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

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.

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

Blood ◽

10.1182/blood.v110.11.3270.3270 ◽

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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Attenuated Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of RORγt.

Blood ◽

10.1182/blood.v116.21.3742.3742 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3742-3742

Author(s):

LeShara M Fulton ◽

Michael J Carlson ◽

James Coghill ◽

Michelle L. West ◽

Angela Panoskaltisis-Mortari ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Graft Versus Host Disease ◽

Allogeneic Stem Cell Transplantation ◽

Graft Versus Host ◽

Donor T Cells

Abstract Abstract 3742 CD4+ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt−/−), a transcription factor crucial for Th17 polarization. Methods: Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 × DBA2 F1 (H-2bxd, B6D2) mice functioned as recipients. Effector T cells (Teffs) were isolated from the spleens of wild type (WT) B6 and RORgt knockout mice backcrossed 7–8 generations onto a B6 background. B6D2 mice were lethally irradiated with 900 rads on day -1 and injected intravenously with 4 × 106 Teffs from WT or RORgt−/− mice supplemented with 3 × 106 WT T cell depleted bone marrow cells (TCD BM) on day 0. For the completely MHC mismatched model, BALB/c mice (H-2d) were lethally irradiated with 800 rads on day -1 and administered 5 × 105 WT or RORgt−/− Teffs supplemented with 5 × 106 B6 TCD BM on day 0. Results: B6D2 mice that received RORgt−/− Teffs displayed significantly attenuated GvHD, recovering from weight loss by day +31 and demonstrating 100% survival on day +60. Conversely, mice that received WT Teffs showed intense disease progression with 100% mortality by day +31 (Figure A, p<0.0001 for survival comparison between WT and RORgt−/− recipients using Fisher's exact test). Similar results were seen using the completely MHC mismatched model, with superior overall survival noted in those animals receiving RORgt −/− Teffs (put in p value here). Recipients of RORgt −/− T cells demonstrated statistically significant decreased TNF in serum compared to WT recipients (Figure B, p=0.001 comparing WT and RORgt−/− recipients using student's t test). Interestingly, despite the decreased severity of GvHD, serum concentrations of IFN-g were increased in recipients transplanted with RORgt −/− T cells. Chimerism studies post-transplant revealed complete donor reconstitution in recipients of both RORgt−/− and WT Teffs. Donor Teffs isolated from recipient livers post-transplant consistently demonstrated an activated phenotype, with low L selectin and high CD25 expression. Conclusions: T cell expression of the Th17 transcription factor, RORgt, is critical for the development of lethal GvHD following allogeneic stem cell transplantation in both the haploidentical and MHC complete mismatch models. GvHD attenuation in the absence of RORgt is not the result of an inability for donor T cells to undergo activation or to engraft in vivo. Interestingly, the absence of RORgt from donor T cells led to enhanced IFN-g in serum. Thus, in vivo, the Th17 pathway is critical for the induction of GvHD. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.144.144 ◽

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.

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Post-Transplant Cyclophosphamide Ameliorates Lethal Thymic GVHD By Restoring Regulatory and Effector T Cell Homeostasis in Recipients with Prior PD-1 Blockade Therapy

Blood ◽

10.1182/blood-2018-99-111142 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 479-479

Author(s):

Shuntaro Ikegawa ◽

Yusuke Meguri ◽

Takumi Kondo ◽

Hiroyuki Sugiura ◽

Yasuhisa Sando ◽

...

Keyword(s):

Overall Survival ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

T Cell Subsets ◽

Post Transplant ◽

Gvhd Prophylaxis ◽

Donor T Cells ◽

The Impact

Abstract Allogeneic HSCT has a curative potential for patients with hematological malignancies. However, graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after HSCT. Regulatory T cells (Tregs) are critical mediator for immune tolerance after HSCT and we recently reported that PD-1 plays an essential role for Treg survival (Asano et al, Blood 2017). Clinical studies suggested that PD-1 blockade prior to HSCT could be a risk of increasing severe GVHD. However, the mechanisms about GVHD induced by PD-1 blockade have largely unclear and there remains a paucity of data on appropriate GVHD prophylaxis for patients who undergo HSCT after PD-1 blockade. To address these issues, we investigated the impact of PD-1 expression on donor T cells on immune reconstitution with murine BMT models. First, lethally irradiated B6D2F1 mice were transplanted with 10 million of C57BL/6-background PD-1+/+ or PD-1-/- spleen cells with 5 million of bone marrow cells from normal C57BL/6, and GVHD scores and overall survival was monitored. Recipients receiving PD-1-/- graft developed severe GVHD resulting in a significant shorter survival than recipients receiving PD-1-/- graft (P<0.0001). We analyzed lymphocytes in spleen and thymus on day3, 7, and 14. We found that CD8 T cells in PD-1-/- group showed markedly higher Ki67 expression and CFSE-dilution until day3. Interestingly, PD-1-/- Tregs increased aggressively at day3 but it could not maintain until day14, while PD-1-/- CD8 T cells and conventional CD4 T cells (CD4 Tcons) continued to increase until day+14, resulting in the significant higher CD8/Treg ratio in PD-1-/- group (P<0.05, vs PD-1+/+ group). PD-1-/- Tregs showed significantly higher expression of Annexin V on day+7 and thymus CD4- and CD8- double-positive (DP) cells were in the extremely low levels in PD-1-/- group on day+14 (P<0.05, vs PD-1+/+ group). Thymic analysis showed that donor PD-1-/- graft-derived CD8 T cells infiltrated thymus in PD-1-/- group, suggesting reconstruction of thymic function was critically disturbed by severe GVHD. These data suggest that loss of PD-1 signaling resulted in unbalanced reconstitution of donor-derived T cell subsets as a consequence of continuous CTL expansion and increased Treg apoptosis. Next, to evaluate the impact of post-transplant cyclophosphamide (PTCy) on the abnormal reconstitution after PD-1 blockade, we administered 50mg/kg of Cy or control vehicle on day3. PTCy efficiently ameliorated GVHD in PD-1-/- group and extended overall survival by safely regulating the proliferation and apoptosis of T cell subsets. Of note, after PTCy, Tregs regained the ability of continuous proliferation in the first 2 weeks, resulting in well-balanced reconstitution of donor-derived T cell subsets. Thymic DP cells on day 14 was markedly increased in PD-1-/- group with PTCy intervention as compared to without PTCy, suggesting PTCy could rescue thymus from PD-1 blockade-related severe GVHD. Finally, to evaluate GVL activity, we performed BMT with co-infusion of P815L tumor cells on day0 and we confirmed that PTCy treatment for PD-1-/- recipients reduced the severity of GVHD with maintaining sufficient GVL effect. In summary, our data suggested three insights about the impact of PD-1 signaling on immune reconstitution. First, PD-1 inhibition influenced graft-derived T cells very differently within T cell subsets. PD-1-/- Tregs increased transiently but it was counterbalanced by accelerated apoptosis, while PD-1-/- CD4+Tcons and CD8 T cells continued the drastic expansion. Second, we found that PD-1-/- donor T cells developed severe GVHD in thymus. Few reports have concentrated on the impact of donor graft PD-1 expression to thymus after BMT and acute GVHD in thymus could lead late central immune disturbance. Third, PTCy successfully ameliorated GVHD induced by PD-1-/- donor T cells preserving GVL effect. Cell proliferation study implied that PD-1-/- graft-derived CD8 T cells might be more susceptible for PTCy because of the high-rate proliferation. In conclusion, PD-1-/- graft cause lethal thymic GVHD and PTCy successfully ameliorated it. The influence of PD-1 inhibition was different within T cell subtypes. PTCy might be appropriate GVHD prophylaxis strategy for patients who had prior usage of PD-1 blockade. Disclosures No relevant conflicts of interest to declare.

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Donor T Cells Maintain Myeloma-Immune Equilibrium after Autologous Stem Cell Transplantation and Concurrent Immunotherapy Promotes Cure

Blood ◽

10.1182/blood-2018-99-118058 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2031-2031

Author(s):

Simone A Minnie ◽

David Smith ◽

Kate H Gartlan ◽

Thomas S Watkins ◽

Kate A Markey ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Median Survival ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Post Transplant

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidation treatment for multiple myeloma (MM) patients, even in the era of novel agents. The prolongation of plateau-phase induced by ASCT is generally attributed to intensive cytoreduction. However, ASCT generates inflammation and profound lymphodepletion, which may result in hitherto unexpected immunological effects. To investigate potential immunological contributions to myeloma control after ASCT, we developed preclinical models of transplantation for MM using Vk*MYC myeloma that generates bony lytic lesions, a serum M band and marrow plasmacytosis that are hallmarks of clinical disease. Myeloma-bearing B6 recipients underwent myeloablative conditioning and were transplanted with naïve B6 bone marrow (BM) grafts with or without T cells from donors that were myeloma-naïve (SCT) or had low M bands at the time of harvest to mimic ASCT. Surprisingly, we demonstrate the broad induction of T cell-dependent myeloma control with enhanced median survival in recipients of grafts containing T cells compared to T cell depleted (TCD) BM alone (SCT= 91 days and ASCT > 100 days post-transplant vs TCD BM alone= 44 days; p<0.0001). Myeloma was most efficiently controlled when recipients were transplanted with memory T cells (CD44+) from autologous grafts (median survival: ASCT-CD44+ T cells >90 days post-transplant vs. CD44─ T cells = 50 days; p = 0.0006). Importantly, T cells adoptively transferred from recipients surviving > 120 days (MM-primed) protected secondary recipients compared to T cells from naïve donors (median survival: MM-primed > 120 days post-transplant vs 65 days naïve T cells; p = 0.0003). Furthermore, MM-primed CD8 T cells were restricted in TCR repertoire and provided protection in a myeloma clone-specific fashion, indicative of a tumor-specific T cell response. Despite this immune-mediated control of myeloma after SCT, progression still occurred in the majority of recipients. We phenotyped CD8+ T cells from the BM of MM-relapsed, MM-controlled and MM-free (that had never seen myeloma) mice 8 weeks after SCT. Expression of the inhibitory receptors, programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on BM CD8+ T-cells strongly correlated with myeloma cell number (r = 0.729, p<0.0001 and r = 0.796, p<0.0001 respectively). Additionally, the co-stimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated as myeloma progressed (r = - 0.865, p<0.0001), as was interferon-γ secretion (r = - 0.76, p = 0.0022). t-SNE analysis confirmed an irreversible exhaustion signature at myeloma progression, characterized by the absence of DNAM-1 and co-expression of PD-1, TIM-3, TIGIT together with CD101 and CD38. Immune-checkpoint inhibition (CPI) early post-SCT, using antibodies against PD-1 or TIGIT facilitated long-term myeloma control (median survival in both treatment arms > 120 days post-SCT vs. 60 and 68 days respectively; p <0.05). Furthermore, TIGIT blockade limited CD8+ T cell exhaustion, increased CD107a and IFNγ secretion and expanded a memory CD8+ T cell population in the BM. Genetic deletion of either IFNγ or the IFNγ receptor from the donor graft resulted in dramatic myeloma progression after SCT. Consequently, treatment with a CD137 (4-IBB) agonist early after SCT profoundly augmented CD8+IFNγ+GranzymeB+ T-cell expansion in the BM, such that majority of treated animals eliminated myeloma and survived long-term. These data provide insights into an unappreciated mechanism of action of ASCT whereby myeloma immune-equilibrium is established and suggest that combination with immunotherapeutic strategies is a rational approach to generate long term disease control. Disclosures Smyth: Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding.

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Nivolumab for Relapsed or Residual Haematological Malignancies after Allogeneic Haematopoietic Stem Cell Transplantation (NIVALLO)

Blood ◽

10.1182/blood-2018-99-112310 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4633-4633 ◽

Cited By ~ 4

Author(s):

Eric Wong ◽

Emily Dawson ◽

Joanne Davis ◽

Rachel Koldej ◽

Mandy Ludford-Menting ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Acute Gvhd ◽

Haematological Malignancies ◽

Advisory Committees ◽

Safety And Efficacy

Abstract Aim: To evaluate the safety and efficacy of nivolumab for the treatment of relapsed or residual haematological malignancies after allogeneic stem cell transplantation (alloSCT). Background: Relapse of haematological malignancies following alloSCT is a major cause of post-transplant mortality. Interaction between programmed cell death protein-1 (PD-1) and its ligand (PD-L1) inhibits T-cell alloreactivity and contributes to immune escape. Nivolumab inhibits PD-1 signalling and augments T-cell cytotoxicity. The safety and efficacy of nivolumab post-alloSCT has not been evaluated in a clinical trial. Method: In this investigator-initiated phase IIa clinical trial, patients with relapsed or persistent haematological malignancies following alloSCT receive nivolumab 3mg/kg for up to 48 weeks. Patients with current graft-versus-host disease (GVHD) or prior grade ≥2 acute GVHD or chronic GVHD are excluded. Results: Six participants have received at least one dose of nivolumab at this interim assessment. Primary haematological malignancies relapsing post-alloSCT included Hodgkin lymphoma (HL, 2 patients), acute myeloid leukaemia (AML, 2), transformed chronic lymphocytic leukaemia (tCLL, 1) and mantle cell lymphoma (MCL, 1). The median time from alloSCT to first dose of nivolumab was 25.5 months. Two participants developed grade 3 acute GVHD at 6 days and 13 days following the first dose of nivolumab. Complete or partial responses were observed in 3 participants (50%). Two participants with HL achieved complete responses. One participant with MCL had a complete nodal response with small volume persistent bone marrow disease. One participant with monosomal karyotype AML achieved initial blast reduction (23% to 13%) however subsequently developed progressive AML. T-cell phenotyping at first AML relapse (prior to nivolumab) demonstrated a high proportion of CD8+ T cells that expressed PD-1 and T-cell immunoglobulin and mucin domain 3 (TIM-3) consistent with T-cell exhaustion. Following treatment with nivolumab there was an increase in TNFα production by CD8+ T-cells at day 7 post nivolumab, demonstrating augmentation of T-cell activity. Despite continued nivolumab treatment TNFα production subsequently declined and correlated with loss of clinical response. TIM-3 expression was further upregulated at post-nivolumab progression suggesting this inhibitory checkpoint receptor may have contributed to nivolumab resistance. Conclusion: Nivolumab treatment after alloSCT results in potent immune stimulation with a high rate of clinical responses, albeit with a risk of GVHD. Acquired resistance to nivolumab may develop via upregulation of alternative inhibitory checkpoints. Disclosures Szer: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support , Research Funding. Grigg:BMS: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

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Donor-Derived T Cells Can Be Rendered Hyporesponsive to Alloantigen without Loss of Pathogen or Tumor Immune Responses.

Blood ◽

10.1182/blood.v110.11.771.771 ◽

2007 ◽

Vol 110 (11) ◽

pp. 771-771 ◽

Cited By ~ 2

Author(s):

Jeff Davies ◽

Dongin Yuk ◽

Lee Nadler ◽

Eva Guinan

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Acute Gvhd ◽

Alloreactive T Cells ◽

Cell Pool ◽

Donor T Cells ◽

Leukemia Antigen

Abstract The prevention of severe acute Graft-versus-Host Disease (GvHD) without impairment of immune reconstitution is the major challenge in HLA-mismatched hematopoietic stem cell transplantation (HSCT). One alternative to experimental strategies to selectively destroy or remove alloreactive T cells from the donor T cell pool is to induce hyporesponsiveness (anergy) in alloreactive T cells within the donor T cell pool and thus preserve the vast majority of T cell repertoire. We previously reported early clinical data of HLA-mismatched HSCT after alloanergization of donor bone marrow via ex vivo allostimulation in the presence of co-stimulatory blockade (CSB) with Cytotoxic T Lymphocyte Antigen-4 Immunoglobulin (CTLA4-Ig). Analysis of a larger cohort of such patients revealed a low rate of severe acute GvHD and very few clinically significant viral infections, with over 30% of patients (pts) surviving long-term without disease relapse. This suggested that CSB might indeed be controlling alloreactivity with preservation of pathogen-specific immunity and a graft-versus-leukemia (GvL) effect. We therefore sought to directly determine the effect of alloanergization of human donor T cells on alloreactivity, pathogen- and leukemia-antigen-specific immunity. After alloanergization via blockade of CD28-mediated co-stimulation with clinical-grade humanized anti-B7.1 and anti B7.2 antibodies, HLA-mismatched alloproliferative responses were reduced by 2 logs, a more efficient reduction in alloreactivity than previously reported with the use of CTLA4 Ig. Using CFSE-based labeling of human responder T cells we have demonstrated directly for the first time that alloanergization efficiently abrogates stimulator-specific alloproliferation in both CD4 and CD8 donor T cells, whereas third party responses are retained (Figure 1). Importantly, the strategy does not diminish the capacity of donor CD4 and CD8 T cells to mount a range of functional immune responses, including proliferation, cytokine production and cytotoxic responses, in response to stimulation with several human herpes viruses. We have also demonstrated that frequencies of WT1-specific IFN-g+ CD4 and CD8 T cells are not diminished after the process of alloanergization, showing that a T cell mediated GvL effect may be retained. Importantly we demonstrated retention of pathogen and leukemia antigen-specific responses to both MHC Class I- and II-restricted antigens and in both HLA-A2+ and non-HLA-A2+ responders. These data confirm that the technique of alloanergization can be used to provide non-alloreactive donor T cells without loss of beneficial CD4 and CD8 donor immunity. The optimal dose of HLA-mismatched alloanergized donor T cells that will improve immune reconstitution whilst controlling acute GvHD after HLA-mismatched HSCT remains to be defined. To answer this question, we have embarked on a dose-escalating clinical study of delayed alloanergized donor T cell infusion to improve immune reconstitution after haploidentical HSCT. Figure Figure

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Impact of Donor IL-7Rα Polymorphism On Recipient Plasma IL-7 levels and Acute Gvhd Following Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v116.21.1464.1464 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1464-1464

Author(s):

Stephanie Thiant ◽

Zaiba Shamim ◽

Lars Peter ◽

Valérie Coiteux ◽

Jean Paul Dessaint ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Cd8 T Cells ◽

Plasma Levels ◽

Acute Gvhd ◽

Memory T Cells ◽

Effector Memory ◽

Post Transplant ◽

Α Chain ◽

Central Effector

Abstract Abstract 1464 IL-7 is one of essential driving forces for homeostatic peripheral expansion of T lymphocytes that are responsible, not only for GVL effects but also for acute GVHD, a major post-transplant complication. High plasma levels of IL-7 in the early phase post-transplant, has been associated with high incidence of severe acute GVHD regardless the intensity of conditioning regimen. Inter-individual variations have also been reported. Here we aimed to identify factors that could have an impact on IL-7 level and, therefore, on acute GVHD. This prompted us to prospectively investigate plasma levels of IL-7, T-cell subsets recovery, T cells’ IL-7Rα chain expression, and IL-7Rα chain polymorphism in 100 pts who underwent fully HLA-matched allogeneic stem cell transplantation in our unit. Pts received either myeloablative (n= 60) or nonmyeloablative (n=40). Forty donors were unrelated. Source of stem cells, was bone marrow in 71 pts and PBCS in 29. Sex ratio (M/F) was (66/34) and median age at transplant was of 49 years. Plasma IL-7 level was determined by ELISA at enrolment, on day 0 before grafting, every three days during the first month, and then on days 60 and 90. CD3+, CD4+, CD8+ T-cells and NK cells counts at day 30, 60 and 90 post-graft were obtained by flow-cytometry-based technique. Expression of IL-7Rα (% and MFI) was evaluated on each subset of naïve and memory T-cells, categorized according to their expression of CD45RA and CCR7 markers. The detection of IL-7Ra single nucleotide polymorphism (SNPs) by sequence specific PCR (SSP), in donors, was carried out as described by Shamim et al, (BMT 2006). IL-7 receptor consisted of γc-chain and specific α-chain. A range of IL7R α-chain SNPs was reported (+510 C/T, +1237 A/G, +2087 T/C which all resulted in amino-acid substitution). At the time of analysis, 40 (40%) recipients had developed grade 2–4 acute GVHD (aGVHD) with a median time of 33 days post transplant. As expected, IL-7 levels peaked around the second week at median of 11.5 pg/mL (0.4-30.2) after transplant. Kinetic courses of plasma IL-7 levels, evolved inversely to lymphocyte counts up to d+30 (p<.001). The cumulative incidence of aGVHD was higher if by day+18 pts had IL-7 levels above the median concentration (p= .046). A higher level of IL-7 at day+18 was confirmed as a predictive factor of subsequent risk of aGVHD (HR= 1,079; 95% CI: 1.022 – 1.139; p= .006). By calculating the area under the curve of IL-7 between d-15 and d+30, we observe that a high exposure to IL-7 during the first month is correlated with the risk of aGVHD (p=.002). IL-7 plasma levels were inversely correlated with IL-7Rα expression only on central/effector memory CD4+ and central/effector memory CD8+, and terminally differentiated CD8+ T-cells (p =.006, .013, .044, .001 and .028, respectively). Of note, at d+30, pts had 85% (34-99) and 86% (23-99) of CD4+ and CD8+ memory T cells, respectively. Contrary to +1237 A/G and +2087 T/C, donor's +510 CC or CT was the only polymorphism to be associated with higher level of plasma IL-7 in recipients during the first month post-transplant in particular at d+18, predictive date for aGVHD (p = .026). In multivariate analysis, pts who received graft from donor with +510CC or CT experienced more often grade 2–4 aGVHD than those with +510 TT (P = .049). Collectively, this study confirms the role of IL-7 in grade 2–4 aGVHD. Indeed, the high level of IL-7 that down regulates IL-7Rα, could suggest activation and consumption of IL-7 by alloreactive T cells, including those involved in aGVHD development. By difference in affinity and cytokine consumption, the polymorphism +510 of donor t-cell IL-7R α-chain might explain, in part, the wide variation of IL-7 level among pts. Disclosures: No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood.v114.22.515.515 ◽

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..

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Definition of a Diagnostic Window Prior to the Onset of Clinically Apparent Acute Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v116.21.3746.3746 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3746-3746

Author(s):

Carina A Bäuerlein ◽

Simone S Riedel ◽

Brede Christian ◽

Ana-Laura Jordán Garrote ◽

Agnes Birner ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Critical Time ◽

Time Points ◽

T Cell Migration ◽

Donor T Cells

Abstract Abstract 3746 Acute graft-versus-host disease (aGvHD) is an immune syndrome after allogeneic hematopoietic cell transplantation (allo-HCT) caused by alloreactive donor T cells that attack the gastrointestinal tract, liver and skin. Thus, early T cell migration patterns to these organs could provide first cues for the onset of aGvHD. Hence, a unique surface marker profile of donor T cells at early time points after allo-HCT may be an indicator for patients at risk of aGVHD. Therefore, we analyzed the course of donor T cell activation, proliferation and homing in a clinical relevant murine MHC minor mismatch (miHAg) allo-HCT model to define critical time points and marker profiles for the detection of alloreactive T cells. Luciferase-labeled C57Bl/6 (H-2b) T cells plus bone marrow cells were transplanted into conditioned (8 Gy) MHC major mismatched Balb/c (H-2d) or miHAg Balb/b (H-2b) recipients. Donor T cell migration was visualized by in vivo bioluminescence imaging (BLI) and cells were characterized by multiparameter flow cytometry for 30 consecutive days after allo-HCT. GVHD scoring was performed by histopathology. Donor T cells proliferated exclusively in secondary lymphoid organs until day+3 (initiation phase) before migrating via the peripheral blood into target organs (effector phase). This occured in both models, MHC major mismatch and miHAg allo-HCT, which resulted in hyper-acute (starting at day+6) or acute GVHD (starting at day+21), respectively. In the hyper-acute scenario one wave of T cell migration starting at day+4 sufficed to cause lethal aGVHD. We detected a 4000-fold increase in CD4 and a 1500-fold increase in CD8 donor T cell numbers in the peripheral blood between day+3 and day+6 in this model. In contrast, in the more clinical relevant miHAg allo-HCT model we found 3 waves of T cell migration with peaks at days +6, +11 and +15 after allo-HCT. In the peripheral blood CD4 T cells increased 20-fold, CD8 T cells 50-fold between day+3 and day+6, but more than 40-fold (CD4) and 400-fold (CD8) between day+3 and day+11. After the third peak on day+15 a period followed when we could only detect very few migrating donor T cells in the peripheral blood before aGvHD became clinically apparent on day+21. Next, we asked whether we could identify alloreactive T cells by testing a large panel of surface markers at the defined migration peaks. Indeed, allogeneic T cells upregulated certain homing receptors at these peaks (e.g. at day+11: α4β7 integrin: 27% of CD4 T cells, 3.4×104/ml, 60% of CD8 T cells, 1.6×105/ml; P-selectin ligand: 28% of CD4 T cells, 3.5×104/ml, 35% of CD8 T cells, 9.1×104/ml). In contrast, syngeneic transplanted mice only showed a constant low expression level of those receptors (e.g. at day+11: α4β7 integrin: 20% of CD4 T cells, 9.6×103/ml, 5% of CD8 T cells, 3.1×103/ml; P-selectin ligand: 17% of CD4 T cells, 8.5×103/ml, 10% of CD8 T cells, 6.6×103/ml). However, other markers such as CD44 could be found on more than 80% of all donor T cells in allogeneic or syngeneic recipients. Our results in this clinical relevant mouse model show accelerating waves of T cell migration consistent with an enhancing feedback loop model of aGvHD pathogenesis. The homing receptor expression profile of donor T cells correlated with critical migration waves and clearly differed between mice with or without aGvHD. The assessment of critical time points frame a diagnostic window for a potential predictive test based on the dynamic change of the T cell homing receptor profile after allo-HCT. This preclinical study now awaits to be evaluated in patients undergoing allo-HCT. Disclosures: No relevant conflicts of interest to declare.

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