Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Abstract Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs. We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities. Next, we employed a well-described murine model of allogeneic BMT (BL/6-->Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro. Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

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Effect of a Novel Nucleoside Analogue, Triciribine Phosphate (TCN-P) on Murine Acute Graft-Vrs-Host Disease (aGvHD)

Blood ◽

10.1182/blood.v118.21.2977.2977 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2977-2977

Author(s):

Edward Dela Ziga ◽

Jaebok Choi ◽

Mark Needles ◽

Julie Ritchey ◽

John F. DiPersio

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nucleoside Analogue ◽

Foxp3 Expression ◽

Negative Control ◽

Jurkat Cells ◽

Acute Graft

Abstract Abstract 2977 BACKGROUND: The successful establishment of donor registries and development of improved conditioning regimens among others, has led to the increased use of hematopoietic stem cell transplant (HSCT) as a key component in the treatment of some malignant and benign hematopoietic/lymphoid disorders as well as some metabolic disorders. Although a potential curative therapy for many hematologic diseases, allogeneic stem cell transplantation is associated with considerable morbidity and mortality primarily from acute graft-versus-host disease (aGvHD). Furthermore, graft-versus-leukemia (GVL) mediated by donor T cells can be abrogated with T cell depletion or suppression in vivo resulting in disease relapse with treatment of aGvHD. Moreso, modern therapies for aGvHD are limited and often toxic, thus there is a need for novel treatments and approaches that control aGvHD without compromising GVL. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor has been shown to decrease the severity of aGvHD (Reddy et al, PNAS 2004) through its effect on pro-inflammatory cytokines while maintaining GVL in a murine GvHD model. Also, previous work from our lab demonstrated that treatment of mice with the hypomethylating agent azacitidine (AzaC) after allogeneic HSCT mitigates aGvHD while preserving GVL by inducing FOXP3 expression in activated non-T regulatory cells (Choi et al, Blood 2010). However, the myelosuppression mediated by AzaC is a potential limitation that results in delayed donor engraftment. This led us to explore alternate options for single or combination drug therapy in the treatment of aGvHD. We screened a library of 2000 chemical agents obtained from the National Institutes of Health. Screening resulted in a single hit identified as Triciribine phosphate (TCN-P), an Akt inhibitor with structural similarity to the nucleoside analogue AzaC. In this experiment, a Foxp3 promoter-luciferase construct was designed and transfected into Jurkat cells. Cells were incubated for 2 days and then treated with three concentrations (0.1uM, 1umM and 10uM) of each chemical agent in the library. Bioluminescence imaging (BLI) was done on day 4 with AzaC as positive control (Choi et al, Blood 2010) and PBS as negative control. Only wells treated with TCN-P 10uM showed a signal, suggesting luciferase activity secondary to the Foxp3-promoter activation. We therefore hypothesized that TCN-P as a single agent or in combination with SAHA and or AzaC would mitigate GvHD by inducing FOXP3 without interfering with engraftment or immune reconstitution. METHODS: Using a C57BL/6(H2b) into Balb/c (H2d) murine MHC mismatch bone marrow transplant (BMT) model, we transplanted 5 × 106 T cell-depleted (TCD) bone marrow cells obtained from C57BL/6 (H2b, CD45.1+) mice into Balb/c (H2d, CD45.2+) mice after 900cGy of TBI. Delayed donor infusions of 2 × 106 pan-T cells/mouse obtained from FOXP3/GFP KI: B6 CD45.2+ H2b mice were infused on Day +11 in order to induce GvHD. Azacitidine 2mg/kg, SAHA 35mg/kg and TCN-P 10mg/kg were injected intraperitoneally every other day from Day +15 to Day +21(total of 4 doses). Acute GvHD was assessed by a standardized scoring developed by Cooke and Ferrara. (Blood, 1996) RESULTS : 1. Using our Foxp3-reporter system, both AzaC and TCN-P induced significant luciferase expression in Jurkat cells. SAHA had no effect. 2. Only AzaC but neither SAHA nor TCN-P induced significant Foxp3 expression in WT bead activated T cells. 3. In vivo, both AzaC 2mg/kg and TCN-P 10mg/kg but not SAHA 35mg/kg significantly improved survival of mice with less weight loss and clinical signs of aGvHD in a MHC mismatched aGvHD model. CONCLUSION: A novel nucleoside analogue TCN-P that was previously FDA approved for treatment of multiple myeloma and structurally related to AzaC, induces Foxp3 using a luciferase reporter construct in Jurkat cells and improves survival in mice after MHC mismatched allogeneic transplant. Though the 100 day survival between TCN-P and PBS (as negative control) in our murine aGvHD model was not quite statistically significant, the findings suggest a therapeutic potential for TCN-P and possibly other Akt inhibitors in the mitigation of aGvHD. Disclosures: No relevant conflicts of interest to declare.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Induction of tolerance to bone marrow allografts by donor-derived host nonreactive ex vivo–induced central memory CD8 T cells

Blood ◽

10.1182/blood-2009-10-248716 ◽

2010 ◽

Vol 115 (10) ◽

pp. 2095-2104 ◽

Cited By ~ 20

Author(s):

Eran Ophir ◽

Yaki Eidelstein ◽

Ran Afik ◽

Esther Bachar-Lustig ◽

Yair Reisner

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Memory Cell ◽

Central Memory ◽

Third Party ◽

High Survival

Abstract Enabling engraftment of allogeneic T cell–depleted bone marrow (TDBM) under reduced-intensity conditioning represents a major challenge in bone marrow transplantation (BMT). Anti–third-party cytotoxic T lymphocytes (CTLs) were previously shown to be endowed with marked ability to delete host antidonor T cells in vitro, but were found to be less effective in vivo. This could result from diminished lymph node (LN) homing caused by the prolonged activation, which induces a CD44+CD62L− effector phenotype, and thereby prevents effective colocalization with, and neutralization of, alloreactive host T cells (HTCs). In the present study, LN homing, determined by imaging, was enhanced upon culture conditions that favor the acquisition of CD44+CD62L+ central memory cell (Tcm) phenotype by anti–third-party CD8+ cells. These Tcm-like cells displayed strong proliferation and prolonged persistence in BM transplant recipients. Importantly, adoptively transferred HTCs bearing a transgenic T-cell receptor (TCR) with antidonor specificity were efficiently deleted only by donor-type Tcms. All these attributes were found to be associated with improved efficacy in overcoming T cell–mediated rejection of TDBM, thereby enabling high survival rate and long-term donor chimerism, without causing graft-versus-host disease. In conclusion, anti–third-party Tcms, which home to recipient LNs and effectively delete antidonor T cells, could provide an effective and novel tool for overcoming rejection of BM allografts.

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Human Bone Marrow Derived Mesenchymal Stromal Cells Enhance the Number and Function of Umbilical Cord Blood Peripheral Tregs during IL-2 Driven Ex Vivo Expansion

Blood ◽

10.1182/blood-2018-99-115069 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1116-1116

Author(s):

Jeong-Su Do ◽

Alex Y. Huang ◽

Daniel Zwick ◽

Fei Zhong ◽

David Askew ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Foxp3 Expression ◽

Ex Vivo Expansion ◽

Mitochondrial Transfer ◽

And Function

Abstract Tumor growth factor β (TGF-β)-induced peripheral regulatory T cells (pTreg) are a promising therapeutic cell source that exhibit Foxp3 expression and suppressive functions similar to natural regulatory T cells. Nonetheless, their clinical potential is limited by the instability of Foxp3 expression and T cell exhaustion that occurs during ex vivo expansion. We postulated that mesenchymal stromal cells (MSCs) could enhance the number, function and Foxp3 expression stability of pTregs during IL-2 driven 21 day expansion due to their diverse immunomodulatory properties. In this study, we observed that use of a human bone marrow mesenchymal stromal cells (hBM-MSC) platform significantly enhanced the number of pTreg during IL-2 driven 21 day ex vivo expansion vs. standard suspension culture condition (MSC platform: 80.2 x 106 vs. IL2/media: 39.3 x 106, n=6; p<0.01). Also the number of pTreg expressing a naive phenotype (CD4+CD45RA+ and CD4+CD62L+ ) were significantly increased (CD45RA+; MSC platform: 74.4 ± 1.6 x 106 vs. IL2/media: 45.9 ± 2.9 x 106, n=6, p<0.001; CD62L+; MSC platform: 79.1 ± 1.3 x 106 vs. IL2/media: 54.5 ± 2.1 x 106, n=6, p<0.001), as well as stability of Foxp3 expression (IL-2/media: 88.2 ± 1.7% vs. MSC platform: 96.2 ± 1.1%, n=7; p<0.05). In addition, pTreg suppressive function was noted to be more potent during 21 day IL-2 driven ex vivo expansion compared to standard IL-2/media culture condition (MSC platform: 79% vs. media: 35% inhibition of T cell proliferation in 10:1 ratio, n=6; p<0.01). pTreg expanded over a hBM-MSC platform exhibited higher surface CD25, CTLA-4, and ICOS MFI expression (CD25; MSC platform: 1410 vs. Media: 774; p<0.001, CTLA-4; MSC platform: 1084 vs. Media: 318; p<0.001, ICOS; MSC platform: 4386 vs. Media: 2641, p<0.01, n=6). Notably, hBM-MSC enhancement of pTreg ex vivo expansion requires direct cell-cell contact, as Foxp3 expression in pTreg was not enhanced by hBM-MSC conditioned media (CM:73.4 ± 6.8% vs. MSC platform: 96.2 ± 1.0%, p<0.001; and IL2/media: 88.8 ± 1.6% vs. MSC platform: 96.2 ± 1.0%, p<0.01) nor in a trans-well culture experiments (Transwell: 83.4 ± 2.5% vs. IL2/media: 88.8 ± 1.6%; and Transwell: 83.4 ± 2.5% vs. MSC platform: 96.2 ± 1.0%, p<0.01). Importantly, optical sectioning microscopy and flow cytometry revealed that hBM-MSC supports Treg number and function via direct contact-dependent mitochondrial transfer (Figure 1A-B). Cytochalasin B treatment blocked mitochondrial transfer, suggesting that tunneling nanotubes (TNT) facilitate mitochondrial transfer from hBM-MSC to pTreg during IL-2 driven ex vivo expansion (Mock: 2208 ± 122.1 vs. Cyto B: 923.8 ± 89 MFI, n=6, p<0.0001). Moreover, the quantity of ATP (n=6; p<0.01) mitochondrial potential of pTreg (MSC platform: 9010 ± 224.5 vs. media: 7316 ± 122.7 MFI, n=6; p<0.01) were significantly enhanced in pTreg during IL-2 driven ex vivo expansion over a hBM-MSC platform. Taken together, hBM-MSC significantly improves the number, maturation, and function of pTreg during 21 day IL-2 driven ex vivo expansion. We have identified one key mechanism of action of hBM-MSC underlying these favorable effects on pTreg during ex vivo expansion to be mitochondrial transfer via TNT. Notably, these studies identify a novel role of hBM-MSC to overcome current limitations in IL-2/media suspension culture conditions including T cell senescence, and loss of Foxp3 expression. Disclosures No relevant conflicts of interest to declare.

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Overcoming Rejection of Bone Marrow Allografts by Treg Cells in a Stringent Mouse Model for T Cell Mediated Rejection: Tolerance Induction by Third Party “Off-the-Shelf” Cells.

Blood ◽

10.1182/blood.v106.11.576.576 ◽

2005 ◽

Vol 106 (11) ◽

pp. 576-576

Author(s):

David Steiner ◽

Noga Brunicki ◽

Esther Bachar-Lustig ◽

Yair Reisner

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Graft Rejection ◽

Ex Vivo ◽

Tolerance Induction ◽

Treg Cells ◽

Third Party ◽

Donor Type

Abstract Recent reports have shown that donor or host CD4+CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x104 purified host type T cells (HTC) and were transplanted one day later with 2x106 BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4+CD25+ cells. C3H (H2k) recipients received BM from Balb- Nude (H2d) donors and the Treg cells were obtained from Balb/c or FVB (H2q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P>0.05). Considering the low levels of CD4+CD25+ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf’ source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.

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Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽

10.1182/blood.v106.11.63.63 ◽

2005 ◽

Vol 106 (11) ◽

pp. 63-63 ◽

Cited By ~ 1

Author(s):

Johannes L. Zakrzewski ◽

Adam A. Kochman ◽

Sidney X. Lu ◽

Theis H. Terwey ◽

Theo D. Kim ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

Growth Factors ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Hematopoietic Stem ◽

T Cell Reconstitution

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of >95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

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Akt Signalling Inhibition Promotes The Ex Vivo generation Of Minor Histocompatibility Antigen-Specific CD8+ Memory Stem T Cells

Blood ◽

10.1182/blood.v122.21.3269.3269 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3269-3269

Author(s):

Anniek B. van der Waart ◽

Noortje van der Weem ◽

Luca Gattinoni ◽

Nicolaas PM Schaap ◽

Robbert van der Voort ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Cd8 T Cells ◽

Memory T Cells ◽

Ex Vivo ◽

Akt Inhibitor ◽

Memory Subset

Abstract Allogeneic hematopoietic stem cell transplantation (allo-SCT) followed by donor lymphocyte infusion (DLI) is a potential curative treatment for patients suffering from a hematological malignancy. Efficacy is attributed to the graft-versus-tumor (GVT) response, during which engrafted donor T cells become activated by recipient minor histocompatibility antigens (MiHA) presented on dendritic cells (DC). Subsequently, these activated T cells expand, acquire effector functions and kill MiHA-positive tumor cells. However, persistence and recurrence of malignant disease is often observed, indicating that insufficient GVT immunity is induced. This imperfect alloreactive response is probably due to insufficient numbers of MiHA-specific effector T cells and/or defective antigen-presentation and costimulation. Therefore, adoptive transfer of potent ex vivo-generated MiHA-specific T cells, restricted to the hematopoietic system, would boost the GVT-effect without increasing the risk for GVHD. Although successful in vitro induction of MiHA-specific CD8+ T cells from naive precursors has been reported, the resulting antigen-experienced T cell population consist of fully differentiated effector-memory T cells (TEM). Over the past years it has been described that this T cell subset is not the most potent memory subset in anti-tumor responses in vivo following T cell transfer. In this regard, the less-differentiated memory subset called stem cell memory T cells (TSCM) with superior in vivo expansion, self-renewal capacity and plasticity to differentiate in potent effectors would generate a stronger GVT response. In this study, we aimed to investigate the in vivo availability and ex vivo generation of TSCM-like MiHA-specific T cells as additive treatment option for allo-SCT patients. First, we investigated whether in allo-SCT patients MiHA-specific T cells could be detected with a TSCM phenotype defined by the expression of CD45RO, CCR7, CD27 and CD95. Though TSCM cells could be clearly detected within CMV-specific CD8+ T cells in allo-SCT patients, similar to healthy controls, no MiHA-specific TSCM cells could be detected. This emphasises the need for more potent adoptive MiHA-specific T cell therapy following allo-SCT. Therefore, we next explored the possibility of generating TSCM-like CD8+ T cells by interfering with the Akt signalling pathway. Emerging findings indicate that the differentiation program of CD8+ T cells is dictated by the strength and duration of AKT activity. Therefore, we explored whether the pharmacological inhibition of this signaling pathway could results in the generation of TSCM-like CD8+ T cells. We stimulated CCR7+CD45RA+ naive CD8+ T cells with CD3/CD28 beads plus IL-2, IL-7 and/or IL-15 in the presence an Akt inhibitor. Interestingly, CD8+ T cells in these Akt-cultures were inhibited in their differentiation stage, expressing higher levels of CD45RA and CCR7 compared to controls. In addition, expression of CD95, IL2Rβ, and IL7Rα was also elevated confirming the TSCM-like phenotype. Although proliferation of the Akt-inhibited CD8+ T cells was decreased as shown by less PBSE dilution, expansion could be significantly preserved. Next, we investigated whether the established culture conditions could be used to generate MiHA-specific TSCM-like cells. Therefore, CD8+ T cells from MiHA-negative donors were primed using autologous MiHA peptide-loaded moDCs in the presence of the Akt-inhibitor. Interestingly, MiHA-specific T cell priming could be induced, consisting of mainly TCM and TSCM-like cells compared to almost entirely TEM cells in the control setting. Akt-inhibited MiHA-specific T cells showed higher expression of CCR7, CD45RA, CD62L, CD28, CD95, and IL7Rα. Importantly, for the Akt-inhibited MiHA-specific T cells, proliferation was reserved, resulting in robust proliferation capacity during restimulation after removal of the Akt-inhibitor. The resulting TEFF cells were highly functional, showing capacity to degranulate and produce IFNγ upon peptide restimulation. In conclusion, by inhibiting the Akt-pathway, in vitro CD8+ T cell differentiation can be reduced. Therefore, Akt signalling inhibition can be exploited for generating TSCM-like MiHA-specific T cells in adoptive immunotherapy after allo-SCT. Disclosures: No relevant conflicts of interest to declare.

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Peripheral Blood T Cells Generated After Allogeneic Bone Marrow Transplantation: Lower Levels of Bcl-2 Protein and Enhanced Sensitivity to Spontaneous and CD95-Mediated Apoptosis In Vitro. Abrogation of the Apoptotic Phenotype Coincides With the Recovery of Normal Naive/Primed T-Cell Profiles

Blood ◽

10.1182/blood.v94.5.1803 ◽

1999 ◽

Vol 94 (5) ◽

pp. 1803-1813 ◽

Cited By ~ 36

Author(s):

Nadia Chafika Hebib ◽

Olivier Déas ◽

Matthieu Rouleau ◽

Antoine Durrbach ◽

Bernard Charpentier ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cell Death ◽

T Cell ◽

Peripheral Blood ◽

Ex Vivo ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Bax Protein

Abstract T-cell reconstitution after bone marrow transplant (BMT) is characterized, for at least 1 year, by the expansion of populations of T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were obtained at various times after transplantation (from 45 to ≥730 days) and were tested for susceptibility to spontaneous apoptosis and anti-Fas triggered apoptosis in vitro. Substantial proportions of CD4+ and CD8+ cells generated during the first year after transplantation, but not by day 730, exhibited in these assays decreased mitochondrial membrane potential (▵Ψm) and apoptotic DNA fragmentation. The apoptotic phenotype tended to disappear late in the follow-up period, when substantial absolute numbers of naive (CD45RA+/CD62-L+) T cells had repopulated the peripheral blood compartment of the BMT patients. The rate of spontaneous cell death in vitro was significantly correlated with lower levels of ex vivo Bcl-2 protein, as assessed by cytofluorometry and Western blot analysis. In contrast, the levels of Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax ratios. Cell death primarily concerned the expanded CD8+/CD45R0+ subpopulation, although CD45R0− subpopulations were also involved, albeit to a lesser extent. These results show that the T-cell regeneration/expansion occurring after BMT is accompanied by decreased levels of Bcl-2 and susceptibility to apoptosis.

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Brief Ex Vivo Incubation with Fas Ligand Selectively Depletes Alloreactive T Cells and Antigen Presenting Cells from Stem Cell Grafts

Blood ◽

10.1182/blood-2018-99-117920 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2033-2033

Author(s):

Hilit Levy-Barazany ◽

Liat Pinkas ◽

Galina Rodionov ◽

Nitzan Marelly ◽

Michal Tzadok ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Fas Ligand ◽

Ex Vivo ◽

T Cell Subsets ◽

Ifn Γ ◽

Antigen Presenting

Abstract Graft versus host disease (GvHD) proceeds to be the Achilles' heel of hematopoietic stem cell transplantation, with clinicians continue facing a classic conflict: too much GvHD and the patient is at risk for transplant-related mortality and decreased quality of life; too little GvHD and the patient is at increased risk of relapse of their malignant disease. T cells and antigen presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cells (PBCs) graft. While GvHD is T cell mediated, the APCs are required for the initiation and maintenance of the GvHD. To reduce the risk for GvHD, grafts are sometimes depleted of their T cells, however, while preventing GvHD, the critically important attributes of graft versus leukemia (GvL) effect and engraftment are reduced significantly. Novel strategies that aim to abrogate or ameliorate GvHD, while preserving engraftment and GvL are of great need. A short incubation (2hr) of G-CSF mobilized PBCs with multimeric Fas ligand (i.e. ApoGraft) selectively induces apoptosis in T cell subsets and APCs (Panels A and B), but not in CD34+ progenitor cells (data not shown). FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory (TSCM), T central memory (TCM), and T effector memory (TEM) cells, as well as the pro-inflammatory T cell subtypes TH1 and TH17 cells, while no apoptotic signal is detected in the non-expressing CD95 naïve T cells (Panel A). The expression of T cells and APCs activation markers; CD25 and HLA-DR, respectively, is significantly reduced following apoptotic challenge in vitro (Panel C), as well as in transplanted mice (data not shown). Furthermore, upon an activation stimulus with anti CD3/CD28 beads in vitro, ApoGraft derived T cells secrete lower levels of IFN-γ, than G-CSF mobilized PBCs derived T cells (Panel D). To gain deeper understanding of the kinetics of GvHD development in vivo, NSG mice were transplanted with ApoGraft or G-CSF mobilized PBCs. Homing, expansion and differentiation of human leukocytes subtypes within the mice bone marrow, spleen and blood, were monitored 3, 7 and 14 days post transplantation. Decreased levels of T and B cells infiltration and expansion were detected in the spleen (Panels E and F), suggesting reduced formation of allo-reactive T cell clones. Reduced proliferation of these cells was associated with lower levels of IFN-γ secreted to the plasma (Panel H) and was in correlation with reduced GvHD and prolonged survival of the ApoGraft transplanted mice (Panel G). Importantly, we have previously demonstrated both in-vitro and in-vivo that ApoGraft has similar GvL and stem cell engraftment capabilities, compared to control G-CSF mobilized PBCs (data not shown). In conclusion, in contrast to conventional T- cell depletion methods, ApoGraft, an ex-vivo FasL-treated graft, affects both the T-cells and APCs, leading to reduced GvHD, while maintaining GvL and engraftment potential (Panel I). ApoGraft is currently being evaluated in a Phase I/II clinical trial (NCT02828878) in subjects with hematologic malignancies undergoing matched related allo-HSCT. Figure. Figure. Disclosures Levy-Barazany: Cellect Biotherapeutics Ltd: Employment. Pinkas:Cellect Biotherapeutics Ltd: Employment. Rodionov:Cellect Biotherapeutics Ltd: Employment. Marelly:Cellect Biotherapeutics Ltd: Employment. Tzadok:Cellect Biotherapeutics Ltd: Employment. Bakimer:Cellect Biotherapeutics Ltd: Employment. Yarkoni:Cellect Biotherapeutics Ltd: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy.

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Infusion of Ex-Vivo Expanded Donor T Cells To Improve Graft-Derived T-Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v110.11.3261.3261 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3261-3261

Author(s):

Nicolas Montcuquet ◽

Sylvain Perruche ◽

Benjamin Shipman ◽

Aliette Marandin-Decock ◽

Francis Bonnefoy ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Immune Reconstitution ◽

Ex Vivo ◽

Murine Models ◽

Hematopoietic Stem ◽

Donor T Cells

Abstract Limitations resulting from the reduced availability of related donors have been solved by the development of haplo-identical transplantation or by the use of cord blood as an alternative source of hematopoietic stem cells (HSC) to the bone marrow or peripheral blood. However, these kinds of transplantation remain associated with an impaired immune reconstitution, leading to an increased risk of infection and require an efficient modulation of post-transplant alloreactivity. In this setting, we and others demonstrated the possibility to control the alloreactivity by suicide gene transfer into donor T cells after ex-vivo T-cell culture. Such ex vivo culture was associated with the acquisition of a memory-like phenotype and with a decreased alloreactivity of gene-modified T cells, leading to an impaired potential of GvHD induction in murine models of allogeneic bone marrow transplantation (BMT). Chen and al. (Blood 2004) showed in an allogeneic BMT murine models that memory T cells were less alloreactive than naive T cells, leading to a less severe GvHD, but improved the immune reconstitution as compared with mice transplanted with bone marrow cells (BMC) only. By analogy with these results, we investigated the potential of ex-vivo expanded T cells (consisting of Con-A-activated splenocytes cultured ex vivo for 12 days in the presence of 500 UI/ml IL-2) to improve immune reconstitution without inducing GvHD. As compared with recipients of T-cell-depleted (TCD) BMC only, the administration of 106ex-vivo-expanded splenocytes (T) from CD45.1 C57Bl/6 mice together with 106 TCD-BMC from CD45.2 C57Bl/6 donors into 8 Gy-irradiated Balb/c allogeneic recipients significantly increased survival of transplanted mice at day 45 (58.3% vs 23.4% for BMC + T vs BMC only; p=0.0012, log rank test). Improved survival was associated with accelerated lymphoid and myeloid reconstitution as evidenced by day 15 lymphocyte and granulocyte blood counts: 212 (median) [range: 15–991]) vs 135 [14–632] lymphocytes/μl (p=0.0220) and 802 [6–5648] vs 114 [5–2411] granulocytes/μl (p=0.0006) for BMC + T (n=61) vs BMC only (n= 55). Importantly, FACS analysis demonstrated that enhanced lymphoid and myeloid reconstitution induced by ex-vivo expanded donor T-cells was due to enhanced donor bone-marrow-derived cells (lymphocyte and granulocyte blood counts: 129 [0–932] vs 11 [0–603] lymphocytes/μl (p=0.0014) and 801 [2–5637] vs 114 [2–2409] granulocytes/μl (p=0.0007) for BMC + T vs BMC only) and not ex-vivo expanded donor cells or residual recipient cells. Within the lymphoid compartment, enhanced reconstitution was observed mainly for CD3+CD8+ cells. Co-infusion of ex-vivo expanded donor T-cells did not induce GvHD (no GvHD-induced mortality or weight loss) while co-infusion of fresh splenocytes from CD45.1 C57Bl/6 mice induced severe GvHD (p<0.001 vs BMC only). Our results establish that ex-vivo expanded donor T-cells have a graft-facilitating effect and that they could be considered as a new cell therapy product allowing improving immune reconstitution after hematopoietic stem cell transplantation. Mechanisms involved in this graft-facilitating effect of ex-vivo expanded donor T cells remain to be elucidated.

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