Immune profile differences between chronic GVHD and late acute GVHD: results of the ABLE/PBMTC 1202 studies

Abstract Human graft-versus-host disease (GVHD) biology beyond 3 months after hematopoietic stem cell transplantation (HSCT) is complex. The Applied Biomarker in Late Effects of Childhood Cancer study (ABLE/PBMTC1202, NCT02067832) evaluated the immune profiles in chronic GVHD (cGVHD) and late acute GVHD (L-aGVHD). Peripheral blood immune cell and plasma markers were analyzed at day 100 post-HSCT and correlated with GVHD diagnosed according to the National Institutes of Health consensus criteria (NIH-CC) for cGVHD. Of 302 children enrolled, 241 were evaluable as L-aGVHD, cGVHD, active L-aGVHD or cGVHD, and no cGVHD/L-aGVHD. Significant marker differences, adjusted for major clinical factors, were defined as meeting all 3 criteria: receiver-operating characteristic area under the curve ≥0.60, P ≤ .05, and effect ratio ≥1.3 or ≤0.75. Patients with only distinctive features but determined as cGVHD by the adjudication committee (non-NIH-CC) had immune profiles similar to NIH-CC. Both cGVHD and L-aGVHD had decreased transitional B cells and increased cytolytic natural killer (NK) cells. cGVHD had additional abnormalities, with increased activated T cells, naive helper T (Th) and cytotoxic T cells, loss of CD56bright regulatory NK cells, and increased ST2 and soluble CD13. Active L-aGVHD before day 114 had additional abnormalities in naive Th, naive regulatory T (Treg) cell populations, and cytokines, and active cGVHD had an increase in PD-1− and a decrease in PD-1+ memory Treg cells. Unsupervised analysis appeared to show a progression of immune abnormalities from no cGVHD/L-aGVHD to L-aGVHD, with the most complex pattern in cGVHD. Comprehensive immune profiling will allow us to better understand how to minimize L-aGVHD and cGVHD. Further confirmation in adult and pediatric cohorts is needed.

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Killer Immunoglobulin-Like Receptor and NOD2/CARD15 Polymorphisms Independently Influence Survival after Allogeneic Hematopoietic Stem Cell Transplanation.

Blood ◽

10.1182/blood.v110.11.2167.2167 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2167-2167

Author(s):

Sebastian Giebel ◽

Aleksandra Holowecka-Goral ◽

Izabela Nowak ◽

Tomasz Czerw ◽

Jerzy Wojnar ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematopoietic Stem ◽

Kir Genes ◽

Increased Risk ◽

Grade Ii ◽

Card15 Gene ◽

Grade Iii

Abstract Background: Activating and inhibitory killer immunoglobulin-like receptors (KIRs) regulate function of NK cells and a subset of T cells. KIR genotype, in particular the content of activating KIR genes is highly polymorphic. NOD2/CARD15 protein is broadly expressed in APCs and lymphocytes. Single nucleotide polymorphisms (SNPs) of this gene have been reported to impair the pathogen elimination and trigger pathologic immunologic reactions like GvHD. The goal of this prospective study was to evaluate the impact of donor’s and recipient’s KIR and NOD2/CARD15 genotypes on outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT). Pateints and methods: One-hundred-two consecutive patients with hematological malignancies, aged 32(18–58)y, treated with alloHSCT from HLA-matched related (n=34) or matched unrelated donor (MUD) (n=68) were included. The conditioning regimen was myeloablative, GVHD prophylaxis consisted of CsA, Mtx, and, in case of MUD-HSCT, pre-transplant ATG. Donors and recipients were tested for 11 KIR genes as well as SNP8,12,13 of the NOD2/CARD15 gene. In addition, immune reconstitution including KIR expression on T cells, was analyzed on days +28, +56, +100, +180, and +360. Results: Overall survival (OS) rate at 2y was significantly lower in alloHSCT with at least one activating KIR mismatch compared to transplants with full compatibility (62% vs. 86%, p=0.01). In particular, the presence of at least one activating KIR in the donor with its absence in the recipient (D+R−) was associated with decreased probability of OS (60% vs. 78%, p=0.01) and DFS (58% vs. 82%, p=0.005), as well as increased incidence of non-relapse mortality (NRM) (27% vs. 7%). KIR2DS1 and KIR3DS1 D+R− mismatches resulted in increased risk of grade II–IV acute GvHD, whereas KIR2DS3 and KIR2DS2 D+R− mismatches were associated with increased risk of chronic GvHD. The presence of at least one activating KIR D+R− mismatch was associated with increased CD8+/CD4+ T cell ratio up to day +100. In all cases of incompatibility regarding KIR2DS1, KIR2DS2 and KIR3DS1, T cells with expression of respective receptors could be detected up to 360 days after alloHSCT. The presence of SNP8 of the NOD2/CARD15 gene in the recipient was associated with decreased probability of OS (20% vs. 70%, p=0.005) and DFS (20% vs. 70%, p=0.01) as well as increased incidence of NRM (60% vs. 17%) and grade III–IV acute GvHD (67% vs. 8%). In a multivariate analysis including KIR and NOD2/CARD15 polymorphisms together with other potential risk factors, increasing number of D+R− activating KIR mismatches as a linear variable appeared to independently influence OS (HR: 1.3, p=0.02), DFS (HR: 1.3, p=0.008), NRM (HR: 1.4, p=0.02), grade II–IV acute GvHD (HR: 1.4, p=0.001), and chronic GvHD (HR: 1.2; p=0.02). Recipient SNP8 of NOD2/CARD15 was predictive for OS (HR: 5.5, p=0.003), DFS (HR: 4.4, p=0.008), NRM (HR: 5.9, p=0.006), grade III–IV acute GvHD (HR: 6.1, p=0.02), and chronic GvHD (HR: 3.7; p=0.03). Conclusions: Both activating KIR D+R− mismatches and recipient SNP8 of NOD2/CARD15 appear to enhance alloreactivity and independently influence survival after alloHSCT. Evaluation of these polymorphisms may contribute to better donor selection and optimization of the alloHCT procedure.

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Direct Intra-Bone Injection of Unrelated Cord Blood Cells Overcomes the Problem of Delayed Engraftment and Improves the Feasibility of Hematopoietic Transplant in Adult Patients.

Blood ◽

10.1182/blood.v110.11.334.334 ◽

2007 ◽

Vol 110 (11) ◽

pp. 334-334 ◽

Cited By ~ 1

Author(s):

Adalberto Ibatici ◽

AnnaMaria Raiola ◽

Marina Podesta ◽

Francesca Gualandi ◽

Nadia Sessarego ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Cord Blood ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Conditioning Regimen ◽

Significant Proportion ◽

Hla Antigen ◽

Hematopoietic Stem ◽

Seeding Efficiency

Abstract Background. Cord blood transplants (CBT) are associated with delayed or failed engraftment in a significant proportion of patients (pts). Two of our previous observations suggested (i) that, in the animal model, direct intra-bone (i.b.) injection improves seeding efficiency and (ii) that the delayed engraftment was not related to an insufficient number of hematopoietic stem cells but rather to some difficulties to differentiate and maturate. Methods. Unrelated CB cells were selected for 29 consecutive pts (18 CB units were 4/6 HLA antigen matched, 10 were 5/6 and one 3/6 antigen matched). Median cell dose infused was 2.3 x10^7/kg (range 1.4 – 4.2). CB cells were concentrated in 4 syringes of 5 ml each and injected in the supero-posterior iliac crest (SPIC) under rapid general anesthesia (10 min. with propofol). Pts’ median age was 38 years (18–63); 25 had acute leukaemia (21 with refractory or relapsed disease and 4 high risk first remission leukemia); 2 chronic myeloid leukemia in advanced phase; 2 refractory Hodgkin’s disease. Most pts (n=24) were prepared with conventional conditioning regimen (TBI-cyclophosphamide). Results. The infusion of cells i.b. in SPIC (11 pts bilaterally; 18 pts monolaterally) was uneventful. Five pts are not evaluable because they died within 14 days from transplant. All pts surviving more than 14 days engrafted (100%). Median time for PMN (>0.5x10^9/l) and platelets (>20x10^9/l) engraftment was day +23 (14–40) and +38 (range 22–60) respectivelly. Four pts died of infection; one patient died of PTLD on day +140. Four patients relasped and 3 died of relapse. Fifteen out of 16 alive patients are in hematologic or molecular remission at a median follow up of 7.5 months (range 2–17). From day +30 full donor chimerism was documented in CD3, bone marrow cells and progenitor cells from both the injected and in non-injected SPIC; from day +30, CFC progenitors had already reached the lower values of the range of normal individuals in bilateral sites. These findings document the colonization of the hematopoietc system and the recovery of stem cell reservoir possibly due to an improvement of seeding efficiency. Only 3 pts (8%) experienced acute GvHD (2 grade II and 1 grade I); 4 pts. have moderate chronic GVHD. It is known that lymphocyte trafficking is one of the crucial factor in immunity. Two combined factors might contribute to the low incidence of acute GvHD: few of the transplanted T cells do not reach/circulate primarily in the lymphatic organs, where they would be immediately confronted with host antigen presenting cells as probably occurs after i.v. injection; injected T cells come immediately in contact with mesenchymal stem cells (MSC) and osteoblasts, known to be potent immunosuppressants. Conclusion. Direct intra-bone transplant of CB cells overcomes the problem of graft failure and is associated with reduced incidence of acute GvHD even when low numbers of HLA mismatched CB cells are transplanted. Nearly all patients searching for a CB unit were able to undergo CBT. This approach may change our policy of hemopoietic cell transplants.

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Impact of the Inhibitory KIR-HLA Receptor-Ligand Model on Unrelated Hematopoietic Stem Cell Transplantation in Patients with Leukemia.

Blood ◽

10.1182/blood.v110.11.5063.5063 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5063-5063

Author(s):

Jun He ◽

Zi-xing Chen ◽

Xiao-jing Bao ◽

De-pei Wu ◽

Xiao-ni Yuan ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Transplantation ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Receptor Ligand ◽

Hematopoietic Stem ◽

Ligand Model

Abstract The interaction between killer cell immunoglobin-like receptor (KIR) and HLA molecule has particular relevance to the unrelated hematopoietic stem cell transplantation (HSCT). The presence of KIR on donor’s NK cells and the absence of the corresponding KIR ligand in the recipient’s HLA repertoire as a receptor-ligand model can be used to predict the outcome of HSCT. To investigate the effect of KIR-HLA receptor-ligand mismatching and the expression of inhibitory KIR (iKIR) on the unrelated Allo-HSCT of leukemia, the following study has been carried out. The KIR genotypes of 36 patients of ALL (n=18), AML (n=10), and CML (n=8) as well as their unrelated donors were obtained from the Database of China Marrow Donor Program. KIR genotyping was performed with PCR-SSP. The KIR and HLA mismatching status (receptor-ligand mismatch model) between recipients and donors was analyzed thereafter. The expression of iKIR was determined by flow cytometry on recipients after HSCT. All cases were followed up closely until July, 2007. Among all the donor/recipient pairs analyzed, 15 pairs displayed the matched pattern with respect to the KIR genotyping though, graft-versus-host (GVH) KIR ligand-mismatched in 13 pairs, and host-versus-graft (HVG) KIR ligand-mismatched in 8 pairs. 26 recipients were lack of HLA-Cw2,4,5,6,15 ligands although iKIR/2DL1 was expressed on their corresponding donors. Similarly, 35 donors with iKIR/2DL2/L3 on their NK cells could recognized HLA- Cw1,3,7,8,12,14 ligands in their recipients. iKIR/3DL1 was expressed on 9 donors although the correlated HLA-Bw4 ligand was absent on the recipients. iKIR/3DL2 was expressed on 24 donors although the correlated HLA-A11 ligand was absent on the recipients. Except for one case, 35 patients were successfully transplanted, in which seven patients were dead, giving the survival rate of 80.0%. The cause of death was either acute/chronic GVHD or relapse. The frequency of acute GVHD (60.0%, 9/15), leading to death (26.7%, 4/15), was the highest in KIR receptor-ligand matched model. The incidence of acute GVHD and death was 30.8% (4/13) and 23.1% (3/13) in the GVH KIR ligand-mismatching, respectively; while the incidence of chronic GVHD was 37.5% (3/8) and no death happened in HVG KIR ligand-mismatching pattern. The expression of iKIR/2DL1 could be detected on ALL (13.0%∼16.6%) and CML (1.8∼6.2%) patients four month after HSCT. The expression of both iKIR/2DL1 (4%) and iKIR/2DL2 (12%) was found only in one patient with CML three month after HSCT, increased to 33.4% and 32.6% accompanied by cGVHD, and dropped to 7.6% and 10.3% one year after HSCT. These results suggested that the deficiency in iKIR/2DL1 based on the KIR-HLA receptor-ligand mismatching model together with the expression of a number of activating KIR in recipients increases the occurrence of GVHD, relapse, and death in unrelated HSCT. Analysis on KIR-HLA gene expression profiling could be useful in predicting the clinical outcome of unrelated Allo-HSCT in leukemia patients. The overall and disease-free survival after HSCT could be improved by preventing the development of GVHD.

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Functionally Altered GPI-Anchor Negative Treg Following Alemtuzumab-Based T-Cell Depletion Are Associated with Acute Gvhd.

Blood ◽

10.1182/blood.v120.21.3059.3059 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3059-3059

Author(s):

Eva M Wagner ◽

Lukas A Schaefer ◽

Tobias Bopp ◽

Matthias Theobald ◽

Wolfgang Herr ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Cell Depletion ◽

Gpi Anchor ◽

Activation Markers ◽

T Cell Depletion ◽

Hematopoietic Stem

Abstract Abstract 3059 Introduction: The monoclonal anti-CD52antibody Alemtuzumab is frequently used for T-cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT) to prevent graft versus host disease (GVHD). We previously demonstrated the long term persistence of functionally impaired glycosylphosphatidylinositol (GPI)-anchor negative effector T-cells in patients receiving high dose (100mg) Alemtuzumab in combination with a dose reduced conditioning regimen (Fludarabin + Melpahlan) (Meyer, Wagner et al. BMT 2010). Despite of Alemtuzumab-mediated TCD, half of our patients developed acute GVHD. Since regulatory T cells (Treg) play a major role for controlling GVHD, we asked whether GPI-anchor negative Treg are present in patients with or without GVHD. Methods: We analyzed peripheral blood samples of 12 patients with acute GVHD (aGVHD), 7 patients with chronic GVHD (cGVHD), and 10 patients who never developed GVHD after Alemtuzumab-mediated TCD. To analyze Treg-subsets, we stained for CD3, CD4, CD25, CD127, FoxP3, CD52 as well as for the activation-markers GARP, HLA-DR and CD45RA. Treg were identified as CD3+CD4+CD25+CD127- or CD3+CD4+CD25+FoxP3+ cells and subdivided according to their CD52-expression. We used FLAER staining to confirm that the loss of CD52 on Treg resulted from the loss of the GPI-anchors themselves. We were able to study Treg subpopulations in the time course of patients who recovered from acute GVHD in comparison to patients with persisting late acute GVHD. In individual patients, we isolated GPI-anchor positive and negative Treg by FACS-Sort, expanded them and performed Treg suppression assays. Results: GPI-anchor negative Treg were observed in all patients, independent of the development of GVHD. However, the frequency of GPI-anchor negative Treg varied considerably between patients with acute GvHD and those with chronic GVHD or without GvHD. The percentage of GPI-anchor negative Treg was significantly elevated in patients with aGVHD: median 80.35% (range 56,2–96,8%) in comparison to 17,4% (range 0–57,8%) in patients with cGVHD or without GVHD. Activated Treg were almost exclusively detected among GPI-anchor positive Treg-subpopulation. Patients who resolved from aGVHD restored GPI-anchor positive Treg and the amount of activated Treg rose. The percentage of GPI-anchor negative Treg populations remained high in patients with ongoing aGVHD. In addition, these patients had no GARP-positive activated Treg even under long term immunosuppressive treatment. Preliminary experiments with sorted and expanded Treg populations suggest that GPI-anchor negative Treg were unable to suppress T-cell proliferation upon IL-2 stimulation. Summary: We demonstrate for the first time the reconstitution of GPI-anchor negative Treg in patients following Alemtuzumab-mediated TCD. These T cells were functionally altered and were less likely to exhibit an activated phenotype in vivo. Ongoing acute GVHD was associated with high percentages of GPI-negative Treg suggesting that their functional alteration might play a role in aGVHD pathophysiology. This is in line with the finding that only in patients who resolved aGVHD, the frequency of GPI-anchor positive Treg increased significantly. Further functional analyses are ongoing to estimate the cellular consequence of missing GPI-anchored proteins. In addition, correlating the reconstitution of GPI-anchor negative T-cell populations with further clinical events is ongoing. Disclosures: No relevant conflicts of interest to declare.

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Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell–depleted reduced-intensity transplantation

Blood ◽

10.1182/blood-2006-03-005769 ◽

2006 ◽

Vol 109 (1) ◽

pp. 374-382 ◽

Cited By ~ 64

Author(s):

Ralf G. Meyer ◽

Cedrik M. Britten ◽

Daniela Wehler ◽

Klaus Bender ◽

Georg Hess ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Good Manufacturing Practice ◽

Hematopoietic Stem ◽

Log Reduction ◽

Reduced Intensity

Abstract Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C–mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

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Perforin Gene Mutations in Patients with Acquired Aplastic Anemia.

Blood ◽

10.1182/blood.v108.11.998.998 ◽

2006 ◽

Vol 108 (11) ◽

pp. 998-998

Author(s):

Elena E. Solomou ◽

Federica Gibellini ◽

Stephen J. Chanock ◽

Daniela Malide ◽

Maria Berg ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Activated T Cells ◽

Hematopoietic Stem ◽

Bone Marrow Failure Syndromes ◽

Prf1 Gene

Abstract Perforin is a cytolytic protein expressed mainly in activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In T and NK cells perforin is stored in cytoplasmic granules and is essential for killing via non-Fas-mediated mechanisms. Perforin regulates the translocation of granzyme B from cytotoxic cells into target cells; after entering the target-cell granzyme B migrates to the target cell nucleus to participate in triggering apoptosis. Functional perforin is essential for normal CTL and NK cell function; without perforin CTL and NK cells show reduced or no cytolytic effect. Inherited perforin mutations account for 20–40% of familial hemophagocytic lymphohistiocytosis, an autosomal recessive fatal disease of early childhood characterized by uncontrolled accumulation of activated T cells and macrophages in many organs, increased Th1 cytokines and absent functional perforin. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in Prf1 occur in AA; peripheral blood DNA samples from 75 patients and 302 controls were analyzed. Three novel nonsynonymous Prf1 mutations among five unrelated patients (ages: 21, 31, 33, 75, and 77 years old), not present in controls, were discovered; two polymorphisms were also identified (H300H, A274A). The mutations were in the coding region of Prf1 gene. In exon 2, arginine was replaced by histidine in one patient (CGT/CAT, R4H) and in 3 patients the same A91V mutation was identified (GCG/GTG, alanine to valine substitution). In exon 3, serine was replaced by isoleucine (S388I; AGC/ATC) in one patient. Germ-line origin of the Prf1 mutations was established by their presence also in DNA from buccal mucosa obtained from affected AA patients. Four of five patients with mutations showed some hemophagocytosis in the bone marrow examination when first diagnosed, but there were no other typical features of hemophagocytic syndrome such as hepatosplenomegaly or altered liver function tests. None of the patients with Prf1 mutations experienced hematologic recovery with immunosuppressive treatment. Perforin protein levels in all patients carrying mutations were very low or absent. By confocal microscopy, CD8 cells from patients with Prf1 mutations had complete absence of perforin granules (perforin and cathepsin D showed the expected pattern of co-localization in controls’ cytotoxic granules). NK cell killing efficiency from patients carrying mutations in a standard Cr51-release cytolytic assay was significantly decreased compared to controls. Prf1 gene mutations may be related to a more severe phenotype of AA associated with marrow hemophagocytosis and failure to respond to immunosuppression. Mutations in the immune regulatory mechanisms identified in young children can manifest in adults without typically associated clinical findings or a suggestive family history. Mechanistically, Prf1 gene mutations help explain the aberrant proliferation and activation of cytotoxic T cells that are destructive of hematopoietic stem cells in AA and may be useful as predictive factors for responses to immunosuppressive treatments and the decision to rapidly undertake stem cell replacement. Prf1 gene mutations are genetic risk factors for bone marrow failure syndromes.

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Cyclosporine, but Not mTOR Inhibitors, Hampers the Reconstitution of Bone Marrow-Derived Tregs in Long-Term Complete Donor chimeras .

Blood ◽

10.1182/blood.v112.11.2331.2331 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2331-2331

Author(s):

Haruko Sugiyama ◽

Yoshinobu Maeda ◽

Hisakazu Nishimori ◽

Koichiro Kobayashi ◽

Miyuki Nishie-Kataoka ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Late Stage ◽

Mtor Inhibitor ◽

Acute Gvhd ◽

Early Stage ◽

Chronic Gvhd ◽

Mtor Inhibitors ◽

Spleen Cells ◽

Hematopoietic Stem

Abstract Chronic graft-versus-host disease (GVHD) is the most common complication in the late stage after allogenic hematopoietic-stem-cell-transplantation (SCT), but the pathophysiology and treatment strategy of chronic GVHD remain poorly defined. Prolonged administration of cyclosporine (CSA) did not decrease the risk of chronic GVHD. Recent studies using a mouse model have shown that regulatory T cells (Tregs) can influence immune responses, and Tregs in the grafts can prevent acute GVHD when injected together with donor T cells. However, it is not known whether Tregs remain in the grafts in the late stage of SCT and play a role in preventing chronic GVHD. First, we examined the origin of Tregs using a major histocompatibility complex (MHC) mismatched mouse SCT model. Lethally irradiated C3H/HeN(H-2k) recipient mice received 10x106 T-cell-depleted bone marrow (BM) cells from B6.Ly-5a(H-2b, CD45.1) mice and 1x106 spleen cells from C57BL/6(B6, H-2b, CD45.2) mice. Spleen cells were collected from SCT recipient mice at serial time points and subjected to fluorescence-activated cell sorting (FACS) analysis. Transplanted mice displayed complete donor hematopoietic chimerism and mild acute GVHD at day14. On day 21 (early stage) after SCT, host type Tregs (CD4+FoxP3+ H-2k) were no longer detectable, and most of the Tregs (83±3%) were derived from donor spleen Tregs (H-2b, CD45.2). However, the homeostatic expansion of spleen Tregs gradually contracted and newly arising donor BM-derived Tregs (H-2b CD45.1) became dominant (93.8±0.5%) in the late stage of SCT (day 120). As in the spleen, BM-derived Tregs reconstitution in the late stage was seen in the thymus and mesenteric lymph nodes. Moreover, in a minor MHC-mismatched SCT model (B6 into C3H.SW), Tregs in the late stage were derived from donor BM cells (97.0±0.2%). These BM-derived Tregs suppress alloreactivity in the same manner as naturally occurring Tregs isolated from naïve mice in the MLR. Next, we compared the effects of CSA and the mTOR inhibitor rapamycin (RAPA) on Tregs reconstitution. Mice receiving CSA or RAPA showed the same Tregs reconstitution pattern: in the early and late stages, Tregs were derived from donor spleen and BM cells, respectively. However, the number of Tregs in the spleen was reduced significantly in mice receiving CSA, as compared to control mice receiving phosphate-buffered saline (PBS; 1.3±0.2x106 vs. 2.4±0.6x106) at day 110. In particular, the number of Tregs in the thymus was reduced dramatically in mice receiving CSA (0.7±0.2 x105 vs. 2.6±0.5x105 , P<0.02). By contrast, the numbers of Tregs in both the thymus and spleen from RAPA-treated mice were the same as those from PBS-treated mice. Mice treated with everolimus, another mTOR inhibitor, also showed no reduction in the numbers of Tregs. Histologic examination revealed that CSA-treated mice showed pathogenic features of chronic GVHD, including sclerodermatous skin changes, bile duct loss, fibrosis in the portal area of the liver and fibrosis and atrophy of acinar tissue in the salivary glands, while RAPA-treated mice showed no sign of chronic GVHD. Our findings indicate that a) Tregs cannot remain in grafts in the late stage, and newly arising donor BM-derived Tregs became dominant; b) CSA hampers BM-derived Tregs reconstitution and may be associated with the development of chronic GVHD; and c) mTOR inhibitors do not hamper Tregs reconstitution and might prove beneficial for the treatment of both acute and chronic GVHD. Figure Figure

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Selective Depletion of T Cell Alloresponses Using a Photodepletion Strategy Preferentially Targets CD4+ T Cells.

Blood ◽

10.1182/blood.v116.21.1467.1467 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1467-1467

Author(s):

Zachariah A. McIver ◽

Andrew Grim ◽

Nicolas Naguib ◽

Hahn Khuu ◽

Minoo Battiwalla ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Nk Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Central Memory ◽

Viral Reactivation ◽

Post Transplant ◽

Selective Depletion

Abstract Abstract 1467 P-glycoprotein (Pgp) is the product of the multidrug-resistance-1 (MDR1) gene and actively transports various molecules across the extracellular membrane. Previous studies demonstrated that activated lymphocytes decrease Pgp activity and preferentially retain the photosensitizing agent 4, 5-dibromorhodamine 123 (TH9402, Kiadis Pharma, NL). We evaluated a photodepletion technique to achieve selective depletion (SD) of graft-versus-host disease (GVHD) alloreacting T cells in 24 HLA-identical sibling stem cell transplants. Donor lymphocytes were activated by 72 hr exposure with irradiated in-vitro expanded recipient T lymphocytes and pulsed with TH9402. Alloactivated T cells preferentially retaining the photosensitizer were eliminated by light exposure. The transfused cell products were then analyzed for T cell subset frequency. After SD preferential CD4+ T cell depletion occurred with an inversion of the CD4+/CD8+ ratio. Within the CD4+ compartment a significant depletion of naïve (CD27+ CD45RO-), central memory (CD27+ CD45RO+), and effector (CD27-CD45RO+) populations was noted. Conversely, CD8+ naïve and effector subsets was relatively conserved with depletion occurring predominantly in the central memory population. Additionally, an 80% reduction in B cells occurred, with a 60% reduction of cytotoxic NK cells (CD56+CD16+) and a relative expansion of regulatory NK cells (CD56+CD16-). We compared outcomes of 24 SD transplants with 34 patients receiving a T-cell depleted transplant. All patients with hematological malignancies were conditioned with fludarabine, cyclophosphamide, and total body irradiation and received a CD34-selected stem cell allograft from an HLA identical sibling. SD recipients also received 5×106/kg SD donor T cells. Low-dose cyclosporine was used as the only post-transplant immunosuppression. Median follow up was 14.5 months. The probability of severe acute GvHD (grade III-IV, 13%) and relapse (25%) was low for all patients. On day 100 post-transplant SD transplant recipients had lower absolute CD4+ counts, and increased rates of CMV reactivation requiring treatment (median 2 vs 1 reactivations within 100 days, p<0.01), and more chronic GvHD (70% vs 31%, p=0.04). Previous studies have shown that Pgp efflux of rhodamine-123 in quiescent T cells varied between subsets, with a preferential retention occurring in the CD4+ and central memory compartments (Br J Haematol. 1998 Jun;101(4):722-7). Our data is in accordance with these observations. In conclusion, while SD appeared to eliminate alloactivated T cells resulting in low frequencies of severe acute GVHD, differences in Pgp activity and dye retention led to the preferential non-specific elimination of CD4+ and central memory T cells which may have contributed to greater viral reactivation and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

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Role of Il-17 Varies at Different Periods After Hematopoietic Stem Cell Transplantation: Protection From Acute Graft-Versus-Host Disease and Exacerbation of Chronic Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v116.21.3741.3741 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3741-3741 ◽

Cited By ~ 1

Author(s):

Rie Kuroda ◽

Ryosei Nishimura ◽

Katsuaki Sato ◽

Hideaki Maeba ◽

Kazuhito Naka ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Immune Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematopoietic Stem ◽

Graft Versus Host

Abstract Abstract 3741 Th17 is a newly identified T cell lineage that secretes the proinflammatory cytokine IL-17. Th17 cells have been shown to play a crucial role in mediating autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), arthritis, and colitis. Anti-IL-17 therapy for some autoimmune diseases in clinical settings has been started and promising results have been reported. However the role of IL-17 on developing acute and chronic GVHD in hematopoietic stem cell transplantation (HSCT) is not yet fully understood. Interaction between IL-17 and IL-17 receptor is complicated because IL-17 is produced in various kinds of immune cells other than CD4+ T-cells, and IL-17 receptors express on not only immune cells but also various epithelial cells, including lung and intestine, both of which are target organs of GVHD. To explore the role of host derived or donor derived IL-17 separately in acute GVHD, lethally irradiated wild type (WT) or IL-17 knockout (KO) Balb/c (H-2d) were given WT or IL-17 KO C57BL/6 (H-2b) bone marrow (BM) cells with WT splenocytes to induce acute GVHD. Infused cell number of WT splenocytes in this study induced acute GVHD, but not lethal in IL-17 WT host mice. In contrast, IL-17 KO host mice receiving WT BM plus WT splenocytes developed severe acute gut GVHD and finally half of them died (p<0.05). To exclude the possibility that alloreactivity of host IL-17 KO derived dentritic cells (DCs) could be much more than that of WT DCs, mixed leukocyte reaction (MLR) was performed using stimulators from WT or IL-17 KO DCs and responders from WT CD4+ T-cells. No significant differences were observed between WT DCs and IL-17 KO DCs in thymidine uptake and percentage of responder cells producing IFN-g or TNF-a. Taken together, host-derived IL-17 has a protective effect against acute GVHD. Moreover similar results were observed when IL-17 KO Balb/c mice were given BM cells from another strain B10.D2 plus splenocytes shown in the figure below (p<0.05). Next, we compared the development of chronic GVHD between the lethally irradiated WT Balb/c mice given IL-17 KO C57BL/6 BM cells or WT BM cells with low dose WT splenocytes to induce sublethal acute GVHD and chronic GVHD subsequently. After day 60 the mice receiving WT BM cells plus WT splenocytes experienced weight loss accompanied by skin histological changes (p<0.05, shown in the figure below), while mice receiving IL-17 KO BM plus WT splenocytes showed minimal signs of GVHD as well as mice receiving IL-17 KO BM or WT BM alone. Increased number of donor-BM derived IL-17 producing cells was observed in the mice showing chronic GVHD compared to BM control (p<0.05). Moreover, a significant increase of T-cell proliferation was observed by adding rIL-17 into MLR culture (p<0.05). These results suggest that donor BM derived IL-17 producing cells involved in the pathogenesis of chronic GVHD by exacerbating the alloimmune response in part. In conclusion IL-17, especially from host-derived, has a protective effect against acute GVHD. On the contrary, donor BM derived IL-17 exacerbates chronic GVHD. Neutralizing IL-17 would be a potent strategy only for preventing chronic GVHD, not for acute GVHD. Disclosures: No relevant conflicts of interest to declare.

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The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v118.21.1984.1984 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1984-1984

Author(s):

Osamu Horie ◽

Kaori Minami ◽

Sawako Toji ◽

Kenji Yonezawa ◽

Hiroshi Gomyo ◽

...

Keyword(s):

Nk Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Polymorphonuclear Cells ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Expression Levels ◽

Proteinase Inhibitor 9 ◽

And Gender

Abstract Abstract 1984 The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

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