Mixed Hematopoietic Chimeras Research Articles

Cutaneous leukocyte lineages in tolerant large animal and immunosuppressed clinical vascularized composite allograft recipients.

Vascularized composite allografts (VCAs) can restore fully functional anatomic units in patients with limb amputations or severe facial tissue loss. However, acute rejection of the skin is frequently observed and underscores the importance of developing tolerance induction protocols. In this study, we have characterized the skin immune system in VCAs. We demonstrate infiltration of recipient leukocytes, regardless of rejection status, and in tolerant mixed hematopoietic chimeras, the co-existence of these cells with donor leukocytes in the absence of rejection. Here we characterize the dermal T cell and epidermal Langerhans cell components of the skin immune system in our porcine model of VCA tolerance, and the kinetics of cutaneous chimerism in both of these populations in VCAs transplanted to tolerant and nontolerant recipients, as well as in host skin. Furthermore, in biopsies from the first patient to receive a hand transplant in our program, we demonstrate the presence of recipient T cells in the skin of the transplanted limb in the absence of clinical or histological evidence of rejection.

INKT cell activation plus T-cell transfer establishes complete chimerism in a murine sublethal bone marrow transplant model.

Transplant tolerance induction makes it possible to preserve functional grafts for a lifetime without immunosuppressants. One powerful method is to generate mixed hematopoietic chimeras in recipients by adoptive transfer of donor-derived bone marrow cells (BMCs). In our murine transplantation model, we established a novel method for mixed chimera generation using sublethal irradiation, CD40-CD40L blockade, and invariant natural killer T-cell activation. However, numerous BMCs that are required to achieve stable chimerism makes it difficult to apply this model for human transplantation. Here, we show that donor-derived splenic T cells could contribute to not only the reduction of BMC usage but also the establishment of complete chimerism in model mice. By cotransfer of T cells together even with one-fourth of the BMCs used in our original method, the recipient mice yielded complete chimerism and could acquire donor-specific skin-allograft tolerance. The complete chimeric mice did not show any remarks of graft versus host reaction in vivo and in vitro. Inhibition of the apoptotic signal resulted in increase in host-derived CD8+ T cells and chimerism brake. These results suggest that donor-derived splenic T cells having veto activity play a role in the depletion of host-derived CD8+ T cells and the facilitation of complete chimerism.

Minor Antigen Vaccine-Sensitized DLI: In Vitro Responses Do Not Predict In Vivo Effects.

BackgroundWe reported on a pilot study of minor histocompatibility antigen vaccination using constructs expressing male-specific gene disparities of selected mouse CDNA on Y and sex determining region Y in the canine model. We performed reduced-intensity hematopoietic cell transplantation with female donors and male recipients, producing stable mixed donor-recipient hematopoietic chimeras. We then performed a vaccine series in three female transplant donors followed by donor lymphocyte infusion (DLI) into their respective mixed chimeras. One mixed chimera experienced a significant shift in the percentage of donor chimerism, but no response occurred in the other 2 recipients. We then hypothesized that inadequate donor sensitization was responsible for these results.MethodsTo test this hypothesis, we added 4 monthly booster vaccinations to 2 of the original hematopoietic cell transplantation donors, including the donor that drove the partial response, followed by a second DLI.ResultsStrong T cell responses were shown by ELISpot and confirmed by intracellular cytokine staining in both donors. A second DLI resulted in a further increase in donor chimerism in the same mixed chimera that experienced the previous increase, but no change in donor chimerism was again seen in the other recipient. Evaluation of RNA expression of the target antigens demonstrated that conversion occurred in the recipient that expressed both selected mouse CDNA on Y and sex determining region Y.ConclusionsT cell responses against Y chromosome-encoded disparities were not necessarily sufficient to drive in vivo female antimale responses. Other factors including the presence of specific haplotypes or the heterogeneous expression of the target antigen may affect T cell responses against minor histocompatibility antigens. These results warrant future vaccine studies in a larger transplant cohort using epigenetic modulation of the recipient to promote target gene expression.

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

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education.

SIRT1 deacetylates RORγt and enhances Th17 cell generation.

The balance of effector and regulatory T cell function, dependent on multiple signals and epigenetic regulators, is critical to immune self-tolerance. Dysregulation of T helper 17 (Th17) effector cells is associated with multiple autoimmune diseases, including multiple sclerosis. Here, we report that Sirtuin 1 (SIRT1), a protein deacetylase previously reported to have an antiinflammatory function, in fact promotes autoimmunity by deacetylating RORγt, the signature transcription factor of Th17 cells. SIRT1 increases RORγt transcriptional activity, enhancing Th17 cell generation and function. Both T cell-specific Sirt1 deletion and treatment with pharmacologic SIRT1 inhibitors suppress Th17 differentiation and are protective in a mouse model of multiple sclerosis. Moreover, analysis of infiltrating cell populations during disease induction in mixed hematopoietic chimeras shows a marked bias against Sirt1-deficient Th17 cells. These findings reveal an unexpected proinflammatory role of SIRT1 and, importantly, support the possible therapeutic use of SIRT1 inhibitors against autoimmunity.

SIRT1 enhances Th17 cell generation by deacetylating RORγt (P1163)

Abstract Although Sirtuin 1 (SIRT1), an NAD-dependent protein deacetylase, has emerged as a critical regulator of the cell cycle, replicative senescence, inflammation, and metabolism, its contribution to immune function remains to be fully investigated. Here we report that SIRT1 regulates the function of IL-17 producing CD4 T helper cells (Th17 cells) by modulating the activity of the Th17 master transcription factor RORγt. We found that Sirt1 interacts with RORγt and increases its transcriptional activity by deacetylating lysine residues K16 and K81. In mixed hematopoietic chimeras, cells from donors in which Sirt1 is deleted in ROR γ+ cells (SIRT1-cKO) exhibited compromised Th17 differentiation. Moreover, SIRT1-cKO mice were resistant to developing experimental autoimmune encephalomyelitis (EAE) upon MOG peptide immunization. Importantly, the SIRT1 specific inhibitor Ex-527 substantially inhibited trans-differentiation to Th17 cells in vitro, and injection of Ex-527 into mice strongly blocked EAE disease progression in vivo. These findings reveal a previously unappreciated proinflammatory role of SIRT1 in CD4 T cells, and provide significant evidence that pharmacological SIRT1 inhibitors might constitute a novel approach in the treatment of Th17-mediated autoimmune diseases such as multiple sclerosis.

Donor Bone Marrow-Derived T Cells Inhibit GVHD Induced by Donor Lymphocyte Infusion in Established Mixed Allogeneic Hematopoietic Chimeras

Delayed administration of donor lymphocyte infusion (DLI) to established mixed chimeras has been shown to achieve anti-tumor responses without graft-vs.-host disease (GVHD). Herein we show that de novo donor BM-derived T cells that are tolerant of the recipients are important in preventing GVHD in mixed chimeras receiving delayed DLI. Mixed chimeras lacking donor BM-derived T cells developed significantly more severe GVHD than those with donor BM-derived T cells after DLI, even though both groups had comparable levels of total T cells at the time of DLI. Post-DLI depletion of donor BM-derived T cells in mixed chimeras, as late as 20 days after DLI, also provoked severe GVHD. Although both CD4 and CD8 T cells contributed to the protection, the latter were significantly more effective, suggesting that inhibition of GVHD was not mainly mediated by CD4 regulatory T cells. The lack of donor BM-derived T cells was associated with markedly increased accumulation of DLI-derived alloreactive T cells in parenchymal GVHD target tissues. Thus, donor BM-derived T cells are an important factor in determining the risk of GVHD and therefore, offer a potential therapeutic target for preventing and ameliorating GVHD in the setting of delayed DLI in established mixed chimeras.

Host Environment Determines the Development of Gvhd but Not the Extent of Activation and Expansion of Gvh-Reactive CD4 T Cells

Transfer of small numbers of naïve allogeneic T cells into freshly irradiated (FI) mice leads to severe, lethal GVHD. In contrast, when large numbers of naïve donor T cells are transferred as delayed donor lymphocyte infusions (DLI) given to established mixed hematopoietic chimeras (MC), a GVH reaction limited to the lymphohematopoietic system (LHGVHR) occurs and does not lead to GVHD. A similar phenomenon can occur when DLI are given to patients in whom mixed chimerism is established with non-myeloablative conditioning. LHGVHR without GVHD across MHC barriers provides an approach to separating GVL and GVHD and an understanding of this phenomenon will be essential to optimize its clinical application. We have previously shown that directly GVH-alloreactive CD8 T cells injected into FI mice or in delayed DLI given to MC show rapid proliferation and accumulation in lymphoid organs and similar modification of homing receptors, but with different kinetics in each type of recipient. However, GVH-reactive CD8 T cells traffic to and accumulate in the epithelial GVHD target tissues only in FI mice and not in MC. In order to determine the influence of the host environment on CD4-mediated GVH responses, we compared the expansion and activation of GVH-reactive CD4 T cells specific for recipient minor (mHA) and MHC antigens in the presence of a full haplotype mismatch (B6→B6D2F1) in MC and FI mice. With a non-myeloablative protocol, we established B6→B6D2F1 MC in transgenic B6D2F1 recipients expressing membrane-bound chicken ovalbumin (OVA-B6D2F1). Six weeks later, a group of MC and a group of OVA-B6D2F1 mice lethally irradiated on the same day received 106 OTII CD4 T cells (specific for OVA peptide 323-339/IAb, representing a mHA) along with 107 polyclonal B6 splenocytes to preserve the strong polyclonal response. FI mice also received B6 bone marrow cells (BMC) to prevent death from aplasia. Activation of OTII CD4 T cells was similar in both MC and FI mice, with no statistically significant differences in the increase in CD44 (average median fluorescence intensity [MFI] 245 and 235, respectively), %CD69+ (average 52% in MC and 36% in FI) and %CD25+ cells (average 18% in MC and 20% in FI) and downregulation of CD62L (average 18% positive in MC and 10% in FI). These changes occurred as early as day 3 in FI mice and were delayed to day 6 in MC. On day 6, OTII CD4 T cells accumulated to a 5-fold greater extent in spleens of MC compared to FI mice (mean±SD of 1.47±0.12 × 106in MC vs 0.30±0.03×106in FI; p<0.001). FI mice died of GVHD by Day 9, whereas GVHD did not develop in MC. Similar studies were performed with directly class II MHC alloreactive CD4 T cells. Six weeks after establishing B6→B6D2F1 MC, a DLI consisting of 106 OTII CD4 T cells (non-specific T cells in this model because they do not recognize any recipient antigen), 106 4C TCR transgenic CD4 T cells (directly alloreactive for recipient IAd class-II antigen) and 107 polyclonal B6 spleen cells was injected into MC and FI B6D2F1 mice that also received B6 BMC. On Days 6 and 9, greater accumulation of 4C CD4 T cells compared to OTII CD4 T cells (p<0.05) was detected in the spleens of both groups. Expression of activation markers on 4C cells occurred in both FI mice and MC, with similar MFI for CD44 (mean 120 in FI mice and 198 in MC; NS), similar %CD69+ (average 54.9% positive in FI and 60.2% in MC; NS) and %CD25+ cells (average 19.6% positive in FI mice and 11.2% in MC; NS). Downregulation of CD62L was also similar but slightly delayed in MC. By day 16, FI mice developed significant weight loss and GVHD, whereas MC did not develop GVHD. These data confirm the influence of the host environment in the development of GVHD and show that, similar to GVH-reactive CD8 T cells, marked antigen-driven activation and proliferation of GVH-reactive CD4 T cells occurs in both FI mice and MC, although with different kinetics.

Proteolytic shedding by ADAM 17 (TACE) functions as a gatekeeper for leukocyte emigration to inflammatory sites

Leukocyte recruitment to inflammatory sites involves sequential adhesive interactions associated with dynamic changes in expression and function of cell surface proteins. We hypothesize that proteolysis of cell surface proteins allows instantaneous uncoupling of adhesive and signaling events, and thus is a key regulator of the inflammatory response. To test this hypothesis, the sterile irritant thioglycollate was injected into the peritoneum of hematopoietic mouse chimeras that lack the sheddase ADAM17 in circulating cells. Thioglycollate injection first initiates neutrophil emigration into the peritoneal cavity, and at 4 hr Ly6G+ neutrophils are increased by 75% in null chimeras. ADAM17‐/‐ chimeras also display a 50% decrease in rolling velocity and a 2‐fold increase in adherent cells visualized by intravital microscopy of the cremaster muscle. In contrast with accelerated neutrophil emigration, monocyte (CD11b+/Ly6G‐) infiltration in null chimeras is reduced by 25% at 48–72 hr, a time of maximal monocyte accumulation. Studies of mixed hematopoietic chimeras (50% wild type, 50% null) further implicate ADAM17 in promoting macrophage exit from the peritoneum. Together these data reveal that ADAM17's function in wild type mice is critical for restriction of neutrophil infiltration, and promotion of monocyte emigration and resolution of the inflammatory response. (Supported by HL67267 &amp; HL18645)

Lymphopenia Promotes the Development of Graft-Versus-Host Disease upon Donor Leukocyte Infusion.

In murine models of hematopoeitic cell transplantation (HCT), a graft-versus-leukemia (GVL) effect can be effectively separated from graft-versus-host disease (GVHD) by giving delayed donor-leukocyte infusions (DLI) to established mixed hematopoietic chimeras (MC) (Mapara, Transplantation 2003). In these models, the absence of conditioning-induced inflammation at the time of DLI prevents trafficking to GVHD target tissues, limiting the GVH response to the lymphohematopoeitic system (Chakraverty, J Exp Med 2006). While clinical trials have shown that the same outcome is possible in humans, some patients develop severe GVHD following delayed DLI (Dey, Blood Biol Marrow Transplant 2003). In contrast to mice, who recover T cell numbers quickly after HCT through de novo thymopoiesis, humans remain lymphopenic for many months. The current study investigates the role of recipient lymphopenia in the development of GVHD following delayed DLI to MC. Allogeneic (B10.A) or syngeneic (B6) lymphocytes (30 × 106 whole splenocytes) and bone marrow cells (10 × 106) were administered to unconditioned RAG–1 knockout (KO) B6 recipients or to established MC, and animals were followed for development of GVHD. By day 7 after injection, the RAG KO recipients of allogeneic lymphocytes (allogeneic group) had lost significantly more weight (p<0.001) and had significantly higher clinical GVHD scores (p<0.0001) than RAG KO recipients of syngeneic lymphocytes (syngeneic group) or MC recipients of allogeneic lymphocytes (MC group). Histologic evaluation on day 7 showed significantly increased GVHD in liver and colon of the allogeneic group. Chimerism analysis on day 7 showed the presence of an ongoing lymphohematopoietic GVH response (LGVHR) in both the allogeneic and MC groups, though the response was slower in the MC group. When followed over time, clinical GVHD in the allogeneic group resolved by day 56, weight differences disappeared by day 65, but mild histologic GVHD persisted as late as day 239. Importantly, the same dose of allogeneic T cells given to freshly conditioned animals results in rapid, lethal GVHD, whereas these unconditioned, lymphopenic animals developed only a relatively mild, largely self-limited GVHD. These results demonstrate that in the absence of conditioning-induced inflammation, lymphopenic recipients of allogeneic T cells do develop mild GVHD, consistent with the possibility that recipient lymphopenia contributes to the GVHD seen in human MC recipients of DLI. The mechanisms by which lymphopenia promotes GVHD in non-conditioned MC recipients of DLI require further investigation, but may include the absence of regulatory T cells, the potential for lymphopenia-induced proliferation of T cells, and the presence of infection-induced inflammation.

Different Mechanisms Control Peripheral and Central Tolerance in Hematopoietic Chimeric Mice

Regulatory T cells (Treg) are important in peripheral tolerance, but their role in establishing and maintaining hematopoietic mixed chimerism and generating central tolerance is unclear. We now show that costimulation blockade using a donor-specific transfusion and anti-CD154 antibody applied to mice given bone marrow and simultaneously transplanted with skin allografts leads to hematopoietic chimerism and permanent skin allograft survival. Chimeric mice bearing intact skin allografts fail to generate effector/memory T cells against allogeneic targets as shown by the absence of IFNgamma-producing CD44(high)CD8+ T cells and in vivo cytotoxicity. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody prior to costimulation blockade prevents chimerism, shortens skin allograft survival and leads to generation of effector/memory cytotoxic T cells. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody two months after transplantation leads to loss of skin allografts even though mice remain chimeric and exhibit little in vivo cytotoxicity. In contrast, chimerism is lost, but skin allografts survive following naïve T-cell injection. We conclude that hematopoietic chimerism and peripheral tolerance may be maintained by different mechanisms in mixed hematopoietic chimeras.

Ex Vivo Generated Cytotoxic T Cells (CTL) for Adoptive Immunotherapy across MHC Barriers Retain Anti-Leukemic Capacity (GVL) without Eliciting Graft Versus Host Disease (GVHD): Crucial Role of Recipient-Derived Antigen Presenting Cells (APC).

BACKGROUND: While recipient-derived APC have proven to be crucial in mediating GVL after allogeneic hematopoietic stem cell transplantation (HCT), this desired effect seemed to be inevitably linked to the risk of developing severe GVHD. It has been elegantly shown in murine models that delayed donor lymphocyte infusions (DLI) into mixed rather than full hematopoietic chimeras produce dramatically improved GVL effects and virtually no GVHD. However, avoidance of GVHD has been more difficult when this intelligent concept was transferred into the clinical setting. Here we demonstrate that donor-derived T cells of MHC-mismatched origin retain strong anti-leukemic activity and lose nonspecific alloreactivity upon specific in vitro priming and consecutive short term expansion.METHODS: We chose a fully MHC-mismatched allogeneic bone marrow transplantation model using B10.A mice (H-2a) as donors and C57BL/6 (H-2b) mice as recipients to produce either full (controls) or stable mixed hematopoietic chimeras. After full hematopoietic recovery mice were challenged with a lethal dose of a C57BL/6-derived leukemic cell line (C1498) and consecutively injected with either naive DLI, ex vivo generated CTL or PBS only. CTL were generated by specifically priming lymphocytes of MHC-mismatched donor origin on bone marrow-derived dendritic cells (DC) (either donor- or recipient-derived) that had been pulsed with recipient-derived leukemia cell lysates. This was followed by expansion using αCD3/αCD28 coated microspheres.RESULTS: We show thatex vivo generated CTL of allogeneic origin can mediate anti-leukemic effects only when primed on recipient-derived DC that had been pulsed with leukemia cell lysates (CTL versus controls, p < 0.05 ). In contrast, loading recipient-derived leukemia lysates on donor-derived DC (T cells and DC syngeneic to each other) abrogates the anti-leukemia effect of adoptively transferred CTL in vitro and in vivo.CTL exhibit a strong proliferative response in vitro when stimulated with irradiated leukemic cells of allogeneic origin (C1498), however, this response is significantly weaker (3-fold) when compared to naive DLI. Remarkably, the nonspecific proliferative response (alloreactivity) of CTL is completely abolished when irradiated C57BL/6 splenocytes are used as stimulators.CTL maintain their responsiveness to leukemia in vivo as demonstrated by cure rates from leukemia in up to 40% of mice. Importantly, upon adoptive transfer into leukemia bearing mice after MHC-mismatched HCT, CTL lose their unspecific alloreactivity in vivo as well as proven on microscopic sections of GVH target organs.Anti-leukemia effects of CTL across MHC-barriers are comparable to naive DLI in leukemia bearing mice (40% versus 50% cure rate).CTL are able to convert mixed hematopoietic chimerism into full hematopoietic chimerism (30% donor →100% donor). In contrast, mixed chimerism remains unchanged when CTL are transferred that had been primed ex vivo on donor-derived DC. This data is encouraging in that it provides further evidence that strong GVL effects can be obtained across MHC barriers without eliciting severe GVHD.

Peripheral CD4 +CD25 + regulatory T cells (T reg) block alloreactive host anti-donor T cells in canine mixed hematopoietic chimeras

CD4+CD25+ Treg cells may be important immune regulators for the maintenance of immune tolerance after allogeneic hematopoietic cell transplantation (HCT). In the well-established canine model of nonmyeloablative HCT, stable mixed donor/host hematopoietic chimerism persists for the lifespan of the recipient following 2 Gray total body irradiation, dog leukocyte antigen (DLA)-identical bone marrow transplantation and combined postgrafting immunosuppression with mycophenolate mofetil and cyclosporine for 4 and 5 weeks, respectively. We hypothesized that CD4+CD25+ Treg are an important component of the active cellular mechanism of peripheral tolerance that maintains stable mixed chimerism in this HCT model. Previously, we showed that depletion of CD4+CD25+ Treg cells from mixed lymphocyte culture (MLC) significantly increased allo-specific proliferation in the DLA-mismatched setting. In the current study, we asked if depletion of CD4+CD25+ Treg from peripheral blood mononuclear cells (PBMC) obtained from dogs with stable mixed chimerism 1 to 4 years after nonmyeloablative HCT could generate minor histocompatibility antigen (mHAg)-specific, host anti-donor cytotoxic T lymphocytes (CTL). Responder PBMC from mixed chimeric dogs were cultured in a modified MLC with irradiated, CD34+ derived dendritic cells (DC) from the respective DLA-identical HCT donor at a 10:1 responder: stimulator ratio. On day 4 of MLC, CD4+CD25+ T cells were depleted by either immunomagnetic selection (Miltenyi) using the anti-CD25 monoclonal antibody (ACT-1) or the addition of 10−11M denileukin diftitox (DAB389IL-2, Ontak) for 18 hours. DAB389IL-2 is a fusion protein consisting of the ADP-ribosyltransferase domain of diphtheria toxin and IL-2. By flow cytometry analysis, depletion of CD4+CD25bright+ was 60%–66% with immunomagnetic selection and 70%–77% with DAB389IL-2. On day 5, culture media was changed to remove residual DAB389IL-2 in the respective flasks. A secondary MLC was established with CD4+CD25+ depleted responder cells and irradiated DC from the respective DLA-identical HCT donor. Peak proliferation of responder cells measured by 3H-thymidine incorporation was on day 4 of secondary MLC. There was a 54%–95% increase in proliferation of cells depleted of CD4+CD25+ T cells with immunomagnetic selection and a 121%–133% increase in proliferation of DAB389IL-2 treated cells compared with non-depleted responder cells. We evaluated mHAg-specific cytotoxic activity of the CD4+CD25+ depleted responder T cells by 51Cr release assay following 3rd MLC stimulation with respective DC. At a 10:1 effector: target ratio, there was 74%–82% specific lysis of the respective HCT donor cells by the CD4+CD25+ depleted responder T cells, while the non-depleted control responder cells had 4%–6% specific lysis. PCR analysis with informative microsatellite markers confirmed the alloreactive CTL were of host origin. In summary, depletion of CD4+CD25+ Treg from PBMC of mixed chimeras permitted the emergence of host anti-donor mHAg-specific CTL. These data suggest that mHAg-specific host anti-donor T cells persist in the peripheral blood of stable mixed chimeric dogs and that peripheral CD4+CD25+ Treg prevent alloreactive immune responses and maintain mixed chimerism. These results support the hypothesis that mixed hematopoietic chimerism established without T cell depletion relies on peripheral immune tolerance mechanisms.

Sequential Blockade and Engagement of Co-Stimulatory Pathways: A Potential Strategy for Amplifying Graft-Versus-Leukemia Responses without GVHD.

One potential approach for the effective disengagement of graft-versus-leukemia (GVL) effects from graft-versus-host disease (GVHD) following BMT is the use of nonmyeloablative conditioning as a platform for the adoptive transfer of donor T cells. In pre-clinical models, donor CD8+ T cells can induce powerful responses against tumors of host origin, but the effect lacks durability such that a re-challenge with tumor inevitability leads to tumor progression and death. This deficit is associated with the failure of functional CD8+ effector/memory T cells (TE/M) to survive long-term post-DLI. To examine the fate of GVH-reactive CD8+ T cells following DLI, we established mixed hematopoietic chimeras (MC) in a parent →F1 model using a nonmyeloablative protocol that incorporates co-stimulatory molecule blockade. B6D2F1 mice received 3Gy TBI and intra-peritoneal injections of anti-CD154 and anti-CD8 mAb on day 0 followed by infusion of 2 x 107 C57BL/6 bone marrow cells. 10 weeks later, when mAb had cleared from the circulation, MC received DLI that included CD8+ T cells from 2C transgenic mice that bear TCR specific for recipient class I MHC Ld. Using a clonotypic marker to monitor the response, we observed expansion of 2C CD8+ cells, peaking in the spleen on day 7 and then rapidly declining such that 2C CD8+ T cells were <0.1% of splenocytes by day 60. The decline in GVH-reactive T cells was associated with marked apoptosis and a sustained reduction in the expression of IL-7Rα. By day 60, no CTL activity against host cells was detectable. We reasoned that strategies that augment the survival of GVH TE/M might enhance the durability of the GVL response and, in the absence of tissue inflammation induced by conditioning, might not lead to GVHD. Co-stimulation through the tumor necrosis family receptor, OX40, which is expressed on activated T cells, is anti-apoptotic and enhances recruitment of TE/M to the memory pool. Following DLI, OX40 expression on 2C CD8+ T cells peaked on day 7 with somewhat earlier and sustained expression on DLI-derived CD4+ T cells. Since OX40 expression was specific for GVH-reactive T cells, we examined the effect of giving agonistic anti-OX40 antibody on day +5 following DLI. This was associated with rapid and complete conversion to full donor chimerism by day +14, whereas DLI + control antibody recipients had only partially converted by day +28. By day 60 post-DLI, anti-host CTL activity was clearly detectable in anti-OX40 recipients but not in controls. No clinical evidence of GVHD was observed, although histological examination revealed transient mild lymphocytic infiltration of the lamina propria on day +13, which resolved completely by day +18. In further experiments, anti-OX40 administration was associated with marked increases in the numbers of 2C CD8+ T cells in spleen, lymph node and bone marrow following DLI. Furthermore, effector differentiation, as assayed by intracellular expression of interferon-γ by 2C CD8+ T cells, was increased in recipients of anti-OX40 antibody. Of note, we observed a complete inhibition IL-7Rα down-regulation that is normally observed on activated CD8+ T cells following DLI. We conclude that OX40 co-stimulation following delayed DLI to established MC represents a potential means to enhance the magnitude and duration of a GVH reaction without the induction of significant GVHD.

Adoptive Transfer of T Cell Help Breaks Tolerance in Endogenous, Tumor-Specific CD8+ T Cells Responding to a Growing Tumor.

Patients who reject allogeneic blood or marrow grafts after non-myeloablative conditioning sometimes develop delayed or sustained clinical disease responses, raising the possibility that a transient graft-versus-host reaction rekindles dormant anti-tumor immunity by recipient T cells. More specifically, we postulate that endogenous tumor-specific CD8+ T cells are not irreversibly tolerant of a growing tumor, and can be activated to effector function by tumor antigen recognition in the presence of adoptively transferred CD4+ T cell help. We have tested this hypothesis by examining the response of influenza hemagglutinin (HA)-specific “endogenous” CD8+ T cells in HA-tumor-bearing mice treated with cyclophosphamide (Cy) and adoptive transfer of allogeneic or tumor-specific CD4+ T cells, with or without concomitant tumor vaccine administration. In these models, this combination therapy induced the clonal expansion of endogenous, tumor-specific CD8+ T cells, their secretion of interferon gamma, and in vivo cytolytic activity against HA-expressing cells. In separate experiments, adoptively transferred, major histocompatibility complex-mismatched CD4+ T cells induced the activation of recipient, CD11c+ dendritic cells, as demonstrated by enhanced expression of the costimulatory molecules CD80 and CD86. The administration of Cy followed by infusion of allogeneic T cells, with or without tumor vaccine administration, also induced regression of B cell leukemia/lymphoma or established prostate cancer. These results highlight the potential cooperation of donor CD4+ T cells and recipient CD8+ T cells in transient or stable mixed hematopoietic chimeras, and open new avenues for the immunotherapy of cancer in the clinic.

ICSBP Gene Deficient Mice Are Highly Susceptible to the Development of Graft-versus-Host Disease (GVHD) Following Administration of Delayed Donor Lymphocyte Infusions (DLI).

Delayed administration of DLI to mixed hematopoietic chimeras following allogeneic bone marrow transplantation (alloBMT) leads to conversion from mixed to full donor chimerism without GVHD. However, in the clinical setting development of GVHD is still the most common complication. Donor T cells are the main mediators of GVHD in alloBMT. However, the precise role of host dendritic cells (DC) and their different subsets needs still to be elucidated. CD8a+ DCs seem to have a regulatory function and to reduce experimental GVHD. Recently, it has been shown that the ICSBP (Interferon Consensus Sequence Binding Protein) gene is essential for the development and activation of CD8a+ DCs. ICSBP-gene deficient mice (ICSBP KO) are characterized by several immune defects and systemic expansion of myeloid cells mimicking human CML and may be therefore an attractive in vivo model to evaluate the role of DCs in alloBMT. The aim of the study was to evaluate the 1. feasibility of inducing mixed hematopoietic chimerism following nonmyeloablative conditioning in ICSBP KO mice using fully MHC-mismatched BMT (Balb/c [H2Dd] to B6 x 129 [H2b]), and 2. susceptibility of ICSBP KO mice to the development of GVHD following administration of DLI. Nonmyeloablative conditioning consisted of in-vivo T cell depletion (anti-CD4-[GK1.5], anti-CD8-[2,43] mAb, d-5), fludarabine (30 mg/kg, d-4 to d-2), cyclophosphamid (200 mg/kg, d-1) and 3 Gy TBI (d0) followed by i.v. injection of 1,5 x 10E7 Balb/c bone marrow cells. Stable mixed chimerism was observed in ICSBP wildtype (wt) mice through week 35 post-BMT without signs of clinical GVHD. In contrast, we observed increasing donor CD4 and CD8 T cell chimerism over time in ICSBP KO mice suggesting a lack of donor T cell tolerance. Furthermore, chimerism was higher in granulocyte, B cells and monocyte compared to ICSBP wt mice. In a second experiment we were able to observe spontaneous conversion to full donor hematopoietic chimerism in 2 of 9 mice after nonmyeloablative BMT. In two separate experiments ICSBP wt or KO mice with mixed chimerism received (DLI) on day 35 post BMT. After injection of DLI all wt mice (n = 10) showed a rapid conversion to full donor chimerism and remained healthy until the end of the observation period without signs of GVHD. Despite myeloid hyperproliferation all ICSBP KO mice showed conversion to full donor chimerism. Furthermore, in ICSBP KO mice conversion to full donor chimerism was associated with development of severe GVHD (n = 9). In vitro studies using mixed lymphocyte reactions showed that splenic stimulator cells from ICSBP KO mice had a higher stimulatory capacity compared to wt stimulators (SI 1,5 versus 2,3). Using the same mixed lymphocyte culture (MLC) conditions, supernatants collected from BALB/c responders with ICSBP KO stimulators had higher levels of TNF-alpha and IFN-g compared to MLC using ICSBP wt stimulators. In conclusion, following alloBMT a defective development of host-specific tolerance and high susceptibility for GVHD in ICSBP KO mice is suggested. Further studies are warranted to delineate the precise underlying mechanism by addressing the potential contribution of defective regulatory host T cells and the lack of host CD8a+ DCs in ICSBP KO mice.

FLT3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease.

Graft-versus-host (GVH) reactions contribute to stable engraftment of allogeneic hematopoietic stem cell transplants. It was hypothesized that the in vivo expansion of recipient dendritic cells (DC) with the administration of ligand for Flt3 (FL) could promote allogeneic engraftment after reduced-intensity conditioning by enhancing the GVH effect. FL was first administered to three nonirradiated healthy dogs for 13 days at a dosage of 100 microg/kg/day. Next, nine dogs received 4.5 Gy total-body irradiation (TBI) and unmodified marrow grafts from dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. FL was administered to the recipients at a dosage of 100 microg/kg/day from day -7 until day +5. In normal dogs, FL produced significant increases in monocytes (CD14+) and neutrophils in the peripheral blood, a marked increase in CD1c+ cells with DC-type morphology in lymph nodes, and increased alloreactivity of third-party responders to peripheral blood mononuclear cells in mixed lymphocyte reactions (P<0.001). Sustained engraftment was observed in eight of nine (89%) FL-treated dogs compared with 14 of 37 (38%) controls (P=0.02, logistic regression). All engrafted FL-treated dogs became stable complete (n=2) or mixed (n=6) hematopoietic chimeras without significant graft-versus-host disease (GVHD). Recipient chimeric dogs (n=4) were tolerant to skin transplants from their marrow donors but rejected skin grafts from unrelated dogs within 7 to 9 days (median, 8 days). In this study, the authors showed that FL administered to recipients promotes stable engraftment of allogeneic marrow from DLA-identical littermates after 4.5 Gy TBI without significant GVHD.

Minor histocompatibility antigen-specific cytotoxic T lymphocytes generated with dendritic cells from DLA-identical littermates.

Donor cytotoxic T lymphocytes (CTL) specific for minor histocompatibility antigens (mHA) mediate the graft-versus-host effect whereas host mHA-specific CTL mediate graft rejection in the setting of major histocompatibility complex identical allogeneic hematopoietic stem cell transplantation. Development of a large animal model from which mHA-specific CTL can be isolated would accelerate translation in clinical studies to improve control of the graft-versus-host effect as well as prevention of graft rejection in sensitized hosts. The aims of the current study were to isolate mHA-specific CTL from dog leukocyte antigen-identical littermate nonsensitized recipients before transplantation, from stable mixed hematopoietic chimeras, and from dogs sensitized to mHA after graft rejection. Donor dendritic cells (DCs) were cultured from bone marrow-derived CD34(+) cells and were used to stimulate recipient T lymphocytes on days 1, 10, and 20 of CTL culture. We reliably generated and expanded mHA-specific CTL ex vivo from sensitized dogs that were given a donor-specific blood transfusion to boost immune recall after graft rejection after a nonmyeloablative transplantation. The mHA-specific cytotoxicity measured by (51)Cr release assay was enriched from less than 5% in the starting population of sensitized peripheral blood mononuclear cells to a median of 63% after 4 weeks in CTL culture. The expanded mHA-specific CTLs were not tissue-specific: hematopoietic cells, fibroblast, and stromal cell lines were lysed in an mHA-specific manner. Allogeneic DCs, but not peripheral blood mononuclear cells, were necessary for stimulating ex vivo expansion of mHA-specific CTL. We were unable to generate mHA-specific CTL from nonsensitized dogs before transplantation, from previously sensitized dogs but without recent recall immunization, or from stable mixed hematopoietic chimeras. We conclude that after recent in vivo sensitization, large-scale ex vivo expansion of mHA-specific CTL was feasible using allogeneic DCs.

Adoptive immunotherapy against kidney targets in dog-leukocyte antigen-identical mixed hematopoietic canine chimeras.

Stable mixed-donor-host-hematopoietic chimerism can serve as a platform for adoptive immunotherapy. Infusions of donor lymphocytes (DLI) sensitized against hematopoietic cells converted mixed hematopoietic into full-donor chimerism in dog-leukocyte antigen (DLA)-identical littermates. Whether sensitization against tissue of solid organs leads to organ-specific immunity that can be transferred by DLI was unknown and was investigated in these experiments using the kidney as target. DLA-identical recipients with established stable mixed-donor-host-hematopoietic chimerism were used. In five pairs, hematopoietic stem-cell transplant (HSCT) donors were sensitized by kidney transplantation from the respective chimeras. In a second group, five HSCT donors received vaccinations that were generated from kidney cells of the respective mixed chimeras. Twenty-eight days after sensitization, DLI were administered to the mixed-hematopoietic chimeras. All HSCT donors rejected their kidney grafts from their mixed-chimeric recipients within 22 to 45 days. DLI caused no sustained graft-versus-kidney effects in the mixed-chimeric recipients. However, DLI donors sensitized by kidney transplantation converted 4 of 5 mixed chimeras into virtually complete (>95%) donor-type chimeras, compared with 1 of 5 mixed chimeras given DLI by vaccination from sensitized donors. Although DLA-identical kidney grafts from mixed-hematopoietic chimeras were readily rejected by their HSCT donors, subsequent transfusions of sensitized-donor lymphocytes into mixed chimeras converted mixed to all-donor chimerism but failed to induce graft-versus-kidney effects. Vaccination strategies in lieu of kidney grafts failed to convert mixed chimerism.

Survival of small bowel transplants in canine mixed hematopoietic chimeras: preliminary results

Survival of small bowel transplants in canine mixed hematopoietic chimeras: preliminary results