Early T Cell Recovery of Thymus-Derived Naive T Cells and NK Cells in Pediatrics Patients after T-Cell Depleted HLA-Haploidentical Stem Cell Transplantation for Thalassemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3816-3816 ◽  
Author(s):  
Antonella Isgro’ ◽  
Pietro Sodani ◽  
Marco Marziali ◽  
Buket Erer ◽  
Cecilia Alfieri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Stromal Cells ◽  
Cd4 T Cells ◽  
Th Cells ◽  
Post Transplant

Abstract Delayed immune recovery post transplant remains a significant obstacle and results in increased risk of infections. T cells are regenerated via 2 pathway, thymus-derived and peripheral expansion, processes for which IL-7 is critical. To analyse the mechanisms involved in immunological reconstitution, we studied six thalassemia patients after 20 and 60 days post T-cell-depleted HLA-haploidentical stem cell transplantation. The mean age ranged from 14 to 5 years. As controls, 6 healthy donors matched by sex and age with the patients were included. We analysed T cell subsets by flow cytometry. Stromal cells, obtained from long term culture of bone marrow mononuclear cells were analysed by immunohystochemistry and the stromal IL-7 production was analysed by ELISA. Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), and this reduced number was mainly observed in CD45RA+CD62L+ (naive phenotype) subset (1.3 ± 2% in patients vs. 52 ± 12% in controls). A significant decrease of peripheral CD45RA+CD31+ Th cells (thymic naive Th cells) (on average 0.5 ± 0.3% in patients vs. 37 ± 10% in controls) was observed, whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD56 bright whereas CD56dim CD16+ NK cells were reduced. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%). Compared with normal subjects, thalassemia patients showed a significant increase of CD4+ cell activation markers (CD95, HLA-DR and CCR5) and this was observed after 60 days post transplant, in parallel with the increase of the CD56dim CD16+ NK cells especially in the patients with full engraftment. Stromal cells secreted lower IL-7 levels (0.3 + 0.1 pg/mL vs. 0.8 + 0.1 pg/mL, in controls) and displayed by immunohistochemistry an altered phenotype (“macrophage-like” morphology). A significant decrease in total lymphocyte counts and depletion of CD4+ T cells expressing predominantly the CD45RA+CD62L+ phenotype were observed after 60 days post transplant. Also the CD4+CD45RA+CD31+ T cell subset was initially reduced but an increase has been observed at day + 60 post transplant, suggesting a thymus involvement in these patients. An IL7/IL7R pathway dysregulation has been also observed, possibly involving bone marrow stromal cells. NK cells were among the earliest lymphocytes to repopulate the peripheral blood, but. CD56dim CD16+ NK cells were increased after 60 days post transplant, especially in the patients with full engraftment, suggesting a role of donor NK cells on bone marrow engraftment. We hypothesize that the recovery of T cell compartment may be due to a deregulated production of new T cells starting from haematopoietic stem cells under the influence of stromal cytokines production.

Immuno Haematological Reconstitution after T-Cell-Depleted HLA-Haploidentical Stem Cell Transplantation for Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1774-1774
Author(s):  
Antonella Isgrò ◽  
Buket Erer ◽  
Pietro Sodani ◽  
Paola Polchi ◽  
Marco Marziali ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Cd4 T Cells ◽  
Normal Subjects

Abstract Background. We evaluated haematological and immunological characteristics of four thalassemia patients after T-cell-depleted HLA-haploidentical stem cell transplantation Methods. We evaluated the clonogenic capability by the colony forming cell assay (CFC) and the long term culture-initiating cell (LTC-IC) assay at baseline and 20 days after transplant. Stromal cells were obtained from long term culture of bone marrow mononuclear cells (BMMCs) and analysed by immunohystochemistry. Lymphocyte subsets were studied by flow cytometry; and stromal IL-7 production by BMMCs was analysed by ELISA. Results. At baseline, no significant differences were observed in haematological and in immunological parameters in thalassemia patients when compared with a group of normal subjects Day + 20 after transplant, a reduced clonogenic capability was observed (4 ± 2 vs. 41 ± 40 CFU-E, 17 ± 9 vs. 109 ± 22 BFU-E, 3 ± 1 vs. 9 ± 6 CFU-GEMM and 16 ± 10 vs. 66 ± 23 CFU-GM). The number of primitive bone marrow (BM) progenitor cells was also decreased (1.8 ± 1.4 vs. 15.4 ± 3.6 LTC-CFC/106 BMMCs). In addition, stromal cells secreted lower IL-7 levels (0.3 + 0.1 pg/mL vs. 0.8 + 0.1 pg/mL, in controls) and displayed by immunohistochemistry an altered phenotype. Upon light microscopy examination, the majority (75%) of these cells appeared as moderately large cells, frequently rounded, with abundant cytoplasm, whereas in control subjects about 90% of the stromal cells exhibited a different morphology characterized by irregular or spindle shape and branching cytoplasmic processes (fibroblast-like). Compared with normal subjects, thalassemia patients showed: reduction of naïve CD4+ T-cells (2 ± 0.5% vs 50 ± 10%), reduction of thymic naïve CD4+ T-cells (1 ± 0.2% vs 40 ± 12%,) and a significant increase of CD4+ cells activation markers (CD95, HLA-DR and CCR5). IL-7 receptor (CD127) expression was also significantly decreased on CD4+ T-cells and on naïve CD4+ T-cells (CD4+/CD45RA+CD62L+/CD127+). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD56 brigh whereas CD16+ NK cells were decreased. Conclusions. Twenty days post transplant, an impaired growth and differentiation capacity of stem/progenitor cells were observed in thalassemia patients, in parallel with an altered homeostasis of T-cells and a reduction of T-cell naïve compartment. We hypothesize that the damage of T cell compartment may be at least partially due to an altered production of new T cells starting from the haematopoietic stem/progenitor cells. CD56+ NK cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factors (IL-2) in the bone marrow. An IL7/IL7R pathway dysregulation has been also observed, possibly involving bone marrow stromal cells. In vitro studies are ongoing about the use of cytokines (IL-2, IL-7, IL-2 plus IL-7) supporting T cell development.

Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2102-2102 ◽  
Author(s):  
Mahesh Yadav ◽  
Cherie Green ◽  
Connie Ma ◽  
Alberto Robert ◽  
Andrew Glibicky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

Graft-Versus-Host Tolerance in Dogs Following T-Cell Depleted Bone Marrow Transplantation with Absorbed ATG or CD6-Antibody.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2338-2338
Author(s):  
Julia Zorn ◽  
Hans Jochem Kolb
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Nk Cells ◽  
Marrow Transplantation ◽  
Graft Versus Host ◽  
Host Tolerance ◽  
Rabbit Complement

Abstract Graft-versus-host disease (GvHD) is the major obstacle of allogeneic stem cell transplantation. Depletion of T-cells from the graft reduces the risk of GvHD, but results in a higher risk of leukemia relapse. Adoptive immunotherapy with donor lymphocyte transfusion (DLT) has been shown to control leukemia in patients after T-cell depleted allogeneic stem cell transplantation. However, GvHD may occur, if DLT is given too early after transplantation. In canine models of DLA-identical and DLA-haploidentical bone marrow transplantation, we compared different methods of T-cell depletion (TCD) and investigated the potential of DLT at different times after transplantation to induce GvHD. T-cell depletion was performed either with absorbed anti-thymocyte globuline (aATG) or with a combination of CD6-antibody and baby rabbit complement. ATG was absorbed with erythrocytes, liver, kidney and spleen for eliminating antibodies against stem cells. CD6-antibody (M-T606) and rabbit complement depleted T-cells effectively without affecting hematopoietic progenitor cells. Unlike aATG, monoclonal CD6-antibody spares natural killer (NK) cells and some CD8-positive cells. Treatment of bone marrow with aATG prevented GvHD in 9 dogs following DLA-identical transplantation. DLT on days 1 and 2 or 21 and 22 induced fatal GvHD in two dogs each. However, it did not induce GvHD when given on days 61 and 62 and later. In DLA-haploidentical bone marrow recipients, non-manipulated marrow produced fatal GvHD in all dogs (n=7), whereas marrow treated with aATG (vol:vol 1:100 and 1:200) produced fatal GvHD in 5 out of 16 dogs only. CD6-depletion prevented GvHD in 3 of 3 DLA-haploidentically transplanted dogs. DLT produced fatal GvHD in one dog each, when given on day 3, 7 or 14 after CD6-depleted haploidentical bone marrow transplantation. However, it produced fatal GvHD in only 2 of 4 dogs transfused on day 20 post grafting. Thus, DLT could be given earlier in DLA-haploidentical animals transplanted with CD6-depleted marrow than in DLA-identical animals transplanted with aATG treated marrow without producing GvHD. These findings support the hypothesis that graft-versus-host tolerance can be induced earlier with grafts not depleted of NK cells. NK cells in the graft may inactivate host dendritic cells necessary for the induction of GvHD. In grafts depleted with aATG, NK cells are depleted as well, because aATG still retains broad specificity despite extensive absorptions. This leaves host DCs unaffected. Transfused donor T-cells encountering this environment will thus be activated which results in severe GvHD. In contrast, monoclonal CD6-antibody spares NK cells, so that donor lymphocytes cannot be activated by host DCs at the time of DLT and thus won’t trigger GvHD. CD6-depletion is the preferred method if adoptive immunotherapy with DLT is planned.

Pentostatin Facilitates Fully MHC-Disparate Murine Mini-Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3539-3539
Author(s):  
Jacopo Mariotti ◽  
Kaitlyn Ryan ◽  
Paul Massey ◽  
Nicole Buxhoeveden ◽  
Jason Foley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Suppression ◽  
Graft Rejection ◽  
Mixed Chimerism ◽  
Post Transplant

Abstract Abstract 3539 Poster Board III-476 Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy. Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. 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.

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

Blood ◽  
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.

scholarly journals Peri-Transplant Steroids Enhance GVL and Attenuate GVHD By Redistributing Alloreactive Donor T Cells from the Gut into the Bone Marrow

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3210-3210
Author(s):  
Takayuki Inouye ◽  
Motoko Koyama ◽  
Ensbey Kathleen ◽  
Nicholas Greene ◽  
Luke Samson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Expansion ◽  
Effector Memory ◽  
Gi Tract ◽  
Donor T Cells ◽  
T Cell Expansion

Leukemia relapse represents a failure of graft-versus-leukemia (GVL) and remains the major limitation of allogeneic stem cell/bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) within the gastrointestinal (GI) tract is the principal determinant of transplant-related mortality and is initiated by a network of alloantigen presentation by professional and non-professional APC that prime donor T cells in the GI tract and related lymphoid structures. Since GVL and lethal GVHD are mediated by donor T cells at spatially distinct sites; bone marrow (BM) and the GI tract respectively, we sought tractable approaches to spatially separate alloreactive responses at these two locations. The administration of high dose steroids in the peri-transplant period is permissive of T cell replete HLA-haploidentical BMT and significant GVL effects (Ogawa H, et al. BBMT. 2006). We utilized murine haploidentical BMT models (B6D2F1 → B6C3F1, B6 → B6D2F1) with recipient background MLL/AF9 primary acute myeloid leukemia (AML), with or without dexamethasone (Dex) administration (5 mg/kg/day i.p., days -1 to +5). Dex-treatment improved transplant survival (from 25% to 68% at day 100, P=0.0012) with significant reductions in GVHD histopathology specifically in the colon (histopathology scores 8.7±1.0 vs 4.6±0.8, P< 0.05), despite excellent leukemia control. To understand this paradox, we analyzed the kinetics of donor T cell expansion after BMT. In the mesenteric lymph node (mLN), Dex treatment significantly suppressed the expansion of both CD4 and CD8 T cells (3.3±0.3 x 105 vs 1.4±0.3 x 105, P< 0.001 and 4.2±0.4 x 105 vs 2.1±0.4 x 105, P< 0.01 respectively) and the activation of CD4 T cells (CD25 MFI: 2021±146 vs 1056±102, P< 0.01). In contrast, donor effector/memory CD44+ CD8 T cells were expanded in the BM of Dex treated recipients (1.9±0.3 x 105 vs 3.1±0.4 x 105, P< 0.05) that demonstrated high per cell cytolytic activity against leukemia (specific lysis: 65±2.4 % vs 62±2.6 % in untreated vs Dex-treated, P> 0.05). Surprisingly, there was no difference in proliferation (cell tracking dye dilution: 63±5.5 % vs 57±5.5 % in untreated vs Dex-treated, P> 0.05) or apoptosis (caspase-3: 6.6±0.4 % vs 6.1±0.6 %, caspase-8: 20±1.6 % vs 17±3.3 % in untreated vs Dex-treated, respectively, P> 0.05) of CD4 T cells in the mLN between the two groups. We undertook experiments with luciferase expressing T cells and noted that Dex-treatment preferentially inhibited T cell accumulation in the GI tract, but not marrow after BMT. Thus, it appeared that Dex treatment preferentially re-distributed donor T cells from the GI tract to the bone marrow. We next determined if Dex exerted effects via direct signaling to the donor T cell. We thus transplanted glucocorticoid receptor (GR)-deficient or intact T cells (GRfl/fl lck-Cre mice). Dex-treatment reduced donor CD4 T cell expansion in the mLN independent of their expression of the GR (untreated vs Dex-treated: 2.8±0.6 x 105 vs 1.2±0.3 x 105, lckCREGRfl/fl and 2.4±0.3 x 105 vs 1.4±0.4 x 105, GRfl/fl littermates, P< 0.05 both groups). Thus steroid effects were mediated indirectly, putatively via effects on recipient alloantigen presentation. There was a marked reduction in recipient dendritic cells (DC) and macrophages expressing the Ea peptide within MHC class II in the GI tract of Dex-treated recipients (terminal Ileum YAe+ DC number 896±93 vs 356±40, P< 0.01, YAe+ macrophage number 1035±136 vs 355±97, P< 0.01). In conjunction with this, expression of the gut homing integrin a4b7 expression was reduced in CD4 T cells from Dex treated recipient mLN (25±1.6 % vs 17±1.7 %, P< 0.01), while the marrow homing integrin VLA-4 (a4b1) was increased (a4: 62±2.2 % vs 75±1.6 %, P< 0.001, b1: 52±2.5 % vs 61±1.6 %, P< 0.05) in donor CD8 T cells from Dex treated recipient BM. Finally, Dex treatment enhanced GVL against a second primary AML (BCR/ABL-NUP98/HOXA9) relative to untreated recipients and those receiving post-transplant cyclophosphamide (PT-Cy) (relapse rate: 0% vs 40% vs 100% at day 35 in Dex vs untreated vs PT-Cy, PT-Cy vs Dex-treated, P< 0.0001; untreated vs Dex-treated, P=0.029). These data suggest a potential therapeutic strategy to modulate antigen presentation in the GI tract and consequent integrin imprinting that minimizes GVHD lethality whilst enhancing GVL within BM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Sebastien Hagmann ◽  
Claudia Rimmele ◽  
Florin Bucur ◽  
Thomas Dreher ◽  
Felix Zeifang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cd4 T Cells ◽  
Surface Marker ◽  
Cd4 T Cell ◽  
T Cell Proliferation

Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis.Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs.Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-αas well as increasing IL-6 secretion.Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches.

scholarly journals Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1706-1715 ◽  
Author(s):  
Holger N. Lode ◽  
Rong Xiang ◽  
Torsten Dreier ◽  
Nissi M. Varki ◽  
Stephen D. Gillies ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Nk Cells ◽  
Natural Killer ◽  
Therapeutic Effect ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

scholarly journals Resistance of Natural Killer and T Cells to Venetoclax Allows for Combination Treatment with Cancer Immunotherapy Agents

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1118-1118 ◽  
Author(s):  
Elisabeth A Lasater ◽  
An D Do ◽  
Luciana Burton ◽  
Yijin Li ◽  
Erin Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Single Agent ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction: Intrinsic apoptosis is regulated by the BCL-2 family of proteins, which consists of both anti-apoptotic (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, BAX, BAK, BAD) proteins. Interaction between these proteins, as well as stringent regulation of their expression, mediates cell survival and can rapidly induce cell death. A shift in balance and overexpression of anti-apoptotic proteins is a hallmark of cancer. Venetoclax (ABT-199/GDC-0199) is a potent, selective small molecule BCL-2 inhibitor that has shown preclinical and clinical activity across hematologic malignancies and is approved for the treatment of chronic lymphocytic leukemia with 17p deletion as monotherapy and in combination with rituximab. Objective: To investigate the effects of BCL-2 inhibition by venetoclax on viability and function of immune-cell subsets to inform combinability with cancer immunotherapies, such as anti-PD-L1. Methods and Results: B cells, natural killer (NK) cells, CD4+ T cells, and CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (n=3) were exposed to increasing concentrations of venetoclax that are clinically achievable in patients, and percentage of live cells was assessed by flow-cytometry using Near-IR cell staining. B cells were more sensitive to venetoclax (IC50 of ~1nM) than CD8+ T cells (IC50 ~100nM), NK cells (IC50 ~200nM), and CD4+ T cells (IC50 ~500nM) (Figure A). CD8+ T-cell subset analysis showed that unstimulated naive, but not memory cells, were sensitive to venetoclax treatment (IC50 ~30nM and 240nM, respectively). Resistance to venetoclax frequently involves compensation by other BCL-2 family proteins (BCL-XL and MCL-1). As assessed by western blot in PBMCs isolated from healthy donors (n=6), BCL-XL expression was higher in NK cells (~8-fold) and CD4+ and CD8+ T cells (~2.5-fold) than in B cells (1X). MCL-1 protein expression was higher only in CD4+ T cells (1.8-fold) relative to B cells. To evaluate the effect of venetoclax on T-cell function, CD8+ T cells were stimulated ex vivo with CD3/CD28 beads, and cytokine production and proliferation were assessed. Venetoclax treatment with 400nM drug had minimal impact on cytokine production, including interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), and IL-2, in CD8+ effector, effector memory, central memory, and naïve subsets (Figure B). CD8+ T-cell proliferation was similarly resistant to venetoclax, as subsets demonstrated an IC50 >1000nM for venetoclax. Taken together, these data suggest that survival of resting NK and T cells in not impaired by venetoclax, possibly due to increased levels of BCL-XL and MCL-1, and that T-cell activation is largely independent of BCL-2 inhibition. To evaluate dual BCL-2 inhibition and PD-L1 blockade, the syngeneic A20 murine lymphoma model that is responsive to anti-PD-L1 treatment was used. Immune-competent mice bearing A20 subcutaneous tumors were treated with clinically relevant doses of venetoclax, murine specific anti-PD-L1, or both agents. Single-agent anti-PD-L1 therapy resulted in robust tumor regression, while single-agent venetoclax had no effect. The combination of venetoclax and anti-PD-L1 resulted in efficacy comparable with single-agent anti-PD-L1 (Figure C), suggesting that BCL-2 inhibition does not impact immune-cell responses to checkpoint inhibition in vivo. These data support that venetoclax does not antagonize immune-cell function and can be combined with immunotherapy targets. Conclusions: Our data demonstrate that significant venetoclax-induced cell death at clinically relevant drug concentrations is limited to the B-cell subset and that BCL-2 inhibition is not detrimental to survival or activation of NK- or T-cell subsets. Importantly, preclinical mouse models confirm the combinability of BCL-2 and PD-L1 inhibitors. These data support the combined use of venetoclax and cancer immunotherapy agents in the treatment of patients with hematologic and solid tumor malignancies. Figure Figure. Disclosures Lasater: Genentech Inc: Employment. Do:Genentech Inc: Employment. Burton:Genentech Inc: Employment. Li:Genentech Inc: Employment. Oeh:Genentech Inc: Employment. Molinero:Genentech Inc: Employment, Equity Ownership, Patents & Royalties: Genentech Inc. Penuel:Genentech Inc: Employment. Sampath:Genentech Inc: Employment. Dail:Genentech: Employment, Equity Ownership. Belvin:CytomX Therapeutics: Equity Ownership. Sumiyoshi:Genentech Inc: Employment, Equity Ownership. Punnoose:Roche: Equity Ownership; Genentech Inc: Employment. Venstrom:Genentech Inc: Employment. Raval:Genentech Inc: Consultancy, Employment, Equity Ownership.

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