Clinical Evidence Of Graft-versus-host Disease Research Articles

Association of Plasma CD163 Concentration with De Novo–Onset Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (GVHD) is the leading cause of long-term morbidity and mortality after allogeneic hematopoietic cell transplantation. To identify prognostic plasma proteins associated with de novo– or quiescent-onset chronic GVHD (cGVHD), we performed a discovery and validation proteomic study. The total study cohort included 167 consecutive patients who had no clinical evidence of GVHD under minimum glucocorticoid administration and had available plasma samples obtained at 80 ± 14 days after transplantation. We first used high-throughput mass spectrometry to screen pooled plasma using 20 cases with subsequent cGVHD and 20 controls without it, and we identified 20 candidate proteins. We then measured 12 of the 20 candidate proteins by ELISA on the same individual samples and identified 4 proteins for further verification (LGALS3BP, CD5L, CD163, and TXN for de novo onset, and LGALS3BP and CD5L for quiescent onset). The verification cohort included 127 remaining patients. The cumulative incidence of de novo–onset cGVHD was higher in patients with higher plasma soluble CD163 concentrations at day 80 than those with lower concentrations (75% versus 40%, P = .018). The cumulative incidence of de novo– or quiescent-onset cGVHD did not differ statistically according to concentrations of the 3 other proteins at day 80. CD163 is a macrophage scavenger receptor and is elevated in oxidative conditions. These results suggest that monocyte or macrophage activation or increased oxidative stress may contribute to the pathogenesis of cGVHD.

Inhibition of Jak1/Jak2 Is More Effective Than Inhibition of Jak3 in Protecting Mice From Acute Graft-Versus-Host Disease (aGvHD) by Significantly Decreasing Alloreactive CD4+ T-Cells.

AbstractAbstract 2997▪▪This icon denotes a clinically relevant abstract Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for patients with hematological malignancies. However, its success is limited by a life-threatening complication: the graft-versus-host disease (GvHD). Although numerous studies have described immunosuppression protocols to mitigate acute GVHD (aGvHD), novel approaches are needed. Chemokines are well known inducers of leukocyte trafficking and activation. Stimulation of the chemokine receptor signaling pathway leads to initiation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway that contributes to the pathogenesis of GvHD. The key role of JAK signaling in normal and abnormal lymphocyte development and function, along with the cytotoxic effects of its inhibitor INCB18424 (Ruxolitinib) on leukemia cells, prompted us to hypothesize that this selective JAK1 and 2 inhibitor could be useful as anti-GvHD agent while maintaining antitumor activity. Since CP-690550, a more selective JAK3 inhibitor, was recently shown to protect against GvHD in mouse models, we also tested whether blocking the JAK1/JAK2 pathway could be more effective in preventing GvHD. Methods:To assess the therapeutic effect of pharmacologic modulation of JAK1 and 2 on GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was used. Recipient BALB/c mice were lethally irradiated and treated either with spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=8), or with spleen and BM cells from B6 donors along with INCB18424 90mg/kg/day at days -1 to 13 (INCB18424 cohort, n=10) or with CP-690550 15mg/kg/day (CP-690550 cohort, n=8) at days -1 to 13. Syngeneic transplants (B6-B6, n=6) and BALB/c recipients treated with B6 BM cells only (control cohort, n=8) were also included as controls. Mice were characterized for GvHD by monitoring overall survival and weight loss. Recipient mice were sacrificed and tissues harvested on day 14 and 30 post transplant and GvHD confirmed by histology. Results:All mice in the GvHD cohort had clinical evidence of GvHD (weight loss, generalized erythema of the skin and poor fur quality) by day 14. The INCB18424 treated mice showed markedly reduced weight loss along the time of observation when compared to the GvHD cohort. Animals in the CP-690550 cohort tended to gain weight during the time of treatment (day-1 to 13), but thereafter they exhibited reduced body weight similar to that observed in the GvHD cohort. The histological examination of the stomach, liver, skin and intestine obtained at day 14 revealed no sign of GvHD in the control group as well as in the INCB18424 group. On the other hand, mild to moderate signs of GvHD were present in the tissues of CP-690550 treated mice and extensive inflammation and disruption of the normal architecture of the tissues was observed in the GvHD group. To determine whether INCB18424 treatment affected alloreactive CD4+ T cells, total spleen T cells were harvested at day 14 from the GvHD cohort and from recipients either of INCB18424 or CP-690550. Total spleen T cells were co-cultured with BM derived BALB/c (recipient-derived) or C57BL/6 (donor-derived) dendritic cells (DCs). After 24h, T cells alloreactivity was determined by IFN-γ production assessed by intracellular staining. As expected, T cells from GvHD mice showed significantly higher alloreactivity against BALB/c DCs compared to the reactivity observed against syngeneic B6 DCs (5.24% and 0.84% respectively, p<0.05). The alloreactivity observed when T cells from INCB18424 treated mice were stimulated with allogeneic BALB/c DCs was significantly lower than that in the GvHD group (0.64% and 5.24% respectively, p<0.05) and eas also lower than that of the CP-690550 group (1.43%). STAT phosphorylation analysis demonstrated that INCB18424 treatment was effective in vitro. Conclusions:The inhibition of Jak/STAT signaling using the sensitive and specific inhibitor of Jak1/Jak2, INCB18424, conferred effective protection from aGvHD in a HSCT mouse model. INCB18424 treatment was more effective than the targeting of JAK3 with CP-690550. In fact, CP-690550 administered during GvHD induction was not completely sufficient to restore the normal weight and to prevent the histological appearance of GvHD whereas INCB18424 was. INCB18424 protected mice against acute GvHD by significantly decreasing alloreactive CD4 T cells. Disclosures:No relevant conflicts of interest to declare.

Acute Gvhd Induced by In Utero Hematopoietic Cell Transplantation Is Reversed by a Regulatory Response Resulting in a Potent Graft Vs. Hematopoietic Effect without Clinical Gvhd

Abstract 1904 Introduction:In Utero Hematopoietic Cell Transplantation (IUHCT) is a potential treatment for congenital hematologic disorders. The rationale is to utilize normal events during hematopoietic and immunologic development to facilitate hematopoietic engraftment. While the risk of graft versus host disease (GVHD) after IUHCT would be anticipated to be high, based on Medawar's tenets, surprisingly little GVHD has been observed after experimental IUHCT and IUHCT induced GVHD has not been previously characterized. To better understand the potential risk of IUHCT induced GVHD prior to clinical application, we attempted to intentionally induce GVHD pathophysiology in the allogeneic murine model of IUHCT. Methods:Bone marrow (BM) cells (10×106) from C57/BL6 (B6, H2kb, GFP−) mice were co-injected with increasing doses of splenocytes from B6-GFP (H2kb, GFP+) mice intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). Control groups included: 1) 10×106 B6 BM cells alone; 2) same doses of B6-GFP splenocytes alone; and 3) IUHCT of congenic donor cell populations into B6 recipients. Surviving pups were assessed using a clinical GVHD scale and separate cohorts were harvested at pre- and post-natal time-points for analysis by fluorescence stereoscopic microscopy (FSM), flow cytometry for assessment of donor and host cell populations in hematopoietic tissues, and histologic assessment of target tissues for GVHD. Results:Prenatal Analysis - In the allogenic groups that received 10×106 BM cells plus increasing doses of splenocytes (0.1×106 to 10×106 splenocytes/fetus) a consistent distribution of GFP positive cells could be seen by whole body FSM in lymphohematopoietic sites (spleen, lymph nodes, thymus and fetal liver) at E16 that did not differ significantly from congenic controls. By E19 – E20 however, marked proliferation of the cells occurred with clear infiltration of GVHD target organs (skin, lungs, liver, intestine). This expansion was proportionate to the dose of splenocytes and was not seen in congenic or BM only controls. Increased mortality was not observed until doses greater than 5 × 106 splenocytes were given (approx. 5 × 1011 splenocytes/kg est. fetal wt.) with perinatal death likely attributable to respiratory insufficiency.Postnatal Analysis - In the allogeneic groups there was minimal clinical evidence of GVHD up to and inclusive of the 5×106 splenocyte dose. A few pups showed poor growth relative to the normal growth curve and mild fur ruffling, but these resolved by P15, and there was no further clinical evidence of GVHD and no postnatal deaths up to P60. In harvested tissues many GFP+ cells persisted in GVHD target organs, however, no GVHD histopathology was observed at any time. Flow cytometric analysis confirmed a proliferation of donor splenocytes that peaked around P5-P10. Lymphocyte subset analysis revealed an expansion of predominantly CD8+ T-cells. Alkaline phosphatase in the host liver, as a marker of GVHD was increased during the same time course and returned to normal by P15. An increase of host and donor BM derived CD4+ cells was also observed that peaked around P15-17. Further analysis confirmed an increase in both T-regulatory cells and activated CD4+ T-cells compared to normal or BM only controls. The most remarkable observation was a dramatic graft versus hematopoietic (GVH) effect with an increase in donor BM derived chimerism at P60 to > 80% with no clinical GVHD. These findings were seen only in the allogenic group that received both splenocytes and bone marrow cells, not in the other groups. In the group that received only bone marrow cells, the chimerism remained at low levels (Figure 1). Conclusion:Our data demonstrates that the fetus is in fact resistant to GVHD relative to myeloablated postnatal BMT recipients. While there is an aggressive alloimmune activation and expansion of donor T-cells, clinical manifestations are mild and are reversed by what appears to be a T-regulatory cell expansion. In our model, the alloimmune activation phase is accompanied by a potent GVH response providing a competitive advantage for donor cells and resulting in marked enhancement of donor cell engraftment without clinical evidence of GVHD. Further work in this model is directed toward defining regulatory and effector mechanisms involved in controlling GVHD and the GVH effect. Disclosures:No relevant conflicts of interest to declare.

Nuclear Translocation of RelB within Host and Donor Antigen Presenting Cells Is Critical for the Initiation and Maintenance of Acute Graft-Versus-Host Disease.

Either donor or host antigen presenting cells (APC) are sufficient for the initiation of CD4 dependent graft versus host disease (GVHD). However the molecular transcription pathways within APC required for this effect are unknown. The NF-kB/Rel family member RelB is associated with dendritic cell (DC) maturation and is critical for the induction of potent APC function. DC from RelB−/− mice had markedly reduced levels of CD40 and to a lesser extent CD80/CD86 following in vitro activation. Following total body irradiation, the number of residual splenic DC with nuclear RelB was increased 5-fold relative to untreated mice. We therefore examined the role of RelB within donor and host APC in GVHD utilizing two well established bone marrow transplant models of CD4-dependant GVHD. To study the requirement of RelB within host APC we generated chimeric mice by transplanting wild-type (wt) or RelB−/− B6 bone marrow into irradiated wt B6 mice. Following immune reconstitution 4–6 months later, the number and frequency of DC (CD11chi and CD11cdimB220+) was equivalent in RelB−/− and RelB+/+ chimeras, although RelB−/− chimeras were specifically deficient in CD11chiCD4+ DC. Chimeras were subsequently transplanted with allogeneic Balb/c bone marrow and purified T cells. The absence of RelB within host APC significantly improved survival (survival day 60: 83% v 19%, P< .0001) and GVHD clinical scores were significantly reduced in RelB−/− chimeras for the first 4 weeks after transplant but subsequently rose to levels equivalent to those in surviving RelB+/+ chimeras. All RelB−/− and RelB+/+ chimeras that received syngeneic grafts survived without clinical evidence of GVHD. Sera from RelB−/− chimera recipients of allogeneic grafts contained reduced IFNg (117 ± 23 vs 253 ± 45 pg/ml; P< 0.02) and increased IL-5 (358 ± 105 vs 112 ± 20 pg/ml; P<0.05) compared to RelB+/+ chimera recipients (mean ± SE). Furthermore, CD4 T cells purified from the spleens of RelB−/− chimera recipients produced 2.6 fold more IL-4 (451 ± 31 vs 168 ± 17 pg/ml; P=0.01) than those from RelB+/+ chimera recipients. Taken together these data suggest the absence of nuclear RelB translocation within host APC abrogates GVHD and this is associated with the induction of donor Th2 differentiation. To study the role of RelB within donor APC we transplanted wt or RelB−/− B6 bone marrow and wt purified T cells into irradiated B6D2F1 recipients. In this model, GVHD severity was identical for the first 4 weeks after transplant but subsequently GVHD clinical scores in the recipients of RelB−/− donor APC returned toward levels seen in syngeneic recipients (clinical scores at day 49: 1.0 ± 0.6; n=6 vs 3.75 ± 0.4; n=6; RelB−/− vs RelB+/+P=0.01). This attenuation of acute GVHD in recipients of RelB−/− donor-derived APC was associated with the reconstitution of donor DC on day 21. These data suggest the inhibition of the nuclear RelB translocation within APC represents a potential new therapeutic target for the prevention of allograft rejection and GVHD.

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.

Normal-looking skin in oncohaematological patients after allogenic bone marrow transplantation is not normal.

Graft-versus-host-disease (GvHD) occurs in one-third or even half of bone marrow transplant (BMT) patients, involving three major target organs: gut, liver and skin. The histopathological and immunohistochemical features of normal-looking skin in oncohaematological patients on day 100 after BMT were studied to find a possible relationship between the histopathological findings and clinical variables (history or clinical evidence of GvHD, previous therapeutic regimens or infections). Fifty-one Caucasian oncohaematological patients, who had had an allogenic BMT, had a biopsy taken from normal-looking skin in nonsun-exposed areas (buttocks or the lumbar region), around the 100th day after BMT. The histology was studied, and the influence of clinical variables on the development of every different histopathological pattern was evaluated through statistical analysis. Histopathological analysis based on morphological criteria revealed the presence of three different patterns: a postinflammatory pattern (45%), changes similar to grade I and II of GvHD (31%) and no significant changes (24%). Statistical analysis revealed that only the presence of peaks of cytomegalovirus (CMV) antigen in the blood within 100 days from BMT was significantly associated with the pattern of GvHD-like changes. Normal-looking skin in 76% of BMT patients is not necessarily histologically normal. The pattern with more prominent changes, the GvHD-like pattern, has been found to be associated with a more frequent history of CMV antigen in the blood within 100 days from BMT.

Encouraging preliminary results in 12 patients with high-risk haematological malignancies by omitting graft-versus-host disease prophylaxis after allogeneic transplantation.

Immunosuppressive therapy, routinely given after allogeneic transplantation to modulate graft-versus-host disease (GVHD) may have an adverse effect on the graft-versus-tumour (GVT) effect. Twelve patients with high-risk haematological malignancies were given cyclophosphamide, total body irradiation and antithymocyte globulin followed by peripheral blood stem cell grafts from HLA-identical siblings without prophylactic immunosuppression. At the earliest clinical evidence of GVHD, patients were treated with high-dose solumedrol and tacrolimus. Prompt haematological recovery [absolute neutrophil count (ANC) > 1.0 x 109/l] was observed (median time 9 d). All patients developed grade III-IV GVHD (median onset 9 d), involving the skin (11), intestine (five) and liver (three). Of nine evaluable patients, seven developed chronic GVHD [extensive (six), limited (one)]. Six patients died 1-6.5 months after transplantation. Three patients died from treatment-related complications, two from acute GVHD and one from relapsing disease. The remaining six patients are alive 5-26 months after transplantation, five in complete remission and one myeloma patient in very good partial remission. In conclusion, omission of post-transplantation GVHD prophylaxis is feasible, does not lead to graft failure or a high incidence of uncontrollable GVHD and appears to be associated with encouraging clinical responses in a group of patients with high-risk disease features.

Treatment of Donor Mice With an αβ T-Cell Receptor Monoclonal Antibody Induces Prolonged T-Cell Nonresponsiveness and Effectively Prevents Lethal Graft-Versus-Host Disease in Murine Recipients of Major Histocompatibility Complex (MHC)-Matched and MHC-Mismatched Donor Marrow Grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57-597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H-2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.

The diagnostic accuracy of the rectal biopsy in acute graft-versus-host disease: A prospective study of thirteen patients

We carried out a coded, prospective analysis of sequential rectal biopsies from 13 patients undergoing allogeneic bone marrow transplantation in order to study the accuracy of these biopsies in the diagnosis of acute graft-versus-host disease (GVHD). We also tested the hypothesis that individual crypt cell degeneration is the initial lesion of intestinal GVHD. Pretransplant biopsies were normal. All biopsies taken 7–10 days after transplantation were diffusely abnormal with nuclear atypia and crypt cell degeneration. These changes were due to the conditioning regimen of total body irradiation and chemotherapy and had resolved by day 20 after transplantation. When acute GVHD was present, rectal biopsies were focally abnormal with crypt cell degeneration, crypt dilation, and crypt abscess. Rectal biopsies were normal after day 20 in those patients who did not have clinical evidence of GVHD. The location of cell degeneration at the base of crypts suggests that an immunologic mechanism is responsible for damage to rectal mucosa in GVHD. The rectal biopsy is an accurate way of detecting intestinal involvement with acute GVHD if done after resolution of radiation/chemotherapy effects.