Graft-versus-host Reaction Research Articles

Molecular Identification of Individual T Cell Clones Specific for Graft- Versus-Leukemia Reaction

Graft-verus-leukemia (GVL) effect in hematopoietic stem cell transplantation (HSCT) is usually complicated by the alloreactivity of donor T cells which leads to acute graft-versus-host (GVH) disease. GVL and GVH reactions are proved to be mediated by different T cell clones. The objective of this study was to identify and characterize T cells clones with specific antileukemia activity without mediating GVHD. We have performed primary mixed leukocyte reaction (MLR) using patient non-leukemic irradiated peripherial blood mononuclear cells (PBMC) as stimulators and donor PBMC as responders. To prepare GVL specific T cells, activated alloreactive T cells were first selectively depleted with an anti-CD25 immunotoxin (Michalek, et al. PNAS 2003, 100: 1180–4). Allodepleted T cells were then stimulated in secondary MLR using irradiated leukemia cells from the same patient. Activated leukemia-reactive cells were purified by immunomagnetic selection or by FACS based on INF-γ or CD25 expression, respectively. Clonotypic assay was used for identification of individual leukemia-specific T cell clones (Michalek, et al. Lancet 2003, 361: 1183–5; Michalek, et al. J Immunol 2007, 178: 6789– 5). This highly sensitive assay is based on detailed analysis of T cell receptor β VDJ unique sequence (TCRB-VDJ). mRNA was extracted from sortred activated cells and cDNA synthetized by anchored reverse transcription. Target TCRB-VDJ gene sequence was amplified by anchor PCR and used to transform bacteria. Bacterial colonies were picked for plasmid isolation and subsequent direct automated sequencing of the TCRBVDJ sequences. We assume that the frequency of particular TCRB-VDJ sequences among bacterial clones after transformation are proportional to the frequency of those sequences in the original population of T cells activated by GVH or GVL reaction. We investigated the presence of individual antileukemic T cell clones in patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), and defined them by the TCRB-VDJ unique sequence. The sequences that occured in more than 10% bacterial colonies are likely to represent the most immunodominant clones. Populations of antileukemic T cell clones were oligoclonal, i.e. we observed limited number of individual immunodominat clones which plays important role in GVL reaction. In first CLL patient who had undergone HSCT, six antileukemic T cell clones were identified, four of them are considered to be immunodominant. In second CLL patient after HSCT, only one highly immunodominat autileukemic T cell clone was observed. This specific clone was further monitored by quantitative real-time PCR in patients peripherial blood.

Blockade of Interleukin 6 Signaling Attenuates Graft Versus Host Disease by Augmenting Regulatory T Cell Reconstitution

Graft-versus-host disease (GVHD) is characterized by a proinflammatory milieu that is attributable to conditioning regimen-induced host tissue damage as well as secretion of inflammatory cytokines by alloactivated donor T cells and other effector cell populations. Interleukin 6 (IL-6) is one of the cytokines that is a component of this proinflammatory environment and is of particular interest with respect to GVHD biology since presence or absence of IL-6 plays a pivotal role in determining the fate of naive T cells as they differentiate to become either proinflammatory or regulatory T cells, respectively. How IL-6 contributes to the pathophysiology of GVHD, however, is not well understood. To address this question, we employed a monoclonal antibody (Ab), MR16-1, that binds to both the soluble and membrane components of the IL-6 receptor to determine whether inhibition of IL-6 signaling affected GVHD severity and what impact antibody blockade had on the reconstitution and expansion of regulatory and effector T cells post transplantation. To identify regulatory T cells (Tregs) in vivo, we used transgenic donor mice in which the foxp3 gene is coupled to the enhanced green fluorescent protein (eGFP) so that eGFP would serve as a surrogate marker for Tregs. In initial studies, lethally irradiated Balb/c (H-2d) mice were transplanted with T cell depleted (TCD) B6 eGFP-foxp3 (H-2b) bone marrow plus splenocytes to induce GVHD. Cohorts of animals were then administered anti-IL-6R or isotype control Ab (0.5 mg/dose) once weekly. Mice treated with anti-IL-6R Ab had significantly less weight loss and reduced pathological damage in the liver, lung and colon when assessed 3–4 weeks post-BMT, demonstrating that protection was generalized and not restricted to a single target organ. Furthermore, antibody-treated animals also had a 12-fold increase in the number of donor-derived Tregs in the spleen compared to control mice (mean 9.5 × 104 versus 0.8 × 104, p=0.0001), indicating that blockade of IL-6 signaling was associated with increased Treg reconstitution. To determine if the increase in Tregs was operative under conditions where thymic function was absent, similar experiments were performed in thymectomized animals. These studies revealed that administration of anti-IL-6R Ab significantly reduced pathological damage in GVHD target organs and increased the absolute number of splenic Tregs. Since one of the mechanisms by which Tregs can be generated in the periphery in the absence of a thymus is through the peripheral conversion of CD4+ foxp3− to CD4+ foxp3+ Tregs, we examined whether blockade of IL-6 enhanced Treg conversion during GVHD. Lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4+ eGFP− T cells so that peripheral conversion could be determined by expression of eGFP. Anti-IL-6R antibody-treated mice had significantly reduced GVHD pathology that was associated with increased absolute numbers of converted Tregs in the spleen. Notably, we also observed a marked reduction in both TH1 and TH17 cells in these same animals, suggesting that IL-6 blockade might also affect expansion of alloreactive donor T cells. To address this question, we employed a murine model (B6® Balb.B) in which donor and recipient animals differ at multiple defined minor histocompatibility antigens. One of these minor antigens, termed H60, is preferentially recognized by donor CD8+ T cells during a GVH reaction. Mice administered anti-IL-6R antibody had a significant reduction in GVHD as assessed by weight loss and overall pathological score when compared to isotype-treated controls. Coincident with the reduction in pathology, there was also a significant decrease in the absolute number of CD8+ H60+ T cells, demonstrating that blockade of IL-6 signaling decreased the expansion of GVH-reactive donor T cells. In summary, we conclude that IL-6 plays a pivotal role in the pathophysiology of GVHD by driving the immune response in a proinflammatory direction. Conversely, blockade of IL-6/IL-6R interactions significantly attenuates GVHD by augmenting Treg reconstitution and decreasing expansion of alloreactive donor T cells which serves to re-balance the immune response. Given the challenges associated with the ex vivo expansion of Tregs for therapy in humans, these results also suggest that anti-IL-6R antibody treatment might represent a novel approach for the expansion of these cells in vivo.

SELDI-TOF-Based Proteomic Analysis of Plasma Samples as a Diagnostic Tool to Predict Acute Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (SCT) offers a curative treatment option for high-risk hematological tumors. Graft-versus-host disease (GvHD) is a severe and potentially life-threatening complication of allogeneic SCT. GvHD is caused by the activation of transplanted alloreactive T-cells and results in severe inflammation of host tissues in skin, liver, and gastrointestinal tract. Manifest GvHD can be diagnosed by clinical criteria and histology only. So far, no diagnostic blood test is able to detect imminent GvHD. GvHD is prevented and treated by systemic immunosuppression which is associated with opportunistic infections and higher relapse rates. Therefore, reliable detection of incipient GvHD would allow to investigate preemptive immunosuppressive therapy tailored to individual patients at appropriate time points. Proteomic profiling is based on the premise that alterations related to pathological states (e.g., GvHD) are accompanied by quantitative and qualitative alterations in plasma protein profiles. The surface-enhanced laser desorption/ionization (SELDI) technology relies on time-of-flight mass spectrometry for accurate measurement of the mass-to-charge ratio (m/z) of proteins that have been preselected on appropriate functional surfaces. We conducted a prospective case-control study based on >3000 serial plasma samples of 236 consecutive patients undergoing allogeneic SCT. 43 patients developed acute GvHD grade I-IV during their sampling period and were matched to control patients remaining GvHD-free. Matching criteria were prior antithymocyte globulin treatment, donor type (related versus unrelated), conditioning protocol, remission status at SCT, diagnosis, age, and sex. These factors were ranked in the listed order. Plasma protein patterns were obtained by SELDI-TOF on day -2 prior to the onset of clinical GvHD and compared to 43 plasma samples obtained on the corresponding day after SCT from the matched GvHD-free patients. In order to enhance the number of analyzable proteins, plasma samples were fractionated to yield 5 different sets of SELDI spectra. Protein peak positions and calibrated peak heights were determined according to Yasui et al (J Biomed Biotechnol, 2003). To identify a limited number of predictive peaks, a sparse logistic regression model was fitted by a boosting approach (Tutz & Binder, Computational Statistics & Data Analysis, 2007), resulting in 4 peaks at 11963, 22363, 22479, and 30977 Da. Prediction performance of a signature derived from this approach on new data was evaluated using bootstrap samples, resulting in an estimated misclassification rate of 38.5%, an estimated sensitivity of 61.0%, and specificity of 60.5% In addition, the identified predictive peaks could be recovered in most bootstrap samples, indicating sufficient stability of the signature. While the performance of this assay strategy is insufficient for use in clinical practice at present, our data nevertheless demonstrate the potential of proteomic plasma analysis in combination with a tailored statistical analysis to identify GvHD prior to its clinically recognizable onset. In contrast to the clinical routine of immunosuppressive GvHD prophylaxis for all patients after allogeneic SCT with treatment escalation in the case of clinically relevant GvHD, this diagnostic tool therefore offers the perspective for preemptive GvHD therapy administered only to patients with an imminent GvH reaction. Such a tailored GvHD strategy may represent a cornerstone to reduce the risk of infectious complications and tumor relapse while maintaining effective GvHD control in patients at risk. Current work addresses the identity of the index peaks, the temporal evolution of the signature, and refinement of the proteomic analysis.

A Novel Approach to Prevent GvHD: Donor Cells Engineered to Display on Their Surface a Recombinant Form of FasL Protein Effectively Prevent Lethal GvHD in a Mouse Model

Allogeneic bone marrow transplantation (BMT) has the potential to cure a series of inherited and acquired hematological disorders and malignancies. BMT can also be used as a cell-based immunomodulatory approach to induce tolerance to foreign and auto-antigens for the prevention and/or treatment of foreign graft rejection and autoimmune disorders. The routine application of allogeneic BMT as a therapeutic intervention in the clinic, however, is complicated by graft-versus-host (GVH) reaction, which is the major cause of graft-versus-host disease (GVHD) with potential life-threatening complications. T cells specific for alloantigens are the primary culprit of GVHD. Although elimination of T cells from the donor bone marrow inoculum can curtail GVH reaction, it results in compromised engraftment. Thus, strategies targeting specific and effective elimination of only the pathogenic T cells may have important implications for routine application of BMT to the clinic for the treatment of a variety of diseases. The main objective of this study was to use a novel and practical approach, designated as ProtEx™, to engineer bone marrow cells to display on their surface a modified form of FasL protein with potent apoptotic activity as an immunomodulatory agent and test the capacity of the engineered cells to engraft in allogeneic recipients without complications of GVHD. ProtEx™ technology involves generation of chimeric molecules with a core streptavidin (SA), modification of cell membrane with biotin, and the display of chimeric molecules on the cell surface taking advantage of strong noncovalent interaction (10−15 M) between biotin and SA. This technology allows for rapid (~ 2 hr) and efficient (100% of the targeted cells) display of exogenous proteins of interest on any cell without compromising the function of the cell or the proteins. In this study, ProtEx™ was used to display SA-FasL protein on C57BL/6 T cells and BMCs and these cells were tested for elimination of alloreactive T cells as well as prevention of acute GVHD following transplantation into lethally (1000 cGy) irradiated F1 (C57BL/6xBALB/c) mice. We hypothesized that mature T cells in the BM inoculum displaying FasL will respond to the host alloantigens, upregulate the death receptor Fas, and undergo apoptosis following the engagement of FasL with Fas on the same or a different cell, resulting in the prevention of GVHD. In support of this hypothesis, we demonstrated specific elimination of C57BL/6 T cells engineered to display SA-FasL on their surface in response to BALB/c antigen presenting cells in mixed lymphocyte cultures. Transplantation of unmodified 10×106 BMCs comixed with 20×106 splenocytes of C57BL/6 mice into lethally irradiated F1 recipients (n=12) resulted in lethal GVHD in all recipients within 34 days. In marked contrast, transplantation of cells engineered to display SA-FasL on splenocytes only (n=9) or splenocytes and BMCs (n=9) effectively prevented acute GVHD in all recipients that survived over 80 days of an observation period. The importance of FasL-mediated apoptosis in immune homeostasis and tolerance combined with our ability to use a practical approach, ProtEx™, to display SA-FasL with potent apoptotic activity on the surface of BMC or mature T cells at the protein level to prevent acute GVHD is significant and may have immediate clinical application.

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.

The T Cell Cytolytic Molecules Fas Ligand and TRAIL, the Trafficking Molecules CCR9, β7 Integrin and PSGL-1, and the Immune Modulating Molecules OX40, CEACAM1, and CTLA4 Are Required for Thymic Graft-Versus-Host Disease

Although thymic graft-versus-host-disease (tGVHD) has been recognized as an important contributor to impaired T cell reconstitution, limited T cell repertoire and increased infection risk in patients with GVHD, the molecular basis of interactions between donor alloreactive T cells, donor bone marrow (BM)-derived thymocytes, and host hematopoietic and non-hematopoietic thymic stromal cells in GVHD has not been well-defined. Here we analyzed the role of molecules relevant for T cell trafficking, cytolytic function, and co-stimulation and co-inhibition of alloreactive T cells in tGVHD. We first demonstrated that thymic output (as measured by RAG2+ splenic recent thymic emigrants) as well as the thymic cellularity (especially of CD4+CD8+ thymocytes) were inversely proportional to numbers of mature donor T cells infused with the allograft, suggesting that tGVHD severity was inversely associated with thymic function. We then studied the migration of alloreactive donor T cells in vivo with bioluminescence imaging (BLI) and found that luciferase-expressing donor T cells infiltrated the thymus within one week after allogeneic bone marrow transplantation (BMT) (Fig. 1). Upon adoptive transfer of CFSE-labeled donor T cells we noted that thymus-infiltrating alloreactive donor T cells were largely fast-proliferating (CFSElo) and highly activated (CD25+ CD44+).We analyzed the importance of T cell trafficking molecules for tGVHD using mice deficient for certain trafficking molecules, and assessed tGVHD by loss of BM-derived CD4+CD8+ thymocytes. We found that CCR9, b7 integrin subunit, and PSGL-1 were all partially required for tGVHD, while L-selectin and aE integrin subunit may be dispensable (Fig. 2A). Similarly, we examined the role of T cell cytolytic pathways for tGVHD, and found that FasL and TRAIL were required for tGVHD, but that perforin and TNF were dispensable (Fig. 2B). Finally, we assessed the role of various T cell co-stimulatory and co-inhibitory molecules for tGVHD, and found that CEACAM1, OX40 and CTLA4 were required, while GITR was partially required and ICOS was dispensable (Fig. 2C).Upon further analysis of donor BM-derived thymocytes, we observed that Bcl-2 expression in donor BM-derived thymocytes was decreased in recipients with GVHD vs. those without GVHD, which suggests that survival of thymocytes is decreased during tGVHD. Hollander and others have previously demonstrated in non-irradiated GVH reaction models that host non-hematopoietic thymic stroma may be an important target for donor alloreactive T cells. We assessed the expression of the death receptors Fas and DR5 in thymic stroma from normal and irradiated (850 cGy) BALB/c mice. We observed that in particular, MHC class II-negative stroma (endothelial cells and fibroblasts), as well as a population of MHC class II-intermediate stroma (epithelial cells) upregulated the expression of both Fas and DR5 after irradiation.Our study defines the specific pathways for cytolysis, trafficking and immune modulation involved in tGVHD and suggests selective therapeutic targets to attenuate tGVHD and improve post-transplant T-cell reconstitution in patients with GVHD. [Display omitted] [Display omitted]

Ex-Vivo Acquisition of Central Memory Phenotype Is Critical for Tolerance Induction by Donor Anti-3rd party CD8 T Cells in Allogeneic Bone Marrow Transplantation.

The robust immunity remaining after reduced intensity conditioning represents a major barrier to durable engraftment of MHC disparate T cell depleted BM transplants (TDBMT). One approach to address this challenge could be afforded by using donor CD8 CTLs, endowed with potent veto activity (the ability of a cell to specifically suppress CTL precursors directed against its antigens, while sparing other CTLs capable of affording protection against infections and malignancies). However, their use is limited due to their marked GVH reactivity. This problem can be addressed effectively by stimulation of donor CD8 T cells against 3rd party stimulators under exogenous IL2 deprivation. The anti-3rd party veto CTLs support engraftment of TDBMT without causing GVHD. Nevertheless, although these cells displayed highly efficient veto activity in vitro, they were less efficient in vivo, requiring administration in conjunction with Rapamycin. In our study we found, using in vivo imaging, that veto CTLs display poor homing to the host lymph nodes (LNs). Therefore, we hypothesized that the relatively low efficiency of veto CTLs in vivo could be attributed to their failure to co-localize with the host T cells (HTCs) at the LNs, during the critical time at which the HTCs are triggered against donor antigens. To enhance LNs homing we generated anti-3rd party CD8 T cells under culture conditions favoring the acquisition of central memory (Tcm) phenotype (CD44highCD62Lhigh). In order to monitor CTLs or Tcm cells adoptively transferred into irradiated recipients of BM allografts, we used (Host x Donor)F1 cells co-expressing host and donor MHC that can be distinguished by FACS. When CD62Lhigh Tcm cells, expanded in the presence of IL15, were administered, the number of cells located at the host LNs two days post transplant was 40-fold higher compared to that found for veto CTLs expanded in the presence of IL2, which exhibit a CD62Llow effector phenotype (p<0.05). Moreover, the total number of Tcm cells harvested from various organs was increased by 9 folds between days 2 to 6 post BMT (p<0.05) in sharp contrast to the CTLs which displayed a non significant proliferation, indicating that Tcm cells possess superior proliferative capabilities. In addition, the Tcm cells exhibited marked durable persistence in-vivo when tested 100 days post transplant, comprising 17%±9% of the total CD8 T cell compartment following infusion of 1x106 cells. When evaluated in a stringent mouse model for T cell mediated BM allograft rejection, Tcm cells displayed marked tolerizing activity. In this model, 3x106 Balb-Nude BM is transplanted into lethally irradiated C3H hosts that were previously inoculated with 1.25x104 HTC (a number sufficient to induce BM rejection and lethality). Thus, administration of 5x106 anti-3rd party Tcm cells derived from (C3H x BALB)F1 mice, with no further immunosuppression, led to overall survival of 83% (19/23) at 100 days post BMT, while administration of 107 anti-3rd party CTLs (bearing an effector phenotype) could only afford survival of 19% (3/16) (p<0.05) in the absence of Rapamycin. To evaluate the risk of GVHD, allogeneic Balb/c derived anti-3rd party Tcm cells or naive CD8 T cells were administered in conjunction with 5x106 Balb/c-Nude BM cells into lethally irradiated C3H hosts. As expected, naive CD8 cells caused lethal GVHD and only 6% (1/16) of the mice which received 2x106 naive cells survived 100 days post BMT. In contrast, the mice receiving Tcm cells displayed 83% survival (10/12 or 5/6 for 2x106 or 5x106 Tcm cells, respectively) with no signs of GVHD. Furthermore, when evaluated in the graft rejection model, 5x106 fully allogeneic donor Tcm cells induced survival, accompanied with complete donor chimerism at 100 days post transplant in 73% (11/15) of the recipients. The similar efficacy of overcoming rejection displayed by both fully allogeneic and F1 derived Tcm cells is in accordance with a veto mechanism, as opposed to potential ablation of host resistance by donor alloreactive clones. Collectively, the ability of anti-3rd party CD8 Tcm cells to expand and persist in vivo without GVHD, their marked LN homing and their capacity to overcome rejection of TDBMT in the absence of further immunosuppression, strongly suggest that these cells hold marked potential for tolerance induction in allogeneic hematopoietic stem cell transplantation.

Effect of Radioprotector Indralin on the Course of Acute GVH Disease

The effect of radioprotector indralin on the graft-versus-host reaction was studied on the model of acute GVH disease induced in mice by intraperitoneal transplantation of 40x10(6)semiallogenic splenocytes. The effect was evaluated by animal mortality from GVH disease. Recipients were male F1(CBAxC57Bl/6) mice exposed to 7 Gy 24 h before transplantation. Donors were male C57Bl/6 mice. Indralin, intraperitoneally injected in a dose of 100 mg/kg 5 min after irradiation attenuated the severity of GVH disease. It eliminated phase I of acute GVH reaction and shifted to the right the dynamics of mortality. Estimated time of 50% mortality (LT50) was prolonged by more than 4 days (the parameter increased by 31.1%). Two (5.7%) animals recovered from acute GVH disease, while all controls died. Indralin treatment after irradiation resulted in a 30% increase in survival of exposed mice.

Late Cerebral Graft versus Host Reaction in a Bone Marrow Transplanted Girl with Hurler (MPS I) Disease

A girl with Hurler disease (MPS IH) underwent allogeneic stem cell transplantation at 13 months of age with her one HLA-B antigen mismatch mother as donor. The procedure was complicated by cerebral hemorrhage and a ventricular-peritoneal shunt device was inserted. Mild GVH reactions were rapidly reversed. One year after transplantation ventriculitis was suspected and the shunt was replaced by a ventricular drainage catheter. Antibiotics had no effect and graft-versus-host disease (GVHD) was diagnosed. All symptoms were reversed by prednisolone and cyclosporine. Increased albumin and pleocytosis in the cerebrospinal fluid (CSF) normalized concomitantly. Electron microscopy of the CSF sediment showed debris consisting of numerous complex aggregates of thin lamellae and electron dense fragments with a tight lamellar texture. Biochemical analysis of the CSF sediment proved that the debris contained galactosylceramide and sulfatide. The electron microscopic and biochemical findings were interpreted to represent stripping of central myelin as a result of subacute GVHD in the central nervous system and its desquamation from the brain parenchyma into the ventricular CSF through the post-hemorrhage defect. From reversal of the GVHD at 2 years of age until follow-up at 10 years of age the clinical condition remained stable with no recurrence or deterioration.

Palifermin Mediates Immunoregulatory Effects in Addition to its Cytoprotective Effects in Mice with Acute Graft-Versus-Host Disease

Treating recipient mice with palifermin (recombinant human keratinocyte growth factor) prevents the development of acute, lethal, graft-versus-host disease (GVHD). This is due, at least in part, to the ability of palifermin to protect epithelial cells from injury. Using the C57BL/6-->(C57BL/6 x DBA/2)F(1)-hybrid model, we previously showed that the protective effect of palifermin was also associated with redirection of the cytokine profile from Th1 to Th2. To study this immunoregulatory effect more directly, we induced acute GVH reactions in which we treated the donors rather than the recipients with palifermin. The recipient mice were protected from GVHD-associated morbidity, and their cytokine profile was predominantly Th2. The palifermin-treated donor mice alone showed a similar Th2 cytokine profile, and we observed elevated levels of thymic stromal lymphopoietin mRNA in the thymus. We further demonstrated that treating the donor mice with palifermin protects against GVHD-associated morbidity, even if the donors are deficient in Valpha14i natural killer T cells. Our findings clearly show that palifermin mediates immunoregulatory effects in addition to its cytoprotective effects and that both are likely to be involved in the mechanism through which palifermin provides protection from acute murine GVHD.

Immunodeficiency in RFM/(T6xRFM)F1 mouse chimaeras with lethal host-versus-graft syndrome.

Rather than central tolerance, the perinatal inoculation of related F1 hybrid spleen cells into inbred mice may result in host-versus-graft (HVG) reactions manifested as transient autoimmunity, or as a lethal immunodeficiency syndrome. RFM/(T6xRFM)F1 chimaeras with lethal disease die in 30 days with lymphosplenomegaly, immune complexes and impaired immune responses. The present studies used in vitro proliferation assays to show that the HVG reaction caused hyperplasia sufficient to account for the lymphosplenomegaly, while also causing severe impairment of splenic and nodal cell responses to concanavalin A (Con A) and to bacterial lipopolysaccharide (LPS). By 25 days, HVG mice could not distinguish between self and non-self as judged by mixed lymphocyte reactions (MLR) to RFM, (T6xRFM)F1 and third party A/J cells. There were no indications that host cells reactive to F1 donor cells had undergone clonal deletion, anergy or expansion. Flow cytometry revealed that donor T lymphocytes achieved stable engraftment, mostly in the nodes, despite the HVG reaction. Taken together with previous observations, these studies showed that HVG reactions in young parent F1/chimaeras can result in an immunodeficiency state which is characterized by an early appearing, profound and persistent impairment of both host and donor T and B cell functions. The results suggest that HVG reactions can contribute directly to immune deficits seen after clinical allogeneic bone marrow transplantation.

Experimental studies of immunologically mediated enteropathy. V. Destructive enteropathy during an acute graft-versus-host reaction in adult BDF1 mice

As a means of investigating further the pathogenesis of intestinal immunopathology, we have attempted to produce a destructive enteropathy by inducing an acute graft-versus-host reaction (GVHR) in mature, immunocompetent mice. Adult (C57b1/10 X DBA/2)F1 (BDF1) mice given C57B1/10(B10) spleen cells develop a severe GVHR which is associated with marked weight loss and high mortality. In the intestine an initial phase of enteropathy characterized by intense crypt hyperplasia is replaced by more severe intestinal damage which includes villus atrophy and loss of intra-epithelial lymphocytes. These pathological alterations are paralleled by the generation of anti-host cytotoxic T lymphocytes (CTL), marked immunosuppression and the loss of natural killer (NK) cells. In contrast to these findings, adult BDF1 mice given DBA/2 donor cells do not develop an acute systemic GVHR and have no CTL or intestinal pathology, despite prolonged splenomegaly and enhanced NK cell activity. Thus, destructive enteropathy can be induced during a GVHR in intact hosts and our results confirm that this enteropathy has a biphasic pattern, with villus atrophy representing the progression of initial crypt hyperplasia in severe forms of disease associated with weight loss and specific CTL.

Hyper IgE in stimulatory graft-versus-host disease: role of interleukin-4.

Intravenous injection of 2 x 10(8) DBA/2 spleen cells into adult intact (C57BL/6 x DBA/2) F1 mice results in a stimulatory graft-versus-host reaction (GVHR) linked to the recognition by donor CD4+ T cells of Ia alloantigens on host B cells. In the experiments presented here, we found that this GVHR is associated with a major increase in IgE serum levels which was already present 7 days after the cell transfer. At 6 weeks, mean IgE levels were more than 200-fold above the control values. Host B cells were responsible for the hypersecretion of IgE in stimulatory GVHR since it was also observed when the DBA/2 donor inoculum was depleted of B cells but not when the F1 recipients were irradiated. The induction of IgE secretion required donor CD4+ T cells as treatment of the donor inoculum with lytic anti-CD4 monoclonal antibody (MoAb) completely prevented the occurrence of the hyper IgE whereas depletion of CD8+ cells had no influence on this parameter. The role played by interleukin-4 (IL-4) in this model was analysed in vivo by the administration of the 11B11 anti-IL-4 rat MoAb (total dose 36 mg) during the first 12 days following induction of stimulatory GVHR by 8 x 10(7) DBA/2 spleen cells. This treatment completely prevented the development of hyper IgE whereas the administration of a control rat MoAb had no significant effect. We conclude that stimulatory GVHR in mice is associated with a major increase in serum IgE which is mediated by IL-4.

Persistence of allospecific helper T cells is required for maintaining autoantibody formation in lupus-like graft-versus-host disease

Induction of a graft-versus-host (GVH) reaction (GVHR) in non-irradiated (C57BL/10ScSn x DBA/2)F1 mice (BDF1) with DBA/2 lymphoid cells leads to chronic GVH disease (GVHD). One of the pathological alterations of this type of GVHD is hyperplasia of host B cells with production of lupus-like autoantibodies. This hyperstimulation of host B cells has previously been demonstrated to be induced by alloreactive donor T helper cells that were also proposed to maintain it. We provide three pieces of experimental evidence in support of this concept. First, treatment of mice with chronic GVHD by injection of monoclonal anti-Thy-1.2 antibodies, performed at week 6 after the injection of C57BL/6 lymphoid cells into (C57BL/6 x C57BL.bm12)F1 mice led to a significant decrease in the titre of anti-nuclear antibodies. Second, CD4+ donor T cells persisted in BDF1 mice with GVHD (GVHF1) for at least 10 weeks after the induction of GVHR; these T cells showed alloreactive helper activity against H-2b MHC determinants of the opposite parent in vitro. Third, T cells of GVHF1 mice, obtained 2 months after the induction of GVHR and transferred into normal secondary recipients, induced signs of chronic GVHD in DBF1 but not in DBA/2 mice. The combined results show that persisting donor T helper cells in GVHF1 mice retain their alloreactivity towards H-2 class II antigens for a long time after the induction of GVHR and they strongly suggest that these T cells are also the driving force behind the production of lupus-like autoantibodies at the late stage of chronic GVHD.

A role for IL-4 in immunologically mediated enteropathy.

A number of clinical enteropathies are associated with a local cell-mediated immune (CMI) response, and experimental evidence indicates that cytokines are responsible for the intestinal pathology. We show here that depletion of IL-4 using MoAb or a soluble form of the IL-4 receptor (IL-4R) prevents the jejunal manifestations of a proliferative form of murine graft-versus-host reaction (GVHR) characterized by crypt hyperplasia and recruitment of intraepithelial lymphocytes (IEL). Depletion of IL-4 did not prevent the appearance of villus atrophy in a destructive model of GVHR, and had no effect on any indices of systemic immunity. These results indicate that IL-4 may play a selective role in mediating proliferative aspects of intestinal immunopathology, and suggest that this cytokine may provide a useful target for immunotherapy.

A high percentage of HLA-DQ+ and HLA-DR+ mononuclear cells is associated with a low incidence of acute graft-versus-host disease after allogeneic bone marrow transplantation (BMT) in children.

In order to discover some biological markers of acute graft-versus-host disease (aGVHD), we have studied the percentage of peripheral monocytes and T lymphocytes bearing HLA-DR and HLA-DQ class II molecules. This study included 25 allogeneic BMT in children, either with (n = 10) or without (n = 15) aGVHD. Within 2 months after transplantation, a higher percentage of DQ+ and DR+ monocytes and of DQ+ T lymphocytes was observed in patients without aGVHD compared with patients with aGVHD. The most discriminating marker was the strong increase in the percentage of DQ+ monocytes in patients without aGVHD (P = 0.001). In a sequential study, we observed a low percentage of DQ+ and DR+ peripheral blood mononuclear cells (PBMC) as long as the clinical manifestations of aGVHD continued. We speculate if the modulation of DQ and DR molecules on PBMC after BMT is a consequence of the action of some lymphokines, and if it plays a role in the regulation of the acute GVH reaction. We conclude that MHC class II molecules on peripheral mononuclear cells may be reliable biological markers for the diagnosis of aGVHD.

Reduction of graft-versus-host reactivity after small bowel transplantation: ex vivo treatment of intestinal allografts with an anti-T cell immunotoxin

A specific T lymphocyte immunotoxin was used to pre-treat small bowel grafts in an attempt to prevent graft-versus-host (GVH) reactivity and GVH disease in a rat transplant model. The immunotoxin used was a conjugate of the anti-CD5 MoAb MRC OX-19 with ricin A chain. The grafts were perfused ex vivo with a standard solution of immunotoxin followed by incubation at 4 degrees C for 1 h before transplantation. In a semi-allogeneic strain combination (parent to F1 hybrid offspring) graft treatment with immunotoxin led to a prolongation of recipient survival compared with groups receiving similar transplants without immunotoxin treatment. An additive effect on survival was observed when the host was treated with cyclosporin. The effect of immunotoxin was greater than that of mesenteric lymphadenectomy in increasing host survival. The effect of graft treatment with the immunotoxin on cellular migration from graft to host lymphoid tissues was assessed in fully allogeneic transplantation (PVG to DA). Host lymphoid tissues were subjected to immunohistochemical analysis using a MoAb specific for donor class I MHC antigens. Graft treatment with the immunotoxin led to a significant decrease in the number of graft cells found in host lymphoid tissues 7 days after transplantation. However, this effect was less marked than that achieved by graft mesenteric lymphadenectomy. With our current protocol graft treatment with a specific T cell immunotoxin can significantly reduce but not abolish GVH reactivity in rat small bowel transplantation.

Correlation of Hsp70-1 and Hsp70-2 Gene Expression With the Degree of Graft-Versus-Host Reaction in a Rat Skin Explant Model

Graft-versus-host disease (GVHD) is the most serious complication after allogeneic hematopoietic stem-cell transplantation. A human skin explant assay has been used to predict the risk of GVHD in patients by histological grading of graft-versus-host reactions (GVHR). New molecular markers of GVHR might help to further increase the predictive value of the assay. A rat skin explant assay has been developed to further aid in identifying potential novel molecular markers. In inbred rat strains GVHR were observed in skin explants co-cultured with allogeneic lymphocytes stimulated against minor or major histocompatibility antigens. The histological signs of GVHR were similar to those observed in human skin explant assays and acute GVHD lesions occurring in rats after experimental bone marrow transplantation. Heat shock protein (HSP) 70 has been shown to be expressed during GVHR. We therefore investigated the expression of the three major histocompatibility complex (MHC)-linked HSP70 genes in rat skin explants. The two major stress-inducible genes Hsp70-1 and Hsp70-2 were found to be upregulated in the allogeneic rat skin explant assays. The increase in mRNA correlated with the GVHR grade (I-IV). Interestingly, the expression of the third MHC-linked Hsp70 gene Hsp70-3 was not found to be augmented during GVHR. The observed induction of the MHC-encoded Hsp70-1 and Hsp70-2 genes might serve as new markers of GVHR and as potentially novel diagnostic tools for GVHD.

Prevention of graft-versus-host disease by intrabone marrow injection of donor T cells: involvement of bone marrow stromal cells

We have developed a new and effective method for bone marrow transplantation (BMT): bone marrow cells (BMCs) are injected directly into the bone marrow (BM) cavity of recipient mice. The intrabone marrow injection of BMCs (IBM-BMT) greatly facilitates the engraftment of donor-derived cells, and IBM-BMT can attenuate graft-versus-host reaction (GVHR), in contrast to conventional intravenous BMT (i.v.-BMT). Here, we examine the mechanisms underlying the inhibitory effects of IBM-BMT on GVHR using animal models where GVHR is elicited. Recipient mice (C57BL/6) were irradiated and splenic T cells (as donor lymphocyte infusion: DLI) from major histocompatibility complex-disparate donors (BALB/c) were injected directly into the BM cavity (IBM-DLI) or injected intravenously (i.v.-DLI) along with IBM-BMT. The BM stromal cells (BMSCs) from these recipients were collected and related cytokines were examined. The recipient mice that had been treated with IBM-BMT + i.v.-DLI showed severe graft-versus-host disease (GVHD), in contrast to those treated with IBM-BMT + IBM-DLI. The suppressive activity of BMSCs in this GVHD model was determined. The cultured BMSCs from the recipients treated with IBM-BMT + IBM-DLI suppressed the proliferation of responder T cells remarkably when compared with those from the recipients of IBM-BMT + i.v.-DLI in mixed leucocyte reaction. Furthermore, the level of transforming growth factor-beta and hepatocyte growth factor in cultured BMSCs from IBM-BMT + IBM-DLI increased significantly when compared with those from the recipients of IBM-BMT + i.v.-DLI. Thus, the prevention of GVHD observed in the recipients of IBM-BMT + IBM-DLI was attributable to the increased production of immunosuppressive cytokines from BMSCs after interaction with host reactive T cells (in DLI).

Down Regulation of CXC Chemokine Receptor 3 Impedes the Ability of Anti-CXCR3 Antibody To Block Graft-Versus-Host Disease.

Graft versus host disease (GVHD) is caused by infused donor T cells that primarily target specific organs, such as skin, liver and intestine. While it has been demonstrated that chemokines are critical to directing in vivo trafficking of host-reactive donor T cells into GVHD target organs, blocking GVHD using antagonist antibody (Ab) against chemokine receptors remains an elusive goal. We found that in vivo administration of anti-CXCR3 Ab for 1 week (named short-term treatment) only partially prevented acute GVHD in B6/SJL mice receiving donor C3H.SW naïve CD8+ T cells, with 45% of them dying from the disease. Flow cytometry analysis showed that CXCR3+ CD8+ T cells occurred in the spleens and livers of these GVHD B6/SJL mice by day 3 and peaked by day 7 after transplantation, and persisted throughout the disease course. When donor CXCR3+ CD8+ T cells were recovered from these GVHD B6/SJL recipients at day 14 after transplantation (named day-14 CXCR3+ CD8+ T cells) and adoptively transferred into secondary B6/SJL mice, they caused severe GVHD in these secondary recipients with 90% mortality. Interestingly, short-term treatment using anti-CXCR3 Ab protected 78% of B6/SJL mice receiving these donor day-14 CXCR3+ CD8+ T cells. Thus, besides CXCR3+ T cells, some other pathogenic T cells have developed during GVH reactions. We found that a substantial population of CXCR3− CD8+ T cells occurred by day 14 (termed day-14 CXCR3− CD8+ T cells) after transplantation in the spleens (71.1%) and livers (81.6%) of B6/SJL recipients with ongoing GVHD. As compared to donor day-14 CXCR3+ CD8+ T cells, donor day-14 CXCR3− CD8+ T cells expressed lower levels of CCR2, CCR3, CCR5, CD127, granzyme B and FasL, and produced less IFN-g and TNF-a. Furthermore, adoptive transfer of donor day-14 CXCR3− CD8+ T cells caused delayed onset of GVHD as compared to those recipients of day-14 CXCR3+ CD8+ T cells, with 55% of them dying from the diseases. Prolonged treatment of B6 mice receiving day-14 CXCR3− CD8+ T cells with anti-CXCR3 Ab up to 3 weeks (named long-term treatment) significantly inhibited the development of GVHD. More than 70% of these recipients survived without clinical signs of GVHD. In vivo experiments showed that 23% of day-14 CXCR3− CD8+ T cells were converted into CXCR3+ by 12 hours after adoptive transfer into B6/SJL recipients, and up to 60% by 24 hours. Interestingly, about 25% of CXCR3+ CD8+ T cells were also converted into CXCR3− by 24 hours after their transfer. We found in ex vivo cultures that both dendritic cells and interleukin-2 accounted for this conversion between CXCR3+ and CXCR3− subsets, which was inhibited by addition of Ab specific to MHC-I or CTLA-4 Ig. Taken together, these results suggest that alloantigen-induced repression of chemokine receptor(s) may present a major barrier to the success of GVHD prevention and treatment when using antagonists against chemokines or chemokine receptors.