogeneic IUHCT and analyzed donor and host thymocyte development. MethodsBone marrow (BM) cells (10×106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal time points the thymocytes were analyzed by multi-color flow cytometry. ResultsThe results demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocytes and from thymocyte development in normal B6 and Balb/c control mice. Though each subpopulation of the host's thymocytes showed comparable levels to the normal untransplanted Balb/c mice, the donor BM-derived thymocytes demonstrated significantly higher proportions of CD4+CD8- and CD4-CD8+ single positive cells, and a dramatically lower proportion of CD4+CD8+ double positive cells compared to their donor-derived counterparts, respectively. These discrepancies increased with each analysis time point up to 12 weeks. Immature single positive cells, including both TCRb-CD4+CD8- and TCRb-CD4-CD8+ cells, were significantly higher in the donor-derived thymocytes than the host, indicating the development of the donor BM-derived thymocytes were impeded at the DN-DP stage in this specific allogeneic IUHCT setting. In addition, we also found higher frequencies and more potent suppressive capacity of regulatory T cells in the donor-derived T cell compartment than in the host T cell compartment. In the reversed allogeneic strain combination, i.e. IUHCT of Foxp3GFP Balb/c BM MNCs into the E14 Foxp3GFP B6 recipients, thymocytes of both origins showed imbalanced intra-thymic development that differed from the normal mouse. ConclusionOur data in the current study suggests that immune reconstitution of the donor bone marrow derived thymocytes differs from that of the host cells and that of normal mice in an allogeneic IUHCT system. The mechanism of the altered thymic development in allogeneic IUHCT, and the potential importance of these observations for clinical IUHCT remains to be determined. [Display omitted] [Display omitted] Bone marrow (BM) cells (10×106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal time points the thymocytes were analyzed by multicolor flow cytometry (Fig. A), and the expression of TCR¦A′ in each individual subpopulation in 4-week old mice was measured (Fig. B). Disclosures:No relevant conflicts of interest to declare.

Lung transplantation research: impact of a new surgical model

Experimental lung transplantation in mice has been deemed ‘infeasible’ for many years and has been cracked technically only a few years ago by some extraordinarily dedicated researchers. Okazaki and colleagues published the first experimental report based on a series of successful murine left lung transplants in 2007 [1, 2]. While other animal models may more closely resemble the human setting, mouse models have huge advantages when it comes to the study of mechanistic questions on the molecular level. Gene knockout mice have revolutionized whole areas of medical and basic science research and are of no lesser value in transplantation research. Above that, reagents needed for in vivo treatment, such as depleting antibodies, proteins or peptides, are easily available is sufficient quantities for mice, but not necessarily for larger animals. The murine heterotopic (non-vascularized) trachea transplantation model was used as an approximation to experimental lung transplantation in mice resembling bronchiolitis obliterans [3], but remains a contraption with severe methodological shortcomings. With the advent of orthotopic mouse lung transplantation, this field of transplantation surgery is now accessible to the modern tools of mechanistic life sciences, and the study by Jungraithmayr et al. [4] in this issue is a good example of this recent development. CD26, also called dipeptidyl peptidase-4 (DPP4) is a protein expressed on many cell types including lymphocytes and lung parenchyma. It is a membrane glycoprotein with a cell surface epitope acting as an antigen detectable by the respective monoclonal antibodies. Its function is the enzymatic cleavage of certain dipeptides from polypeptides such as growth factors, chemokines and neuropeptides. This degradation inactivates the respective polypeptides. Apparently, CD26/DPP4 cleaves a wide variety of substrates, interfering with many biological pathways including, for example, glucose metabolism and suppression of cancer development. Stromal cell-derived factor 1 (SDF-1, also called CXCL12) is a substrate of CD26/DPP4. Jungraithmayr et al. deem SDF-1, together with its receptor CXCR4, important in the modulation of stem cell homing in response to ischaemic injury of the lung. In their experimental setting, they make use of CD26/DPP4 knockout mice, of in vivo inhibition of CD26/DPP4 activity using vildagliptin and of a murine orthotopic lung transplantation model to induce ischaemia-reperfusion injury. The main findings of the study are: 2 days after transplantation, lung isografts from mice undergoing CD26/DPP4 inhibition by vildagliptin showed lesser signs of ischaemia-reperfusion injury in histological samples when compared with untreated control grafts. CD26/DPP4 inhibition in experimental animals abrogated SDF-1 degradation, leading to elevated SDF-1 concentrations in plasma and lung grafts. This was adjoined by a concomitant increase in expression of the SDF-1 receptor CXCR4 on leukocytes in peripheral blood and in lung grafts. Together with CXCR4, important for homing of haematopoietic stem cells, T cells and other CD45+ leukocytes, CD34, the regenerative progenitor marker Flt-3 or the regenerative stem cell marker c-kit (CD117) were coexpressed in transplanted lung tissue harvested 2 days following surgery from CD26/DPP4-inhibited mice. The authors conclude that targeting the SDF-1/CXCR4 axis by CD26/DPP4 inhibition improves recovery from ischaemia reperfusion injury. Further, they state that this intervention ‘may be a promising strategy to intensify sequestration of regenerative stem cells and thus emerges as a novel therapeutic concept’. There are two obvious criticisms. SDF-1 is only one among many, known and unknown, substrates of CD26/DPP4 and improved recovery from ischaemia-reperfusion injury through enhanced regenerative stem cell engrafting in lung transplants is only one among many biological effects of SDF-1. There could be many other, potentially detrimental, effects of SDF-1 overexpression. To name only one example, increased homing of effector leucocytes including T cells to the graft through SDF-1/CXCR4 interaction could have the result of aggravated rejection. Additional transplant experiments with longer follow-up, including isogeneic but also allogeneic strain combinations, are necessary in the future to further validate this approach in the setting of lung ischaemia-reperfusion injury after transplantation. Taken together, this study showcases the exciting possibilities resulting from merging a new surgical animal model, i.e. murine orthotopic lung transplantation, with the established field of mechanistic molecular science in mice. It would be my prediction that we have yet only seen a very small fraction of the exiting experimental results to come from murine orthotopic lung transplantation models in future. Entire fields of lung transplantation research await thorough application of this new model, not only ischaemia-reperfusion injury, but also chronic

Transgene number-dependent, gene expression rate-independent rejection of Dd-, Kd-, or DdKd-transgened mouse skin or tumor cells from C57BL/6 (DbKb) mice

Recipient cells migrating into the transplantation site of an allograft recognize histocompatibility antigens on the grafts and are cytotoxic against the grafts. Although the alloreactive immune response is predominantly directed at the major histocompatibility complex (major histocompatibility complex [MHC]; H-2 in mice) class I molecules, the basic mechanisms of allograft rejection (e.g., ligand-receptor interaction) remain unclear, because of the polymorphism and complexity of the MHC. To examine the role of MHC class I molecules in allograft rejection, D(d) , K(d) or D(d) K(d) -transgenic skin or tumor cells we established on a C57BL/6 (D(b) K(b) ) background and transplanted into C57BL/6 mice. Skin grafts from allogeneic (i.e., BALB/c, B10.D2, and BDF1) strains of mice were rejected from C57BL/6 mice on days 12-14 after grafting, whereas isografts were tolerated by these mice. Unexpectedly, skin grafts from D(d) -, K(d) -, and D(d) K(d) -transgenic C57BL/6 mice were rejected on days 12-14 in a transgene expression rate-independent manner from 9/19 (47%), 20/39 (51%), and 12/17 (71%) of C57BL/6 mice, respectively. Similarly, intradermally transplanted allogeneic (i.e., Meth A), but not syngeneic (i.e., EL-4), tumor cells were rejected from C57BL/6 mice; the growth of D(d) - or K(d) -transfected EL-4 cells was delayed by 10-13 days; and 4/10 (40%) of D(d) K(d) -transfected tumor cells were rejected from C57BL/6 mice. These results indicate that D(d) and K(d) genes are equivalent as allogeneic MHC class I genes and that C57BL/6 (D(b) K(b) ) mice reject D(d) -, K(d) -, or D(d) K(d) -transgened skin or tumor cells in a transgene number-dependent, gene expression rate-independent manner.

Orthotopic vascularized right lung transplantation in the mouse

Orthotopic left lung transplantation in the mouse, as recently developed by our laboratory, represents a physiologic model for studies in transplantation biology. However, because of the limited overall respiratory contribution of the murine left lung, left lung transplant recipients remain healthy despite immune-mediated graft necrosis. We sought to develop a lung transplantation model in which animal survival depends on graft function. Orthotopic vascularized right lung transplantations using cuff techniques were performed in syngeneic and allogeneic strain combinations. Grafts were assessed histologically or functionally by measuring arterial blood gases from 7 to 28 days after transplantation. In a parallel set of experiments, syngeneic and immunosuppressed allogeneic hosts underwent a left pneumonectomy 2 weeks after right lung transplantation, with assessment of graft function 1 week later. We performed 40 right lung transplantations, with a survival rate of 87.5%. Syngeneic grafts remain free of inflammation as far as 28 days after transplantation. On day 7, arterial oxygen levels in syngeneic recipients (481 +/- 90 mm Hg) are equivalent to those in naive mice (503 +/- 59 mm Hg) after left hilar occlusion. Alternatively, allogeneic grafts develop histologic evidence of acute rejection, and arterial oxygen levels are significantly decreased after left hilar clamping (53.3 +/- 10.3 mm Hg). Both syngeneic and immunosuppressed allogeneic right lung recipients tolerate a left pneumonectomy. Right lung transplantation followed by left pneumonectomy represents the first survival model of vascularized lung transplantation in the mouse and will therefore allow for the design of novel studies in experimental lung transplantation.

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.

Antigen‐specific clone selection in vivo

THYMUS NEGATIVE SELECTION In 1959–1961, I was far from Burnet’s influence studying the pathogenesis of virus-induced mouse leukaemia in London, UK. As a result of this work, I discovered the immunological function of the thymus.1 How that came about has been recounted elsewhere.2 Grafting thymus tissue reversed the immune defects of neonatally thymectomized mice but when thymus came from a strain of mice allogeneic to the thymectomized recipients, these were able to reject skin grafts from the allogeneic strains tested but not from the thymus donor strain (Figure 1). This led me to suggest, ‘when one is inducing a state of immunological tolerance in a newly born animal, one is in effect performing a thymectomy, not a complete thymectomy but a partial, selective or immunological thymectomy. In accordance with selective theories of antibody formation, injected antigenic material might make contact with certain cell types differentiating in the thymus and in some way prevent these cells from maturing to a stage when they would be capable of reacting immunologically.3 In other words, the thymus could negatively select antigenspecific clones in vivo and might be the seat where self-tolerance was induced. It was, therefore, a great surprise to me when Burnet asked me to see him during his visit in London in early 1962 and I was immensely pleased to show him the thymus graft results that were in press in the Proceedings of the Royal Society.3 He was, at that time, one of the few immunologists convinced of the immune function of the thymus, as was evident in the lecture he later gave in London.4 The finding that thymus-deficient mice were much more prone than normal mice to develop tumours after treatment with certain carcinogenic chemical agents and oncogenic viruses5,6 provided some experimental support for Burnet’s concept of immunological surveillance.7 Although T cells can, in some experimental situations, act as killer cells against tumour cells, I believe that if tumour antigens do cross-react with self-antigens that are ectopically expressed in thymus medullary epithelial cells, one will not be able to prime the immune system to these antigens.2 POSITIVE SELECTION OF ANTIGEN-SPECIFIC T-CELL CLONES In 1966, I headed the Experimental Pathology Unit of the Walter and Eliza Hall Institute of Medical Research following the invitation of the then director, Gustav Nossal. With my first PhD student, Graham Mitchell, we gathered more evidence for in vivo selection of antigen-specific clones. As admirably recounted by Graham Mitchell in this symposium,8 we used genetically marked cells and adoptive transfer to established unequivocally and for the first time: (a) that thymusderived cells could be activated specifically by antigen9 (b) that they were not the precursors of antibody-forming cells,10,11 (c) that they were essential to help, via some type of collaboration, other lymphocytes derived from bone marrow to respond to antigen by producing antibody.11 These results were initially greeted with surprise and scepticism (for example, ref12), a common criticism being that two rare, clonally individuated

Allogeneic Differences in the Dependence on CD4+T-Cell Help for Virus-Specific CD8+T-Cell Differentiation

CD4(+) T-cell help enables antiviral CD8(+) T cells to differentiate into fully competent memory cells and sustains CD8(+) T-cell-mediated immunity during persistent virus infection. We recently reported that mice of C57BL/6 and C3H strains differ in their dependence on CD28 and CD40L costimulation for long-term control of infection by polyoma virus, a persistent mouse pathogen. In this study, we asked whether mice of these inbred strains also vary in their requirement for CD4(+) T-cell help for generating and maintaining polyoma virus-specific CD8(+) T cells. CD4(+) T-cell-depleted C57BL/6 mice mounted a robust antiviral CD8(+) T-cell response during acute infection, whereas unhelped CD8(+) T-cell effectors in C3H mice were functionally impaired during acute infection and failed to expand upon antigenic challenge during persistent infection. Using (C57BL/6 x C3H)F(1) mice, we found that the dispensability for CD4(+) T-cell help for the H-2(b)-restricted polyoma virus-specific CD8(+) T-cell response during acute infection extends to the H-2(k)-restricted antiviral CD8(+) T cells. Our findings demonstrate that dependence on CD4(+) T-cell help for antiviral CD8(+) T-cell effector differentiation can vary among allogeneic strains of inbred mice.

Biological features of intrahepatic CD4+CD25+ T cells in the naturally tolerance of rat liver transplantation

The biological features of intrahepatic CD4(+) CD25(+) T regulatory cells in the naturally tolerance of rat liver transplantation were explored. Orthotopic liver transplantation was performed in two allogeneic rat strain combinations, one with fatal immunosuppression despite a complete major histocompatibility complex mismatch. The subjects were divided into three groups according to different donors and recipients [Tolerance group: LEW-to-DA; Rejection group: DA-to-LEW; Syngegnic group (control group): DA-to-DA]. The proportion of intrahepatic CD4(+)CD25(+) T cells from three groups was determined by flow cytometry (FCM) in different time. The intrahepatic CD4(+)CD25(+) T cells were isolated by magnetic activated cell sorting (MACS) method and identified by FCM. The Foxp3 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). And their suppression on the proliferation of CD4(+)CD25(-) T effector cells was analyzed by cell proliferation assay in vitro. Beginning immediately after transplantation, the proportion of Treg cells increased over time in both allogeneic groups but was significantly greater in the Rejection group. The proportion of Treg cells declined after day 5, and such reduction was more dramatic in the Rejection group than in the Tolerance group. Animals in the Tolerance group showed a second increase in the proportion after day 14. Intrahepatic CD4(+) CD25(+) T cells isolated from spontaneous tolerance models inhibited the proliferation of mixed lymphocyte reaction. The purity of CD4(+)CD25(+) T cells sorted by MACS was 86%-93%. The CD4(+)CD25(+) T cells could specifically express the Foxp3 gene compared with CD4(+)CD25(-) T cells. In vitro, the spleen cells from LEW rats can irritate the proliferation of CD4(+)CD25(+) T cells more obviously than the syngegnic spleen cells. CD4(+)CD25(+) Tr cells could suppress the proliferation of CD4(+)CD25(-) T cells, but the inhibition was reversed by exogenous IL-2 (200 U/mL). The CD4(+)CD25(+) T regulatory cells specifically express the Foxp3 gene, which may play an important role in the induction of liver transplantation tolerance by suppressing the reaction of effective T cells.

Maintenance of Airway Epithelium in Acutely Rejected Orthotopic Vascularized Mouse Lung Transplants

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the anti-apoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli may be required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide a new and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts.

Undifferentiated Murine Embryonic Stem Cells Cannot Induce Portal Tolerance but May Possess Immune Privilege Secondary to Reduced Major Histocompatibility Complex Antigen Expression

Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.

Busulfan-conditioned bone marrow transplantation results in high-level allogeneic chimerism in mice made tolerant by in utero hematopoietic cell transplantation

In utero hematopoietic cell transplantation (IUHCT) is a non-ablative approach that achieves mixed allogeneic chimerism and donor-specific tolerance. However, clinical application of IUHCT has been limited by minimal engraftment. We have previously demonstrated in the murine model that low-level allogeneic chimerism achieved by IUHCT can be enhanced to near-complete donor chimerism by postnatal minimally myeloablative total body irradiation (TBI) followed by same-donor bone marrow transplantation. Because of concerns of toxicity related to even low-dose TBI in early life, we wondered if a potentially less toxic strategy utilizing a single myelosuppressive agent, Busulfan (BU), would provide similar enhancement of engraftment. In this study, mixed chimerism was created by IUHCT in a fully allogeneic strain combination. After birth, chimeric mice were conditioned with BU followed by transplantation of bone marrow cells congenic to the prenatal donor. We demonstrate that: 1) low-level chimerism after IUHCT can be converted to high-level chimerism by this protocol; 2) enhancement of chimerism is BU dose-dependent; and 3) BU reduces the proliferative potential of hematopoietic progenitor cells thus conferring a competitive advantage to the non-BU-treated postnatal donor cells. This study confirms the potential of IUHCT for facilitation of minimally toxic postnatal regimens to achieve therapeutic levels of allogeneic engraftment.

Administration of granulocyte colony stimulating factor after liver transplantation leads to an increased incidence and severity of ischemic biliary lesions in the rat model

Recently it has been reported that granulocyte colony stimulating factor (G-CSF) can induce hypercoagulability in healthy bone marrow donors. It is conceivable that the induction of a prothrombotic state in a recipient of an organ graft with already impaired perfusion might cause further deterioration in the transplanted organ. This study evaluated whether G-CSF treatment worsens liver perfusion following liver transplantation in the rat model. A non-arterialized rat liver transplantation model was employed to evaluate the effect of G-CSF treatment on the liver in a syngeneic and allogeneic strain combination. Study outcomes included survival time and liver damage as investigated by liver enzymes and liver histology. Observation times were 1 d, 1 wk and 12 wk. Rats treated with G-CSF had increased incidence and severity of biliary damage following liver transplantation. In these animals, hepatocellular necrosis was accentuated in the centrilobular region. These lesions are indicative of impaired perfusion in G-CSF treated animals. G-CSF should be used with caution in recipients of liver transplantation, as treatment might enhance preexisting, undetected perfusion problems and ultimately lead to ischemia induced biliary complications.

Bacterial translocation in acute rejection after small bowel transplantation in rats

Acute rejection after small bowel transplantation (SBTx) may facilitate bacterial translocation (BT) and subsequent changes in the liver, spleen, and lungs. This study investigated whether BT occurs after acute rejection and whether this is followed by changes in the structure of the intestine and the phagocytic organs interposed between the gut and the general circulation. Orthotopic SBTx was performed in allogeneic (ALLO) rat-strain combinations (BN-Wistar, n=5). For comparison we used syngeneic SBTx (SYN) (BN-BN, n=6) controls. Animals were sacrificed on postoperative day 7. Mesenteric lymph nodes and portal and caval blood were cultured for aerobes and anaerobes. Escherichia coli beta-galactosidase DNA was assessed by polymerase chain reaction in the blood samples. Intestine, liver, spleen, and lung protein and DNA contents were measured. Histologic changes were graded according to standard criteria of acute rejection. For comparisons we used chi(2) and nonparametric Mann-Whitney test with a threshold of significance of p<0.05. ALLO rats lost more weight after SBTx than SYN rats (-13.02+/-4.39% vs. -8.04+/-5.08% of preoperative weight), although the difference was not significant (ns). A variable degree of graft rejection was histologically demonstrated in all ALLO rats, and DNA/protein content in the graft was significantly higher in this group (0.245+/-0.85 vs. 0.134+/-0.21, p<0.05). Gram-negative enteric bacteria were found in 4/5 ALLO and 4/6 SYN rats (ns), and aerobic Gram-positive bacteria in 2/5 and 3/6 (ns), respectively. Anaerobic growth occurred in mesenteric lymph nodes in one ALLO rat and in the bloodstream in another one. E. coli DNA was isolated in none of the ALLO but in two SYN rats (ns). BT was frequent after SBTx in both syngeneic and allogeneic strain combinations. Contrary to our expectations, BT after SBTx was not higher in ALLO group rats. However, anaerobic germs were isolated only in this group.