Minor Histocompatibility Barriers Research Articles

Increased Activity in the T Cell Effector Phase and Enhanced T Cell Repertoire Target-Tissue Stability Distinguish Alloreactivity Across Major Versus Minor Histocompatibility Barriers

Graft-versus-host disease (GVHD) occurs when transplanted donors' T cells recognized the recipients' antigens and damaged host tissues and cells, particularly the skin, gut and liver in the acute setting. Although it is well known GVHD is more aggressive and manifests more quickly across major versus minor histocompatibility barrier, little is known comparatively about the donor T cell activation and T cell repertoire changes.To investigate temporal and spatial events of GHVD development, side-by-side transplants were conducted into major and minor-mismatched murine recipients (Balb.c and Balb.b) using hematopoietic cells from the same donor strain(B6). In both models, T cells home to nodal sites by day 3, proliferate, and exit to GVHD target tissues by day 6. Additionally, expression of homing and activation markers was equivalent for all markers examined on day 3. However, tissue migration and proliferation were reduced in the minor model. By day 6, minor-mismatched T cells had increased CD62L retention and reduced P-selectin and CD44 expression. We also found fewer MHC-matched T cells producing IFN-g and TNF-a.Our data show that early events of donor T cell activation are similar in both models, suggesting that the delayed onset and attenuated disease GVHD seen across minor barriers arise from temporal differences in the effector phase, rather than the initiation phase, of GVHD.To further understand the differences across major versus minor histocompatibility barriers on the T cell repertoire and patterns of T cell alloreactivity, we collected a sample of T cells from donor mice used for transplantation, and also sampled gut tissues from syngeneic, major and minor- mismatched transplanted mice on day 9, 9 and 30 respectively at times when the allogeneic groups of mice showed severe GVHD symptoms.To reduce the background of high percentage of TCR pseudogenes in mouse genome, 5'RACE starting from RNA samples and deep sequencing of TCRa and TCRb were applied to investigate whether TCR repertoire of the major and minor-mismatched mice were skewed with clonal expansion, and how among the major and minor-mismatched mice. While we hypothesized that the major MHC mismatched group would have lower diversity because of expanded clones associated with the GVHD, the shannon index of TCRa indicated gut TCR repertoire of major and minor mismatched mice have greater diversity than syngeneic group(P<0.05). We did not see differences in the shannon index of diversity on examination of the TCRb repertoire.Contrary to our expectation, that the TCR repertoire of major mismatch would be skewed towards representation of a few highly expanded clones in the gut, we in fact saw that the TCR repertoire in these mice appeared less skewed. The TCR repertoire of the gut was more highly related among individuals in the major mismatch groups than among or between the other groups. This strongly suggests a more reproducible repertoire structure.To examine if certain T cell clones were more likely to appear reproducibly in the major and/or minor mismatch setting, we compared the shared clones across the major-mismatched transplanted mice. Bhattacharyya coefficients showed that the major-mismatched mice shared more clones with the minor-mismatched group than the syngeneic groups(P<0.05). A total of eight shared TCRa clonotypes among major-mismatched mice are also detected. The common CDR3 clonotypes might be associated with GVHD. We found 7 of them are also in donor CD4 memory cells' repertoire; and one is present in both donor's CD4 and CD8 memory cells. One of the complementarity determining regions sequences is "AASYQGGRALI" which is also in common among minor-mismatched mice.We also measured the repertoire similarity between donor T cells and the gut TCR repertoires of three groups. Bhattacharyya coefficients of TCRa and TCRb between donor T cells and major-mismatched gut repertoire is greater than donor T cells with either syngeneic or minor-mismatched repertoire(P<0.05), which suggest that the major-mismatched mice has more T cells homing to gut which might be associated with GVHD.Our sequencing data showed that major and minor-mismatched transplantation might not cause the clonal expansion in the gut TCR repertoire, but their repertoire patterns are different from the syngeneic groups, which might be associated with T cell alloreactivity and GVHD. DisclosuresNo relevant conflicts of interest to declare.

IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease after Bone Marrow Transplantation

Regulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4+CD25+Foxp3+ natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17–producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17–deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation.

Recombinant CD95-Fc (APG101) prevents graft-versus-host disease in mice without disabling antitumor cytotoxicity and T-cell functions

AbstractGraft-versus-host disease (GVHD) induced by transplant-derived T cells represents a major complication after allogeneic bone marrow transplantation (BMT). However, these T cells support engraftment, early T-cell immunity, and mediate the graft-versus-tumor (GVT) effect. Cytotoxic effector functions by transplanted T cells are predominantly mediated by the perforin/granzyme and the CD95/CD95L system. APG101, a novel recombinant human fusion protein consisting of the extracellular domain of CD95 and the Fc domain of an IgG1 antibody inhibited CD95L-induced apoptosis without interfering with T-cell function in vitro and was therefore tested for its ability to prevent GVHD in murine BMT models across minor or major histocompatibility barriers. Starting APG101 treatment either 1 day before or 6 days after transplantation effectively reduced clinical GVHD and rescued survival between 60% and 100% if GVHD was CD95L mediated. APG101 did not interfere with the GVT effect, because P815 mastocytoma and most importantly primary Bcr-Abl–transformed B-cell leukemias were completely eradicated by the alloantigen-specific T cells. Phenotype and homing of alloantigen-specific T cells or their perforin/granzyme-mediated cytotoxicity and proliferative capacity were not affected by APG101 treatment suggesting that APG101 therapy might be useful in GVHD prophylaxis without impairing T-cell function and most importantly preserving GVT activity.

Corneal Transplantation and Immune Privilege

Corneal transplants have been successfully performed in human subjects for over 100 years and enjoy an immune privilege that is unrivaled in the field of transplantation. Immune privilege is defined as the reduced incidence and tempo in the immune rejection of corneal allografts compared to other categories of organ allografts performed under the same conditions. Skin allografts transplanted across various MHC or minor histocompatibility barriers undergo rejection in approximately 100% of the hosts. By contrast, orthotopic corneal allografts experience long-term survival in 50% to >90% of the hosts, depending on the histocompatibility barriers that confront the host. The capacity of corneal allografts to evade immune rejection is attributable to multiple anatomical, physiological and immunoregulatory conditions that conspire to prevent the induction and expression of alloimmunity.

IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease Following Bone Marrow Transplantation,

Abstract 4037Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for many hematologic malignancies such as leukemia, lymphoma and myelodysplastic syndromes. A potentially fatal complication of HCT is graft-versus-host disease (GVHD), where conventional T cells of alloreactive-specificities (CD4+ and CD8+ T cells; Tcon) infiltrate and destroy target organs such as the liver, gut and skin. While immunomodulation is necessary to control the adverse GVHD reactions, an effective immune response is required for successful tumor eradication, named graft-versus-tumor effect (GVT).Following allogeneic bone marrow transplantation, our and other laboratories have shown that the transfer of highly purified regulatory T cells (Treg), a key immunoregulatory cell population involved in the maintenance of immune tolerance, can prevent lethal GVHD induced by the donor-derived Tcon in multiple models across both major and minor histocompatibility barriers. Notably, when transferred into recipient mice with established leukemia or lymphoma, Treg cells effectively suppress Tcon cell proliferation and prevent lethal GVHD, while preserving GVT activity. We have recently adapted our pre-clinical model to an ongoing clinical trial, and are currently assessing the efficacy of highly purified human Treg cells in a clinical setting. However, recent evidence suggest that, under the influence of intense inflammatory signals, the Tregsuppressive phenotype can become unstable, a phenomenon that can culminate in Tregconversion into IL-17-producing pro-inflammatory cells. IL-17 is a proinflammatory cytokine that has been reported to promote GVHD in different models for human HCT. We hypothesized that under the influence of the intense proinflammatory signals, such as those released during an ongoing alloreaction, a fraction of the transferred Treg might be redirected to the Th17 cell fate, thereby losing immunosuppressive potential and acquiring proinflammatory capacity. Our preliminary in vitro studies demonstrated that Treg cells do indeed upregulate Il17 gene expression following activation by allogeneic antigen presenting cells (APCs). We, therefore, sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model, in order to ultimately assess the possible therapeutic potential of IL-17 targeting for the enhancement of Treg-mediated GVHD suppressive ability following HCT.On the basis of our preliminary studies we anticipated that Treg cells purified from Il17 knockout (KO) donor mice would have enhanced immunomodulatory capacity compared to wild-type (WT) Treg, given their inability to produce IL-17. To monitor ongoing GVHD our laboratory has developed an imaging system that allows for the in vivo visualization of T cell homing and proliferation over time, without the necessity to sacrifice the experimental animals. Specifically, the allogeneic Tcon cells are isolated from donor mice where the gene coding for the enzyme luciferase (luc) is expressed by all hematopoietic cells. Importantly, the proliferation of the transferred luc+ Tcon can be directly assessed by the number of photons detected by the bioluminescent imaging system (BLI), which gives a measure of ongoing GVHD. Treg were purified from both WT and IL-17 KO animals and their GVHD-suppressive ability was directly compared in vivo following the transfer of luc+ Tcon. IL-17 KO Tregcells were as proficient as WT Treg in suppressing GVHD. Similarly, the GVHD-inhibitory capacity of Treg cells isolated from IL-1R KO mice, that were previously shown to be resistant to Th17 conversion, was comparable to that of WT Treg. Furthermore, upon ex vivo re-isolation of Tregthat were previously transferred into transplanted recipients, we could detect little or no IL-17 production by intracellular cytokine staining. Thus, we conclude that freshly isolated and highly purified Treg do not convert into IL-17 producing cells in our model, suggesting that IL-17 production is not a factor affecting Tregstability during ongoing GVHD, and excluding IL-17 targeting as a valuable strategy to improve Treg immunotherapy following bone marrow transplantation. Disclosures:No relevant conflicts of interest to declare.

A Novel Model of Pre-Emptive ECP Treatment Shows Significant Reduction of Gvhd-Related Mortality In Mice,

Abstract 4025Graft versus host disease (Gvhd) remains the major cause of morbidity and mortality after bone marrow transplantation (BMT). To prevent Gvhd the graft has to be rendered tolerant to avoid alloreactivity towards the host. One promising approach to induce tolerance in the graft is to administer apoptotic cells to the host. Apoptosis can be induced by extracorporeal photopheresis (ECP), a therapy based on exposure of cells to photoactivable 8-methoxypsoralen (8MPO) and UVA irradiation. The effect of ECP treatment is at least in part, due to alteration of dendritic cell (DC) maturation, leading to reduced antigen presentation and Tcell activation. Moreover, ECP treatment increases the number of regulatory Tcells (Treg), further reducing Tcell activation.In contrast to clinical practice where ECP is only applied to established Gvhd, we have developed a pre-emptive murine model with the aim of inhibiting the initiation phase of acute Gvhd and improving survival. Here we describe our model and characterization of the mechanisms of action.Apoptotic host-type splenocytes were infused into MHC matched or mismatched recipients 5 and 2 days prior to lethal irradiation and BMT. Apoptotic cells were generated by incubation with 8-MPO and UVA light (UVAR light set, Therakos). Mice were followed for Gvhd score and survival. ECP-treated mice survived longer both across major (median survival 51 versus 26 days, p<0.0001) and minor (median survival 56.5 versus 9 days, p<0.002) histocompatibility barriers. Importantly, even though ECP treatment promoted tolerance against the host it did not impact the graft versus tumor effect in a B-cell lymphoma model.To address the mechanisms of this beneficial outcome we investigated Tcell proliferation in vivo (i), ex vivo (ii) and in vitro (iii). (i) Luciferase-expressing Tcon were used to quantify Tcell proliferation in vivo by bioluminescent imaging. ECP-treated mice showed reduced CD4+Tcell proliferation during the initiation phase of Gvhd (day+4) compared to mice receiving Tcon only. (ii) Re-isolation of CFSE labeled Tcon on day+4 after BMT showed a reduction in CD4+Tcell proliferation. (iii) FACS-sorted DC incubated with apoptotic cells for 2 days in vitro showed a diminished capability to induce Tcell proliferation.To evaluate the impact of ECP treatment on the expression of homing (P-Selectin, α4β7) and activation (CD44, CD69) markers on donor Tcells we performed re-isolation experiments on day+3 and day+7. At day +3 ECP-treated groups showed a reduced expression of these markers in comparison to control groups. These studies demonstrate slower trafficking to Gvhd target organs. Furthermore, at day+7 ECP-treated groups showed increased FoxP3-expressing host Tregs in comparison to control groups whereas donor Treg were similar in both groups. Ongoing experiments will utilize a mouse model in which it is possible to selectively deplete either donor or host Tregs to examine the role of these two subpopulations of Treg in improving Gvhd outcome.In summary, ECP treatment prior to transplantation led to (i) a significant survival benefit with no impact on graft versus tumor effect; (ii) reduced CD4+ Tcell proliferation; (iii) delayed expression of homing and activation markers during the initiation phase of Gvhd and (iv) an increase in host Treg.Overall our work suggests that pre-emptive ECP of host cells prior to BMT may be a clinically relevant strategy to prevent acute Gvhd. Disclosures:No relevant conflicts of interest to declare.

Kinetics of Murine GVHD Induction Across Minor and Major Histocompatibility Barriers

Kinetics of Murine GVHD Induction Across Minor and Major Histocompatibility Barriers

Similarities In the Initial Events of Murine Gvhd Across Minor. as Compared to Major, Histocompatibility Barriers.

AbstractAbstract 3750Graft-vs-Host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation (HCT). Murine models have been critically important to define the biological mechanisms and potential pathways of intervention of GVHD prevention and treatment. Although it is well recognized that GVHD occurs in response to minor histocompatibility antigens, little is know about the kinetics of donor T cell proliferation and homing in minor mismatch models. This is in contrast to models across major histocompatibility barriers where the early development of GVHD has been more thoroughly characterized. In prior studies across major barriers, we have defined an initiation phase within the first 3 days where conventional CD4+ and CD8+ T cells (Tcon) home to secondary lymphoid tissues, proliferate and up-regulate key homing markers allowing for entry into GVHD target tissues during the effector phase (Beilhack, et al. Blood 106:1113, 2005). Since minor models are more similar to clinical HCT, it is critical to understand the timecourse of GVHD development across minor histocompatibility barriers. Since the manifestations of GVHD in recipients of minor mismatch transplants are delayed, it is possible that disease development has altered kinetics. To investigate the temporal and spatial events of donor T cell activation and homing, side-by-side transplants were conducted using T cell depleted bone marrow (TCD BM) and Tcon from donor C57BL/6 (H2b) mice into either major mismatched Balb.c (H2d), or minor mismatch Balb.b (H2b) recipients. Balb.c mice received 1×106 Tcon while Balb.b mice were given 15×106 Tcon, based on previous titration experiments. Recipient mice were regularly scored for GVHD symptoms and monitored for at least 100 days for survival. Additionally, donor Tcon proliferation and migration were monitored longitudinally using in vivo and ex vivo bioluminescent imaging (BLI) by quantitating photons emitted by luciferase (luc+) expressing donor Tcon isolated from luc+ transgenic mice. Donor Tcon were also labeled with CFSE to determine proliferation kinetics at selected timepoints. The upregulation of T cell activation and tissue specific homing markers was examined using flow cytometric analysis of donor CD4+ and CD8+ T cells re-isolated from the secondary lymphoid tissues of transplanted mice. In both models, T cells initially home to secondary nodal sites by 3 days post-transplant, with an exodus into the tissues by day 6, albeit to a lesser extent in recipients of minor mismatch transplants. Additionally, similar levels of global donor CD4+ and CD8+ T cell proliferation between the models were observed using both BLI and CFSE staining as early as 3 days after transplant (BLI, p>0.05, n=9). More noticeable reductions in minor mismatch recipients were apparent by day 6 (BLI p<0.0001, n=9). The expression profiles of several T cell activation and tissue homing markers, such as CD44, CD69, a4b7, and P- and E-selectin, were also quite similar when measured at timepoints within one week of transplant, although there was some variability in expression between tissues as well as between CD4 and CD8 T cells. However re-isolated donor T cells from recipients of minor mismatched transplants did consistently show delays in the up-regulation of CD25 and the down-regulation of CD62L, as compared to that noted in major mismatch transplant recipients. Together, these data support the conclusion that the early events of donor T cell activation in the initiation phase of GVHD, particularly spatially, are quite similar to those previously documented in major mismatch models of GVHD, reinforcing the usefulness of both models as translational research tools. More importantly, the data suggest that the delay in visible GVHD onset noted in transplants across minor barriers arises from temporal differences in the effector phase of T cell migration and proliferation, rather than delays in the initiation phase. As such, these findings support the targeting of very early events in T cell activation as the most effective method of reducing GVHD in the clinical setting. Disclosures:No relevant conflicts of interest to declare.

Common Gene Deletions and Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation.

Graft-versus-host disease (GVHD) is a serious and common complication of allogeneic hematopoetic stem cell transplantation (HSCT). GVHD results from genetic incompatibilities between donor and host. Although matching of donor and recipient human leukocyte antigens (HLA) is critical to transplant outcomes, GVHD is common even in transplants involving HLA-identical sibling donors. The additional determinants of graft compatibility are poorly understood, though a small number of minor histocompatibility antigens have been reported. The human genome has recently been found to show large-scale structural variation, including large gene deletions sufficiently common to appear as homozygous gene deletions in many patients. We investigated whether donor-recipient disparity for common gene deletion polymorphisms (homozygous gene deletion in donor but not in recipient) was associated with GVHD following allogeneic HSCT in 821 HLA-identical sibling donor-recipient pairs. Patient samples and clinical outcomes data were assembled collaboratively from institutions in Finland and North America, in strict compliance with ethical standards and IRB-approved protocols. Donors and patients were typed for the presence of six common gene deletions using real-time PCR. Donor-recipient disparities were then assessed for association with acute and chronic GVHD. Donor-recipient disparities in the expected direction (-/- in donor, +/- or +/+ in recipient) for two common gene deletions, UGT2B28 and UGT2B17, were associated with chronic GVHD (OR > 4, P=0.001; and OR=2.0, P=0.05 respectively). Donor-recipient disparity for UGT2B17 was also associated with acute GVHD (OR=2.0, P=0.05). In the cohorts assembled for this study, we also observed an expected robust association for sex mismatch with chronic GVHD (OR=1.6, p=0.001) and a non-significant trend toward association with acute GVHD (OR=1.1, p=0.30). No significant association with acute or chronic GVHD was observed for the other four common gene deletion polymorphisms (GSTT1, GSTM1, OR51A2, and LCE3C). Consistent with prior studies of minor histocompatibility antigens on the Y-chromosome, we observed evidence of serologic immunoreactivity to a nine residue peptide on UGT2B17 in an HSCT recipient with mismatch at this locus. Because disparities for UGT2B17 and UGT2B28 occur in fewer than 6% of sibling-donor transplants in populations with European ancestry, they can explain only a fraction of the genetically attributable risk of GVHD. Of note, deletion of UGT2B28 appears to be more common in individuals with African and Latin American ancestry (allele frequency of 25–35%), and therefore merits investigation as a potential contributor to transplant outcome in those populations. We conclude that variation in genome structure associates with graft-versus-host disease and merits further investigation as a cause of minor histocompatibility barriers to hematopoietic stem cell transplantation. For the Transplant Structural Genetics Consortium: David Altshuler, Shannon Chilewski, Steven McCarroll (Broad Institute of Harvard and MIT, Cambridge, MA); Joseph Antin, James Bradner, Stephanie Lee, Jerome Ritz, Robert Soiffer (Dana-Farber Cancer Institute, Boston, MA); Aarno Palotie (Finnish Genome Center, Helsinki, Finland); Jukka Partanen, Hannu Turpeinen (Finnish Red Cross, Helsinki, Finland); Tapani Ruutu, Liisa Volin (Helsinki University Central Hospital, Helsinki, Finland); David Ginsburg, David Siemeniak (University of Michigan, Ann Arbor, MI).

Studying Human Regulatory T Cells In vivo

In this issue of Clinical Cancer Research , Mutis et al. ([1][1]) use a model of xenogeneic graft-versus-host disease to assess the suppressive function of human regulatory T cells in vivo . This model is based on the transfer of human peripheral blood cells into RAG-2−/−γc−/− mice that are

Induced peripheral regulatory T cells: The family grows larger

In order to control immune reactions and to prevent autoimmunity, the immune system relies on a multitude of regulatory T cells (Treg). Most work in this field has focussed on Treg of the CD4(+) T cell subset because of the central role CD4(+) T cells play as initiators and regulators of immune responses. One discovery of particular importance was the identification of naturally occurring CD4(+)CD25(+) Treg that arise in the thymus and express the transcription factor Foxp3; however, Treg can also be induced in the periphery after immune activation. A paper in this issue of the European Journal of Immunology identifies a novel type of induced murine CD8(+) Treg that arise in TCR HY transgenic female mice after injection of MHC class I tetramers loaded with HY peptide. These Treg prevent subsequent rejection of male skin grafts suggesting that their manipulation may represent a new way of improving graft rejections against minor histocompatibility barriers.

Sequential Expression of Adhesion and Costimulatory Molecules in Graft-versus-Host Disease Target Organs after Murine Bone Marrow Transplantation across Minor Histocompatibility Antigen Barriers

Graft-versus-host disease (GVHD) is a potentially fatal complication after allogeneic bone marrow transplantation. However, few data exist thus far on the molecular signals governing leukocyte trafficking during the disease. We therefore investigated the sequential pattern of distinct adhesion, costimulatory, and apoptosis-related molecules in GVHD organs (ileum, colon, skin, and liver) after transplantation across minor histocompatibility barriers (B10.D2 --> BALB/c, both H-2d). To distinguish changes induced by the conditioning regimen from effects achieved by allogeneic cell transfer, syngeneic transplant recipients (BALB/c --> BALB/c) and irradiated nontransplanted mice were added as controls. Irradiation upregulated the expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-l, and B7-2 in ileum, as well as VCAM-1 and B7-2 in colon, on day 3 in all animals. Whereas in syngeneic mice these effects were reversed from day 9 on, allogeneic recipients showed further upregulation of VCAM-1, ICAM-1, B7-1, and B7-2 in these organs on day 22, when GVHD became clinically evident. Infiltration of CD4+ and CD8+ donor T cells was noted on day 9 in skin and liver and on day 22 in ileum and colon. Surprisingly, the expression of several other adhesion molecules, such as ICAM-2, platelet-endothelial cell adhesion molecule 1, E-selectin, and mucosal addressin cell adhesion molecule 1, did not change. Proapoptotic and antiapoptotic markers were balanced in GVHD organs with the exception of spleen, in which a preferential expression of the proapoptotic Bax could be noted. Our results indicate that irradiation-induced upregulation of VCAM-1, ICAM-1, and B7-2 provides early costimulatory signals to incoming donor T cells in the intestine, followed by a cascade of proinflammatory signals in other organs once the alloresponse is established.

Mechanism of action of glatiramer acetate in multiple sclerosis and its potential for the development of new applications.

Glatiramer acetate (GA, Copaxone, Copolymer 1) is an approved drug for the treatment of multiple sclerosis and is highly effective in the suppression of experimental autoimmune encephalomyelitis in various species. The mode of action of GA is by initial strong promiscuous binding to MHC molecules and consequent competition with various myelin antigens for their presentation to T cells. A further aspect of its action is potent induction of specific suppressor cells of the T helper 2 (Th2) type that migrate to the brain and lead to in situ bystander suppression. Furthermore, the GA-specific cells in the brain express the antiinflammatory cytokines IL-10 and transforming growth factor beta, in addition to brain-derived neurotrophic factor, whereas they do not express IFN-gamma. Based on this immunomodulatory mode of action, we explored the potential of GA for two other applications: prevention of graft rejection and amelioration of inflammatory bowel diseases. GA was effective in amelioration of graft rejection in two systems by prolongation of skin graft survival and inhibition of functional deterioration of thyroid grafts, across minor and major histocompatibility barriers. In all transplantation systems GA treatment inhibited the detrimental secretion of Th1 inflammatory cytokines and induced beneficial Th2/3 antiinflammatory response. GA was effective also in combination with low-dose immunosuppressive drugs. Inflammatory bowel diseases are characterized by detrimental imbalanced proinflammatory immune reactivity in the gut. GA significantly suppressed the various manifestations of trinitrobenzene sulfonic acid-induced colitis, including mortality, weight loss, and macroscopic and microscopic colonic damage. GA suppressed local lymphocyte proliferations and tumor necrosis factor alpha detrimental secretion but induced transforming growth factor beta, thus confirming the involvement of Th1 to Th2 shift in GA mode of action.

Toll-like receptor-4 and allograft responses.

Signaling through toll-like receptors (TLRs) is believed to be the critical first step in the activation of antigen presenting cells and the initiation of adaptive immune responses. Of these receptors, TLR-4 particularly recognizes endogenous agonists and may be important for allograft responses. We tested this concept using mice with defective function and structure of TLR-4 as recipients of grafts across major and minor histocompatibility barriers. The kinetics of rejection was the same in mutant mice and wild-type controls. Our results highlight an important difference between alloimmune and conventional immune responses.

Allogeneic cell-mediated immunotherapy of leukemia with immune donor lymphocytes to upregulate antitumor effects and downregulate antihost responses.

Donor lymphocyte infusion mediates most effective graft- versus-leukemia (GVL) effects following induction of host-versus-graft tolerance by transplantation of donor stem cells. This study was designed to maximize GVL effects across both major (MHC) and minor (mHgs) histocompatibility barriers in recipients inoculated with murine B-cell leukemia (BCL1), using specifically immune donor lymphocytes. GVL effects were induced with donor spleen cells from mice immunized across MHC or mHgs barriers with BCL/1 cells or normal BALB/c spleen cells. Our data suggest that spleen cells from donor mice immunized against murine B-cell leukemia of BALB/c origin, or to a lesser extent against normal host alloantigens, induce better therapeutic GVL effects with less great-versus-host disease (GVHD) across both mHgs and MHC. The cytokine profile of effector cells inducing predominantly GVL effects with reduced GVHD across MHC and mHg barriers consisted preferentially of upregulated IFN-gamma, IL-2, IL-10 and IL-12 in donors, implying a Th-1 to Th-2 cytokine shift. We hypothesize that immunotherapy with immune donor lymphocytes sensitized in vivo or in vitro with allogeneic tumor cells or normal host cells together with allogeneic BMT may provide an effective approach for amplifying GVL effects, while reducing procedure-related morbidity and mortality due to uncontrolled GVHD.

Fate of donor cells in vascularized bone grafts: identification of systemic chimerism by the polymerase chain reaction.

Systemic chimerism, or the movement of cells from a transplanted tissue into host organs, is a phenomenon known to occur in association with development of immunological tolerance in allotransplantation. However, little is known about the fate and movement of cells into or out of autogenous free tissue transfers, including vascularized bone grafts. The purpose of this study was to identify systemic chimerism in vascularized bone grafts by transplantation of a vascularized tibiofibular graft from isogenous (inbred) male Lewis rats to female recipients. Donor (male) cells could be identified in the recipient (female) tissues by semiquantitative polymerase chain reaction analysis for a Y chromosome-specific DNA sequence. Chimerism was assessed at 1, 12, 18, and 24 weeks after transplantation. Competitive polymerase chain reaction study using the specific primers for a Y-chromosome marker ( gene) and an autosomal gene (GAPDH) allowed detection of small amounts of male cells in a large pool of female cells and measurement of their relative proportions as a function of time. Of 19 nonimmunosuppressed recipients, nine animals (47 percent) showed low-level chimerism (<0.1 percent) in the peripheral blood. Nine (47 percent), three (16 percent), and two (11 percent) recipients showed high-level chimerism (>1 percent) in the spleen, liver, and thymus, respectively, at final assessment. Donor cells were detected in all bone grafts and in six contralateral tibial bones (i.e., 67 percent of sampled contralateral tibial bones) at 18 and 24 weeks after transplantation. Twenty-four recipients were immunosuppressed with FK506 (tacrolimus) to suppress reaction to a minor histocompatibility barrier present on the Y chromosome. In this group, 14 animals (58 percent) showed low-level chimerism in peripheral blood and 12 (50 percent), eight (33 percent), and one (4 percent) recipients showed high-level chimerism in the spleen, thymus, and liver, respectively. Transplanted cells were detected in nine contralateral tibial bones (i.e., 60 percent of sampled contralateral tibial bones) at 12 and 18 weeks after surgery. The results indicate that polymerase chain reaction for the Y chromosome is a useful tool for differentiating between donor and recipient cell populations experimentally using sex-mismatched tissues in a rat model. This study demonstrated that systemic chimerism occurs after successful vascularized bone transplantation. Transplanted cells not only survive in the graft but also gradually migrate into the recipient's body.

A two-phase pathogenesis of graft-versus-host disease in mice.

Activation of donor T cells is required for the development of graft-versus-host disease (GVHD), a major complication of bone marrow transplantation. We investigated a murine model of GVHD across major and minor histocompatibility barriers. BALB/c recipients were lethally irradiated and transplanted with 10(7) bone marrow and 5 x 10(6) spleen cells from C57BL/6 donors. There were two separate phases of clinical disease. The first phase was most severe on day 7 after transplant. Weight and condition improved until day 12 and then a second phase of clinical GVHD commenced, which persisted until euthanasia. IL-2 mRNA expression, as a measure of T cell activation, was determined by quantitative PCR. The two phases of clinical GVHD were preceded by two separate peaks of IL-2 mRNA in the spleen. Host MHC class II(+) cells became undetectable by flow cytometry 7 days after transplantation, whereas donor MHC class II(+) cells increased during the first 9 days after transplantation. Removal of donor MHC class II(+) cells from the graft had no effect on the first phase. Possible roles for host and donor antigen-presenting cells (APC) in the two phases of the disease are discussed.

No evidence of BO in miniature swine following lung transplantation across minor histocompatibility barrier

No evidence of BO in miniature swine following lung transplantation across minor histocompatibility barrier

Copolymer 1 inhibits manifestations of graft rejection.

Copolymer 1 (Cop 1) was previously shown to prevent graft-versus-host disease and interfere in various manifestations of immune rejection. In this study, we tested whether Cop 1 can also hinder the reactivity of host against graft and inhibit graft rejection. Cop 1 was tested in two transplantation systems: skin and thyroid grafting assays. The effect of Cop 1 on T cell reactivity was investigated by proliferation and cytokine secretion of spleen and lymph node cells from transplanted mice, as well as the T cell lines generated therefrom. Cop 1 treatment prolonged skin graft survival and inhibited the functional deterioration of thyroid grafts, in various strain combinations, across minor and major histocompatibility barriers, similarly to the potent immunosuppressive drug FK506. Cop 1 inhibited the proliferation of graft-specific T cell lines, as well as their interleukin (IL)-2 and interferon-gamma (IFN-gamma) secretion, when incubated in vitro with the stimulating allogeneic cells. Spleen and lymph node cells from Cop 1-treated mice, as well as the T cell lines generated from them, demonstrated a pronounced inhibition of proliferation and secretion of T helper (Th)1 cytokines in response to graft cells. In addition, cells from Cop 1-treated mice secreted high amounts of Th2 cytokines in response to Cop 1 and small but significant amounts of Th2 cytokines, mainly IL-10, in response to allograft cells. Cop 1 treatment inhibited the Th1 response to graft and induced a Th2 cytokines secretion in response to both Cop 1 and graft cells, leading to improved survival and function of the transplanted grafts.

IL-6 deficiency allows for enhanced therapeutic value after bone marrow transplantation across a minor histocompatibility barrier in the twitcher (globoid cell leukodystrophy) mouse.

Bone marrow transplantation (BMT) has therapeutic value for twitcher (globoid cell leukodystrophy) mice, which suffer from a genetic deficiency of the lysosomal enzyme galactosylceramidase that leads to progressive demyelination and early death. Preliminary investigations indicated that a semiallogeneic BMT resulted in graft vs. host disease (GVHD) in twitcher mice but not normal mice. Increased production of the cytokine IL-6 has been demonstrated in twitcher mice, and it has been linked with induction of GVHD. We investigated the effects of BMT in twitcher/IL-6 deficient mice and compared these findings with those from transplanted twitcher and control mice. After a semiallogeneic BMT, 11.4% of controls died within few weeks while the rest survived >100 days without GVHD. In contrast, 85% of the transplanted twitcher mice died by 70 days and 65% developed clinical signs of GVHD, e.g., alopecia and weight loss. In transplanted twitcher/IL-6 deficient mice, only 21% died by Day 70, none had alopecia, and 23% had weight loss. There was no difference in the onset day and severity of twitching between twitcher and twitcher/IL-6 deficient mice after BMT. In transplanted twitcher/IL-6 deficient mice, there was improvement of BBB integrity and a decrease in globoid cell number compared with nontransplanted twitcher/IL-6 deficient mice. In summary, these results demonstrate that an underlying pathology like globoid cell leukodystrophy leads to activation of GVHD responses in a donor-host combination that would not normally induce GVHD. Furthermore, IL-6 seems to play a key role because a deficiency of IL-6 results in a better prognosis.