Expression Of PD-L1 Is Required To Control Acute GvHD In Allo-HSCT Recipients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2008-2008
Author(s):  
Mohammad Sohrab Hossain ◽  
Vicky F El-Najjar ◽  
David L Jaye ◽  
Rafi Ahmed ◽  
Edmund K. Waller
Keyword(s):  
Weight Loss ◽  
T Cells ◽  
Acute Gvhd ◽  
Hematopoietic Cells ◽  
Post Transplant ◽  
Radiation Chimeras ◽  
Donor T Cells ◽  
Host Tissues ◽  
Activation Status

Abstract Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with both malignant and non-malignant hematologic disorders. But life-threatening graft-vs-host diseases (GvHD) caused by alloreactive donor T cells limits its clinical use. Alloreactive T cells are also required for graft-vs-leukemia (GvL) and to fight opportunistic infections. Hence, a method that modulates donor T cells activity to reduce GvHD but to retain GvL effect is highly desirable. The inhibitory receptor programed cell death-1 (PD-1) reduces T cell activation through binding with its ligand PD-L1 or PD-L2. Interaction between PD-1 and PD-L1 induces cardiac allograft tolerance and expression of PD-L1 is upregulated in presence of inflammatory stimuli. Here, we studied the role of PD-L1 expression on hematologic and non-hematologic tissues and PD-1 - PD-L1 binding in the development of GvHD. Methods Wild type C57BL/6 (WT B6), PD-L1 knock out B6 (KO) and PD-L2 KO B6 mice were transplanted with 2 x106 splenic T cells and bone marrow (BM) cells from H-2K B10.BR donors. The average acute GvHD scores were determined by combining the GvHD scores obtained from the histological tissue sections of small intestine, large intestine and liver, and weight-loss, posture, activity, fur texture and skin integrity data following standard published procedures. The activation status of splenic T cells was analyzed by flow cytometry. Serum cytokines were determined by using 26 plex Luminex assay. The requirement of hematopoietic and or non-hematopoietic tissues expressing PD-L1 to reduce GvHD was investigated by generating radiation chimeras using WT B6 mice engrafted with PD-L1 KO BM and vice versa. Two months later radiation chimeras were transplanted again with 2 x106 splenic T cells along with 2 x106 BM cells from congenic na•ve H-2K donors. The role of PD-L1 expressing donor hematopoietic cells on the development of acute GvHD was tested by transplanting B10.BR mice with donor PD-L1 KO B6 and WT B6 splenocytes. Results PD-L1 KO B6 recipients had significantly increased acute GvHD (scores 1.68 ± 0.07) compared with WT B6 GvHD (0.78 ± 0.024, p<0.0005) and B6 PD-L2 KO B6 (0.86 ± 0.14, p<0.0005) within day 8 after transplant. All PD-L1 KO B6 recipients had severe GvHD with >25% weight loss on day 8 after transplant and were sacrificed. The WT B6 and PD-L2 KO B6 recipients survived 75% and 80%, respectively until 34 days of transplantation with similar levels of chronic GvHD. To test whether excessive activation of donor T cells caused severe acute GvHD in PD-L1 KO B6 recipients, we determined the activation status of donor T cells in the spleen. The numbers of donor CD4+ and CD8+ T cells expressing ICOS-1 and PD-1 in the spleen were significantly higher (p<0.005) in PD-L1 KO B6 recipients compared with the WT B6 and PD-L2 KO B6 recipients. Additionally, significantly increased levels of serum inflammatory cytokines (IFN-g and TNF-a) were also detected in the PD-L1 KO B6 recipients compared with the WT B6 recipients on day 8 post transplant (Figure 1). Using WT B6 or PD-L1KO hematopoietic cell radiation chimeras as allo-HSCT recipients, we further confirmed that both allo-HSCT radiation chimeras having PD-L1 expressing hematopoietic (10% survival, open square) and non-hematopoietic cells (10% survival, closed triangle) were required to protect from GvHD (Figure 2). We next investigate whether PD-L1 KO donor cells cause increased GvHD. The B10.BR recipients transplanted with donor PD-L1 KO B6 splenocytes had 70% survival while the same recipients transplanted with WT B6 donor splenocytes had only 20% survival until 100 days post transplant. These data suggest that only PD-L1 expressed by host tissues is required to inhibit the development of GvHD. In summary, our data suggest that PD-L1 expressed by host tissues are required to control GvHD and method(s) that enhance expression of PD-L1 in allo-HSCT recipients may represent a novel strategy to control GvHD. Disclosures: No relevant conflicts of interest to declare.

Alloantigen Expression on Host Target Epithelium Inhibits Graft-Versus-Leukemia (GVL) Effects.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 596-596
Author(s):  
Shoji Asakura ◽  
Daigo Hashimoto ◽  
Ken-ichi Matsuoka ◽  
Yukimi Sakoda ◽  
Mitsune Tanimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Hematopoietic Cell Transplantation ◽  
Cellular Therapy ◽  
Hematopoietic Cells ◽  
Target Cells ◽  
Host Epithelium ◽  
Host Target

Abstract We previously demonstrated that alloantigen expression on host target epithelium is not necessary but augment acute GVHD (Nat Med 2002). Here, we tested the role of alloantigen expression on host target epithelium on the GVL effect. We created BM chimeras, [B6 → C3H.Sw] where only host hematopoietic cells express multiple minor histocompatibility antigens (mHAs) allogeneic to the donors but not on host target epithelium. Identically treated [B6 → B6] chimeras were created as controls where both hematopoietic and target cells express mHAs allogeneic to the donors. Four month later, these chimeras were reirradiated and were injected with 5×106 BM and 1×106 CD8 T cells harvested from C3H.Sw donors. Acute GVHD developed in [B6 → C3H.Sw] chimeras but was less severe in these chimeras than controls (Table). Next, animals were transplanted, as above, with the addition of 2500 B6-derived EL4 thymoma to the donor inoculum. The cause of death was determined by postmortem examination to be either GVHD or leukemia (presence of hepatic and/or splenic nodules). All [B6 → B6] recipients of C3H.Sw CD8+ cells died from leukemia, although their survival time was significantly prolonged compared to syngeneic controls (P<.01). Surprisingly, [B6 → C3H.Sw] chimeras displayed more potent GVL effects than controls (Table), in spite of reduced GVHD in these chimeras. Similar results were obtained in the other sets of chimeras [DBA → Balb/c] when injected with BM and T cells isolated from Balb/c donors together with DBA-derived P815 mastocytoma and [Balb/c → DBA] when injected with BM and T cells isolated from DBA donors and Balb/c-derived A20 lymphoma. These animals displayed more potent GVL effects compared to control chimeras (Table). To elucidate the mechanisms of this superior GVL effect in these chimeras, analysis of the spleen was performed 3 weeks after BMT. Expansion and activation of donor CD8+ T cells were greater in [B6 → C3H.Sw] recipients than those in [B6 → B6] recipients in spleens (P<.05). Thus, alloantigens expressed on host antigen-presenting cells stimulate host-reactive T cells but in the absence of alloantigen expression on host epithelium contraction of host-reactive T cells may be impaired, resulting in a superior GVL effect. These results provides a complete picture of the role of alloantigen expression on host epithelium in allogeneic hematopoietic cell transplantation; alloantigen expression on host target cells i) does not always necessary to induce acute GVHD, ii) augment GVHD, and iii) suppress GVL effects. These imply that allogeneic cellular therapy targeting at mHA preferentially expressed on hematopoietic cells may induce potent GVL effects while inducing less severe GVHD. Donor Recipients GVHD score (d21) GVHD mortality (d50) Leukemia mortality (d50) *P<.05. **not examined B6 CD8 [B6 → B6] 1.0 +/− 0.4 0% 100% C3H.Sw CD8 [B6 → B6] 3.7 +/− 0.3 33% 95% C3H.Sw TCD [B6 → C3H.Sw] NE** 0% 100% C3H.Sw CD8 [B6 → C3H.Sw] 2.1 +/− 0.4* 0%* 69%* Balb/c TCD [DBA → DBA] 0.2 +/− 0.3 NE** 100% Balb/c T [DBA→ DBA] 3.2 +/− 0.6 NE** 60% Balb/c TCD [DBA→ Balb/c] NE** NE** 100% Balb/c T [DBA→ Balb/c] 1.3 +/− 0.3* NE** 10%* DBA TCD [Balb/c→ Balb/c] 0.0 +/− 0.0 NE** 100% DBA T [Balb/c→ DBA] 1.8 +/− 0.6 NE** 100% DBA TCD [Balb/c→ Balb/c] NE** NE** 100% DBA T [Balb/c→ DBA] 0.8 +/− 0.3 NE** 30%*

Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 144-144
Author(s):  
Mohammad S Hossain ◽  
David L Jaye ◽  
Brian P Pollack ◽  
Alton B Farr ◽  
John Roback ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Viral Immunity ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Radiation Chimeras ◽  
Donor T Cells

Abstract Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus. To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

Upregulation of Immune Checkpoint Blockade Molecules Post Allogeneic Stem Cell Transplantation Is Necessary but Insufficient to Prevent GvHD

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1101-1101
Author(s):  
Mohammad Sohrab Hossain ◽  
Ghada M Kunter ◽  
Vicky Fayez Najjar ◽  
David L. Jaye ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
T Cell ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Time Points ◽  
Post Transplant ◽  
Donor T Cells ◽  
Over Time

Abstract Donor T-lymphocytes are effective adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but life threatening complications related to GVHD limits its clinical application. Recent advancement in the field of immunotherapy has directed our interest to enhancing the anti-tumor response of donor T cells by modulating expression of checkpoint blockade molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and foxp3, the transcription factor associated with regulatory T cells. The two ligands of PD-1, PD-L1 or PD-L2 are highly expressed in the presence of inflammatory signal induced by infection or cancer and PD-1/PD-L1 interaction negatively regulates T-cell antigen receptor (TCR) signaling and dampen T cell cytotoxic activity. Herein, we studied the role of PD-1, CTLA-4 and transcription factor foxp3 expressing donor CD4+ and CD8+ T cells in the development of GVHD. Methods: We have used two established allo-HSCT murine GvHD models. Lethally irradiated wild type (WT) B6, PD-L1 knock out (KO) B6 and PD-L2 KO B6 mice were transplanted with 2 x 106 splenic T cells and 2 x 106 T cell depleted bone marrow (TCD BM) cells from H-2Kdonors. Lethally irradiated CB6F1 recipients were similarly transplanted with splenocytes and TCD BM cells from B6 donors. Acute GvHD scores were determined by combining scores obtained from histological tissue sections and weight-loss, posture, activity, fur texture and skin integrity following standard published procedures. The activation status of donor T-cells and BM and host-derived non-T cells in GvHD target organs was analyzed by flow cytometry. Data from allo-HSCT recipients were compared with the respective data obtained from B6 à B6 syngenic HSCT (syn-HSCT) recipients. Serum cytokines were determined by Luminex assay. Results: PD-L1 KO B6 allo-HSCT recipients had significantly increased acute GvHD scores compared with WT B6 allo-HSCT recipients (p<0.0005) and B6 PD-L2 KO allo-HSCT recipients (p<0.0005) measured on day 8 after transplant. All PD-L1 KO allo-HSCT recipients died within 10 days post transplant while WT B6 and PD-L2 KO allo-HSCT recipients had 20% mortality until 36 days post transplant. Increased acute GvHD was associated with increased amount of serum inflammatory cytokines and increased numbers of activated PD-1+CD69+CD4+ donor T cells. Interestingly, PD-1 expression on donor CD4+ T cells significantly increased in the spleen of transplant recipients but not in BM, while PD-1 expression was significantly increased on donor CD8+ T cells in both spleen and BM compartments of allo-HSCT recipients compared with the syn-HSCT recipients. CTLA-4 expression on CD4+ and CD8+ donor T cells were significantly increased in spleen in the first two weeks post transplant but decreased at later time points compared with syn-HSCT. Again, CTLA-4 expression on CD4+ donor T cells in the BM remained significantly higher measured on 100+ days post transplant in allo-HSCT recipients compared with the syn-HSCT but similar levels of CTLA-4 expression on CD8+ T cells were measured in BM between these two HSCT recipients. Foxp3 expression on donor T cells and the numbers of CD4+CD25+foxp3+ regulatory T (Tregs) were markedly suppressed in donor T cells on day 4 post HSCT of allo-HSCT recipients compared with the syn-HSCT recipients. Although total numbers of donor T cells in the spleen of allo-HSCT recipients remained low over time, the percentage of PD-L1-expressing donor T cells in spleen were significantly higher (p<0.005) at early time points (day 4) in allo-HSCT recipients compared with the syn-HSCT. While total numbers of host-derived cells in spleen decreased over time in mice that developed GvHD, host-derived PD-L1 expressing CD3+ T cells persisted at higher levels through day 36 post transplant. Additionally, PD-L1 expression was also increased in donor BM-derived T cells and non-T cells populations over time. Collectively, these data indicate that severe GvHD occurs in allo-HSCT recipients in spite of increased numbers of PD-1, CTLA-4 and PD-L1 expressing donor and host cells. The occurrence of severe GvHD in these allo-HSCT models systems was associated with markedly reduced levels of CTLA-4 and foxp3 transcription factor expressing Tregs indicating that these pathways may be more relevant to controlling GvHD than PD-1:PD-L1 expression. Disclosures No relevant conflicts of interest to declare.

Genomic Loss of the Mismatched HLA Locus in Leukemia Is a Major Mechanism of in Vivo Escape from T Cell Immunosurveillance Following Haploidentical HSCT

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 828-828 ◽  
Author(s):  
Luca Vago ◽  
Serena Kimi Perna ◽  
Monica Zanussi ◽  
Benedetta Mazzi ◽  
Maria Teresa Lupo Stanghellini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Direct Evidence ◽  
De Novo ◽  
Patient Specific ◽  
Post Transplant ◽  
Donor T Cells ◽  
Leukemia Relapse

Abstract Hematopoietic Stem Cell Transplantation (HSCT) from haploidentical family donors is a promising therapeutic option for nearly all patients suffering from high-risk leukemia. Until now, its application has been limited by the prolonged immunodeficiency that patients suffer as a consequence of graft T cell depletion, used to prevent severe Graft versus Host Disease (GvHD). When efficient strategies to control GvHD are applied, adoptive immunotherapy with donor T cells grants a significant advantage for immune reconstitution. However, direct evidence for the role of haploidentical donor T cells in controlling leukemia relapse is still missing. Here we report on the in vivo selection of de novo mutant variants of acute myeloid leukemia (AML), accounting for relapse after haploidentical HSCT and adoptive transfer of donor T cells. These novel variants of AML were observed in 5 out of 17 (29%) patients suffering from disease relapse in a series of 43 patients transplanted at the San Raffaele Hospital in Milan from 2002 to 2008. All patients received a myeloablative conditioning regimen and high doses of haploidentical donor stem cells (median 10.2×106 CD34+ cells/kg, range 4.6–15.5). Donor T lymphocytes were infused as part of the graft (n=21, median 438×106 CD3+ cell/kg, range 179–796) or as post-transplant add-backs (n=22, median 111×105 CD3+ cell/kg, range 1–900). Human Leukocyte Antigen (HLA) genomic typing was routinely used for post-transplant donor-recipient chimerism assessment. The five patients with de novo mutant variants of the original leukemia came to our attention because patient-specific HLA alleles could not be detected in bone marrow samples harvested at disease relapse, nor in subsequently sorted AML blasts. A Loss of Heterozygosity (LOH) study was performed on purified blasts from these patients, and demonstrated that patient-specific HLA alleles were lost due to extensive events of homologous recombination, encompassing a region of chromosome 6 comprising the entire HLA locus. We show that donor T cells capable of recognizing the original, HLA-heterozygous, leukemia were efficiently transferred from the haploidentical donor to the patient, granting an in vivo cytotoxic, cytokine and proliferative anti-tumor response by specific recognition of the mismatched HLA molecules. However, consistent with genomic loss of the patientspecific HLA locus in disease recurrence, the same alloreactive T cells were unable to recognize the mutant variant of the leukemia, harvested at the time of relapse. This observation strongly suggests that the genomic rearrangements we identified granted the disease an in vivo selective advantage in escaping from an established donor T cell response. Taken together, our data show that adoptive transfer of alloreactive donor T cells in haploidentical HSCT is efficient in providing a patient-specific antileukemic effect, and that the loss of this effect is an important mechanism underlying the outgrowth of relapsing disease. The frequency we documented for this phenomenon calls for routine assessment of the leukemia HLA genotype in the post-transplant follow-up and for careful consideration in the choice of a putative second haploidentical donor in case of leukemia relapse. Ultimately, our data provide the first direct evidence for the role of donor T cell alloreactivity in controlling minimal residual disease after haploidentical HSCT, favoring the use of donor T cell-based immunotherapeutic strategies to exploit alloreactivity for the cure of high-risk leukemia.

Allografts Selectively Photodepleted of GvHD Causing T Cells and Followed by Low-Level Immunosuppression: A Novel Method to Improve Disease Control After HLA-Matched Sibling Transplantations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 515-515
Author(s):  
Stephan Mielke ◽  
Aarthi Shenoy ◽  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Zachariah A. McIver ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
High Risk ◽  
The Netherlands ◽  
Acute Gvhd ◽  
Study Cohort ◽  
Post Transplant ◽  
Current Trial ◽  
Donor T Cells ◽  
Grade Iii

Abstract Abstract 515 We established a highly efficient GMP-grade, ex-vivo selective allodepletion process where host-activated donor T cells are eliminated based on their preferential retention of the photosensitizer 4,5-dibromorhodamine 123 (TH9402) and exposure to visible light (Kiadis Pharma, The Netherlands). As relapse of disease largely impairs the overall success of allogeneic stem cell transplantation we aimed to improve this outcome by using selectively T cell depleted allografts in order to reduce post transplant immunosuppression and thereby enhance graft versus malignancy effects. To determine the appropriate level of post transplant immunosuppression we designed a three sequential de-escalation stage trial with grade III-IV acute GvHD as the primary endpoint involving 17 patients per study cohort. Here we report on the first completed study cohort of NIH trial 07-H-0136 where seventeen patients (median age 44 (28-68) years) with hematological malignancies received a CD34-selected (Miltenyi, Germany) stem cell allograft together with 5 × 106/kg selectively depleted donor T cells following an age-adapted, radiation-based preparative regimen (FluCyTBI). Eleven patients had high risk disease (including ALL (Ph+, CR>1), refractory NHL, AML/MDS, AML with chloroma and blast crisis CML). Low-dose cyclosporine was used as sole immunosuppression for 90 days post transplant in the absence of GvHD. At a median follow-up of 385 (119-714) days actuarial probabilities (±SEM) of acute GvHD were 35±12% for grade II-IV and 0% for grade III-IV. Non-relapse mortality (NRM) was low with 17±11%. Overall survival (OS) was 73±12% and relapse-free survival (RFS) was 65±13% with a relapse probability of 21±11% (Figure). A low relapse incidence in a high-risk population suggests functionality of selectively allodepleted T cells. The absence of severe GvHD reflects the efficacy of the allodepletion process. Based on these findings we have initiated recruitment for the next study cohort where post transplant immunosuppression will be limited to 45 days only. Ultimately the aim is to achieve further reduction in immunosuppression in the absence of severe GvHD in order to enhance graft versus malignancy effects and thereby improve the outcome after allogeneic stem cell transplantation especially for patients at high risk for relapse. Disclosures: Mielke: Kiadis Pharma, The Netherlands: Research Funding, The current trial is supported under a clinical trial agreement between NHLBI and Kiadis.. Savani:Kiadis Pharma Inc., The Netherlands: Consultancy. Barrett:Kiadis Pharma, The Netherlands: The current trial is supported under a clinical trial agreement between NHLBI and Kiadis..

MiR-146a Regulates the TRAF6/TNF-Axis in Donor T Cells during Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 843-843
Author(s):  
Natalie Stickel ◽  
Gabriele Prinz ◽  
Dietmar Pfeifer ◽  
Annette Schmitt-Graeff ◽  
Marie Follo ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Negative Regulator ◽  
Snp Analysis ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract Introduction: Acute graft-versus-host disease (GvHD) arises from the attack of recipient tissues by donor allogeneic T cells and represents one of the major limitations of allogeneic hematopoietic cell transplantation (allo-HCT). In spite of many clinical trials, the standard immunosuppressive regimens for prevention of acute GvHD have improved little in the last two decades. Hence, a better understanding of the biology of acute GvHD may improve therapeutic options. MicroRNA-146a (miR-146a) was found to be increased in the sera of patients with GvHD. Therefore, we aimed to decipher the role of miR-146a in allogeneic donor T cells during GvHD by functional studies and in patients undergoing allo-HCT by single nucleotide polymorphism (SNP) analysis. Methods: We used two different murine major MHC mismatch models for acute GvHD. Recipient mice were conditioned with irradiation before transplantation of bone marrow and either wildtype or miR-146a deficient T cells from allogeneic donor mice. Furthermore, genomic DNA from 289 patients that underwent allo-HCT and their respective hematopoietic stem cell donors was isolated in order to determine their miR-146a rs2910164genotype. Results: We observed miR-146a upregulation in T cells of mice developing acute GvHD compared to untreated mice in a major MHC and a minor histocompatibility antigen mismatch model. Transfer of miR-146a deficient T cells caused increased GvHD severity, elevated TNF serum levels and reduced survival. Conversely, the phytochemical induction of miR-146a or its overexpression in donor T cells using a specific miR-146a mimic reduced GvHD severity. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was upregulated in miR-146a-/- T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased NF-κB activity and TNF production in miR-146a-/- T cells, while other pro-inflammatory cytokine levels were unaffected. The detrimental effect of miR-146a deficiency in T cells could be antagonized by TNF blockade in vivo. Moreover, in contrast to WT T cells, over expression of miR-146a in Tnf deficient T cells had no effect on their alloreactivity. In the human system, the minor genotype of the SNP rs2910164, which causes reduced miR-146a expression, was more frequent in patients developing acute GvHD grade III/IV compared to all other allo-HCT recipients (n=289). Conclusions: Taken together we show that miR-146a functions as a negative regulator of the TRAF6/TNF-axis in allogeneic donor T cells during GvHD, leading to reduced TNF transcription. Given our observation on the predictive role of the SNP leading to decreased miR-146a expression in acute GvHD in patients and the possibility to exogenously enhance miR-146a expression, we provide a novel and targeted molecular approach to mitigate GvHD. Disclosures No relevant conflicts of interest to declare.

Deletion of AMP-Activated Protein Kinase (AMPK) in Donor T Cells Protects Against Graft-Verus-Host Disease through Control of Regulatory T Cell Expansion and Target Organ Infiltration

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 806-806 ◽  
Author(s):  
Kevin Beezhold ◽  
Nathan Moore ◽  
Pailin Chiaranunt ◽  
Rebecca Brown ◽  
Craig A. Byersdorfer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lower Percentage ◽  
Cd4 Cells ◽  
Post Transplant ◽  
Alloreactive T Cells ◽  
Target Organs ◽  
Donor Cells ◽  
Donor T Cells

Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) represents a curative treatment for high-risk leukemia and a number of non-malignant hematologic disorders. However, the therapeutic use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that the alloreactive T cells responsible for GVHD increase their dependence on the oxidation of fat relative to either syngeneic or naive T cells. To explore this adaptation mechanistically, we studied the role of AMPK, an intracellular energy sensor and known driver of fat oxidation, in donor T cells during GVHD. Alloreactive T cells increased phosphorylation of AMPK as early as day 3 post-transplant, with up-regulation in pathways both up- and downstream of AMPK. Changes in phosphorylation were up to 8-fold higher in alloreactive T cells compared to naive T cells or syngeneic controls (p=0.0003). We then investigated the role of AMPK during GVHD pathogenesis using donor cells deficient in AMPK (from AMPKα1fl/flα2fl/fl x CD4-Cre mice). AMPK-/- T cells caused significantly less GVHD in both major-MHC and minor-histocompatibility mismatch models of GVHD (Figure 1A), with a coordinated decrease in the number of donor T cells recovered on day 7 post-transplant (3.15 +/- 0.49x106 versus1.87 +/- 0.53x106, p=0.0006, wildtype (wt) versus AMPK-/- respectively). Importantly, expansion of syngeneic T cells was unaffected by AMPK deficiency (Figure 1B). We next investigated the ability of AMPK-/- T cells to mount effective cytotoxic and anti-leukemia responses. AMPK-/- T cells demonstrated equivalent cytotoxicity against MHC-mismatched targets both in vitro and in vivo and differentiated in similar proportions into cytokine-producing cells (IFN-γ, TNFα, IL-17, and IL-4). We then assessed graft-versus-leukemia (GVL) potential in AMPK-/- cells using a GVL model with high tumor burden. AMPK-/- T cells exhibited equivalent clearance of p815 leukemia cells on day 13 post-transplant (Figure 2A), and extended survival of recipient mice similarly to wt T cells (Figure 2B). To elucidate possible mechanisms underlying this separation of GVL and GVHD responses, we evaluated metabolic pathways in wt and AMPK-/- T cells recovered on day 7. To our surprise, rates of fatty acid oxidation were identical between wt and AMPK-/- T cells and loss of AMPK did not impact alloreactive T autophagy, nor impair signaling downstream of mammalian target of rapamycin. To define the mechanism underlying AMPK-/- benefits, we quantitated levels of regulatory T cells (Treg) on day 7 post-transplant. In contrast to expectation, both the percentage and total number of Treg increased in mice receiving AMPK-/- T cells (0.85 +/- 0.32x104 vs. 1.69 +/- 0.34x104, wt vs. AMPK-/-, p=0.004). Loss of AMPK facilitated donor Treg expansion, as elimination of FoxP3+ cells prior to transplantation abrogated differences between wt and AMPK-/- donors on day 7. Importantly, Treg levels were equal in wt versus AMPK-/- donors prior to transplantation. Finally, we assessed the ability of AMPK-/- T cells to infiltrate into GVHD target organs. As shown in Figure 3, peri-portal infiltration of AMPK-/- cells was significantly reduced compared to wt T cells, and infiltrates in recipients of AMPK-/- cells contained many fewer CD3+ T cells per high-powered field, with CD3+ cells representing a lower percentage of cells overall. Decreased hepatic infiltration correlated with a lower percentage of circulating CD4+ cells and lower levels of the integrin pair α4β7 (55.2 +/- 1.4% versus 47.2 +/- 2.8% α4β7Hi cells, p=0.0017, wt vs. AMPK-/-). In conclusion, deletion of AMPK in donor T cells decreases GVHD severity but spares anti-leukemia responses and preserves homeostatic immune reconstitution. Mechanistically, this occurs through a decrease in pathogenic T cell numbers, an increase in the number and percentage of Treg cells, fewer circulating CD4+ cells, and decreased infiltration of donor cells into target organs. From these findings, we conclude that AMPK represents a clinically relevant target in donor T cells pre-transplant and are actively exploring ways to translate this exciting therapy into clinical practice. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Critical Role for Donor-Derived IL-22 in Cutaneous Chronic Gvhd

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 69-69
Author(s):  
Kate H Gartlan ◽  
Hemamalini Bommiasamy ◽  
Katelyn Paz ◽  
Andrew Wilkinson ◽  
Mary Owen ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Acute Gvhd ◽  
Chronic Gvhd ◽  
Protein Detection ◽  
Cytokine Gene Expression ◽  
Cell Transplant ◽  
Cutaneous Manifestations ◽  
Post Transplant

Abstract Graft-versus-host disease (GVHD) is the major cause of non-relapse morbidity and mortality after allogeneic stem cell transplant (allo-SCT). Chronic cutaneous manifestations of GVHD are common late after allo-SCT with limited treatment options beyond protracted steroid therapy. Cytokines are critical mediators of inflammatory processes during GVHD and both IL-17A and IL-22 have been found to have dual pathogenic and protective roles, which are largely dependent upon their cellular source. We have demonstrated in mice that whilst recipient-derived IL-17A and IL-17R signaling suppresses inflammatory responses and prevents gut dysbiosis, donor T cell-derived IL-17A is pathogenic and drives GVHD in both lung and skin. Similarly, contrasting roles have also been described for IL-22 in mice, such that recipient-derived IL-22 helps maintain gut epithelial integrity after allo-SCT, but donor IL-22 can exacerbate acute GVHD pathology. To date, investigation of the effects of donor-derived IL-22 in allo-SCT has focused on acute GVHD, with no data regarding the role of IL-22 in chronic GVHD. Given the complexities and the potential competing risks of targeting IL-22 early post-transplant, we examined IL-22 deficiency in murine models of chronic GVHD following allo-SCT. In the absence of donor-derived IL-22, we observed a significant decrease in skin GVHD clinical scores and chronic GVHD skin pathology (histopathology scores in WT donors: 10.2±2.1, vs. IL-22-/- donors: 2.6±0.9, p &lt;0.01), which is in line with the pathogenic role of IL-22 in autoimmune skin diseases. Intriguingly, lung manifestations of chronic GVHD (i.e. bronchiolitis obliterans) were not IL-22-dependent (histopathology scores in WT donors: 1.6±0.5, vs. IL-22-/- donors: 1.5±0.3, p &gt;0.05), despite the fact we have recently defined their IL-17-dependency. These data demonstrate a relationship between IL-22 and IL-17 and the distribution of chronic GVHD. We identified CD4+ T cells as the major source of donor IL-22 by both direct protein detection and cytokine reporter systems, where we observed co-expression of IL-22 with a range of pro-inflammatory cytokines including IL-17A, IFNγ, GM-CSF and TNF. We identified IL-22+IL-17A+ and IL-22+IL-17A- CD4+ T cells as two distinct sources of donor-derived IL-22 post-transplant (2.2±0.1% and 6.5±0.3 % of LN CD4+ T respectively), both of which were highly dependent upon IL-6 for their development (IL-22+IL-17A+: IgG 1.3±0.2% vs. anti-IL-6Rα 0.08±0.01% of splenic CD4+ T, p &lt; 0.001; IL-22+IL-17A-:IgG 3.4±0.2 vs. anti-IL-6Rα: 0.6±0. 1% of splenic CD4+ T, p &lt; 0.001). Since we have previously identified significant cytokine plasticity within IL-17A+ T cells after allo-SCT, we performed lineage assessment to determine if these two populations arose independently. Using IL-17creRosa26YFP fate-mapping mice that permanent label any cell that has expressed IL-17A, we found that IL-22+IL-17A- CD4+ T cells did not have a history of IL-17A production and were therefore identified as a definitive Th22 population after allo-SCT. Since IL-22 and IL-17A have been reported to induce synergistic responses in the skin, we also explored the possibility of interdependence between Th17 and Th22 development. Using both IL-17RC-/- mice and an IL-17creRosa26YFP/iDTR reporter-deleter system we demonstrate that IL-17 signaling to donor Th22 directly promotes their development in allo-SCT (WT donors: 6.5±0.4 %, Th/Tc17 deleter donors: 3.6±0.2, IL-17RC-/- donors: 3.7±0.3 % Th22 in LN CD4+ T, p &lt;0.001). Finally, we observed a similar pro-inflammatory cytokine gene expression signature (Il22, Il17, Ifng) in the skin of patients who developed cutaneous GVHD &gt; 100 days following allo-SCT but not in those after autologous SCT. These data demonstrate a key role for donor-derived IL-22 in chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation. Together these findings suggest that IL-22 intervention late post-transplant may reduce cutaneous chronic GVHD, whilst maintaining the protective effects of IL-22 in the gut early after transplant. Disclosures Serody: Merck: Research Funding.

Paradoxical effects of interleukin-18 on the severity of acute graft-versus-host disease mediated by CD4+ and CD8+ T-cell subsets after experimental allogeneic bone marrow transplantation

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3393-3399 ◽  
Author(s):  
Chang-Ki Min ◽  
Yoshinobu Maeda ◽  
Kathleen Lowler ◽  
Chen Liu ◽  
Shawn Clouthier ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Interleukin 18 ◽  
Graft Versus Host ◽  
Donor T Cells ◽  
Paradoxical Effects

Abstract Administration of exogenous interleukin-18 (IL-18) regulates experimental acute graft-versus-host disease (GVHD) in a Fas-dependent manner when donor CD4+ T cells are required for mortality after experimental allogeneic bone marrow transplantation (BMT). However, CD4+ and CD8+ T cells can induce acute GVHD after clinical allogeneic BMT, and the role of IL-18 in CD8+-mediated acute GVHD is unknown. We, therefore, determined the role of IL-18 in GVHD mediated by CD4+ or CD8+ T cells across major histocompatibility complex (MHC) class II- and class I-disparate allogeneic BMT, respectively. Administering IL-18 significantly increased survival in CD4+-mediated GVHD but reduced survival in CD8+-mediated GVHD. This increase in deaths was associated with significantly greater clinical, biochemical, and histopathologic parameters of GVHD damage and was independent of Fas expression on donor T cells. Administering IL-18 significantly enhanced allospecific cytotoxic function and expansion of CD8+ cells. Endogenous IL-18 was critical to GVHD mediated by CD8+ donor T cells because IL-18 receptor-deficient donors caused significantly less GVHD but exacerbated CD4+-mediated, GVHD-related death. Furthermore, administering anti-IL-18 monoclonal antibody significantly reduced CD8+-mediated, GVHD-related death. Together these findings demonstrate that IL-18 has paradoxical effects on CD4+ and CD8+ cell-mediated GVHD. (Blood. 2004;104:3393-3399)

Mechanisms of Migration and Generation of Allo-Reactive Donor T Cells That Cause Organ Specific Acute GvHD in Allogeneic BMT Recipients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3270-3270
Author(s):  
Mohammad S. Hossain ◽  
Ned Waller
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Lymph Nodes ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Post Transplant ◽  
Allogeneic Bmt ◽  
Donor T Cells ◽  
Organ Specific

Background: Allo-reactive donor T cells are primarily responsible for GvHD in allogeneic BMT. A number of studies have shown that increased allo-reactivity is found among the CD62L+ subset of donor T-cells, but the mechanisms for organ specific allo-reactivity are poorly defined. Our hypothesis is that rapid proliferation and migration of CD62L+ naive donor CD4+ and CD8+ T cells to specific organs leads to acute GvHD. Methods: We used a parent (C57BL/6) to (C57BL/6 × BALB/c) CB6F1 allogeneic BMT model with a combination of T cell depleted BM (TCD BM) and splenocytes. 30 × 106 congeneic donor splenocytes labeled with CFSE were transplanted with 5 × 106 TCD congeneic BM into lethally irradiated (11Gy) CB6F1 mice. Recipients were sacrificed within 3.5 days of transplant and FACS was used to measure proliferation of CFSE-labeled donor T-cells isolated from blood, spleen, liver, lungs, thymus, BM, lymph nodes, and peritoneal exudates cells (PEC). Syngeneic C57BL/6 recipients served as controls. At least 5 mice per group were used in each experiment. Results: There was increased homing of CFSE-labeled donor T-cells to most organs in allogeneic compared to syngeneic BMT recipients. CD45.1+ donor cells were 4-fold higher in spleen, p=0.01; 9-fold higher in liver, p=0.002; 14-fold higher in PEC, p=0.017; 136-fold higher in lung, p=0.0006; 126-fold higher in BM, P=0.002, 1482-fold higher in thymus p=0.002 compared to syngeneic recipients. Allogeneic and syngeneic recipients had equivalent numbers of donor CFSE-labeled lymphocytes in PBMC and lymph nodes. The tissue specific homing of CD4+ and CD8+ donor T-cells was also found significantly higher in most organs except the PBMC and LNs. Donor splenocytes were 80% CD62L+ before transplant, but the frequency of CD62L+ donor T-cells had declined to 15–16% in BM, 4–10% in liver, 17–30% in spleen and 10 to 25% in the thymus within 3.5 days post-transplant. In syngeneic recipients, 80% of donor T-cells remained CD62L+ within 3.5 days post-transplant. Most donor T-cells that divided rapidly lost expression of CD62L, while non-replicating donor CD4+ and CD8+ T cells remained predominately CD62L+. The expression of CD44 on donor T-cells were the opposite, with CD44+ cells undergoing less, and CD44− cells dividing more in allogeneic transplant recipients. In syngeneic BMT, donor CD4+ and CD8+ T-cells underwent minimal proliferation within the first 3.5 days post-transplant. Intracellular cytokine staining showed that high levels of IFN-g and TNF-a synthesis was seen among CD62L+ CD4+ and CD8+ T cells that had yet to divide (and had un-diluted CFSE staining). Conclusion: Migration of allogeneic donor T cells to tissues and local proliferation occurs rapidly after allogeneic BMT compared to recipients of syngeneic transplants. The dissociation of CD62L expression from lymph node homing suggests lack of the CD62L-receptor expression in lymph node HEV following irradiation, or a dominant effect of other chemokine receptors in directing donor T-cell preferentially to other organs. The marked and preferential homing of donor T-cells to the recipient thymus and bone marrow may play a role in achieving donor hematopoietic and T-cell chimerism in recipients of allogeneic BMT. Tissue specific homing of naive CD62L+ donor T-cells, with a high proliferative capacity, is likely responsible for the initiation of acute GvHD at these sites.

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