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 ◽  
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.

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

Blood ◽  
2010 ◽  
Vol 115 (10) ◽  
pp. 2095-2104 ◽  
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.

A Role for Regulatory Cells in Tolerance Induction in Recipients of Haploidentical Combined Non-Myeloablative Bone Marrow and Kidney Transplantation?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3255-3255
Author(s):  
Giovanna Andreola ◽  
Meredith Chittenden ◽  
Juanita Shaffer ◽  
A. Benedict Cosimi ◽  
Tatsuo Kawai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Conditioning Regimen ◽  
Tolerance Induction ◽  
In Vitro Assays ◽  
Mixed Chimerism ◽  
Post Transplant ◽  
Hla Dr

Abstract Following an in vivo T cell depleting non-myeloablative conditioning regimen, 5 patients, aged 22–49, received combined kidney and bone marrow transplantation from a haploidentical related donor. Rituximab was included in the conditioning for patients 4 and 5. All patients developed initial mixed chimerism but lost it by day 21; no patient developed GVHD. Four patients discontinued immunosuppression from 240 to 422 days after BMT and have remained off immunosuppression for 9 to 52 months with no evidence of allograft rejection. Flow cytometry was used to assess lymphocyte subsets recovering after transplant. CD3 counts recovered slowly, exceeding 500 cells/μl at days +271, +365, +640 and +450. While memory CD45RO+ cells were most prevalent among CD4+ cells, naïve-type CD4+CD45RA+ cells, presumably arising from the recipient thymus, ranged from 8% to 56% at the time when total CD4 counts recovered to >100 cells/μl (days +165, +21, +352, +240). Notably, a very high proportion of initially recovering T cells were CD3+CD4+ expressing CD25 in all patients as early as day 7 and persisted over 1 year in 2 patients. At approximately day +120 and +365, we further characterized these cells for CD127, FOXP3, CD45RO, CD45RA, HLA-DR and CD62L expression. At Day +120, all 4 patients showed increased frequencies (10.7±4.6%) of CD25+CD127-FOXP3+ regulatory T cells (Treg) within the CD4 population compared to healthy subjects (3.8±0.4%). Expression of CD45RO, CD45RA, CD62L and HLA-DR was variable. By 1 year post-transplant, frequencies of Treg had decreased to levels similar to those in normal subjects. In vitro assays for CD8 and CD4 T cell-mediated alloreactivity (CML/MLR) showed development of long-lasting donor-specific unresponsiveness by 3 months after transplant in Patients 2, 4 and 5, and by 9 months in Patient 1. Responses to 3rd party recovered in all patients after a period of unresponsiveness. In Patient 1, in whom anti-donor CML reactivity declined gradually to become unresponsive by 9 months, depletion of CD4+CD25+ cells revealed a residual anti-donor CML and MLR response at 1year but not at 18 months. In 2 other patients, depletion of CD4+CD25+ cells did not reveal an anti-donor response at time points analyzed from day +122 to 2 years. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, loss of killing activity against donor RTEC was observed post-transplant. The high percentage of Treg recovering early after transplant suggests that they may play a role in initial tolerance induction. This regulatory mechanism may be followed by later deletion of donor-reactive T cells. The variable ability to detect regulation of anti-donor reactivity may reflect the strength of the initial response, as patients with weak pre-transplant anti-donor responses and rapid post-transplant development of donor unresponsiveness did not reveal anti-donor response when Treg were depleted. In addition, infiltration of Treg at the graft site, not revealed by the assays described, might be responsible for tolerance in these patients.

scholarly journals Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells

2016 ◽  
Vol 113 (12) ◽  
pp. 3329-3334 ◽  
Author(s):  
Maurizio Perdicchio ◽  
Juan M. Ilarregui ◽  
Marleen I. Verstege ◽  
Lenneke A. M. Cornelissen ◽  
Sjoerd T. T. Schetters ◽  
...  
Keyword(s):  
T Cells ◽  
Sialic Acid ◽  
T Cell ◽  
De Novo ◽  
Ex Vivo ◽  
Treg Cells ◽  
Sialic Acids ◽  
Antigen Uptake

Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.

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 ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1803-1813 ◽  
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.

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 ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1803-1813 ◽  
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

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.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2029-2029
Author(s):  
Ivan Odak ◽  
Solaiman Raha ◽  
Saleh Tavil ◽  
Christian R Schultze-Florey ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Clonal Expansion ◽  
Tcr Repertoire ◽  
Suppression Assay

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.

In vitro–expanded donor alloantigen–specific CD4+CD25+ regulatory T cells promote experimental transplantation tolerance

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 827-835 ◽  
Author(s):  
Dela Golshayan ◽  
Shuiping Jiang ◽  
Julia Tsang ◽  
Marina I. Garin ◽  
Christian Mottet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Skin Graft ◽  
Graft Rejection ◽  
Treg Cells ◽  
Transplant Tolerance ◽  
Cba Mice

Abstract CD4+CD25+ regulatory T (Treg) cells play a critical role in the induction and maintenance of peripheral immune tolerance. In experimental transplantation models in which tolerance was induced, donor-specific Treg cells could be identified that were capable of transferring the tolerant state to naive animals. Furthermore, these cells appeared to have indirect allospecificity for donor antigens. Here we show that in vivo alloresponses can be regulated by donor alloantigen-specific Treg cells selected and expanded in vitro. Using autologous dendritic cells pulsed with an allopeptide from H2-Kb, we generated and expanded T-cell lines from purified Treg cells of CBA mice (H2k). Compared with fresh Treg cells, the cell lines maintained their characteristic phenotype, suppressive function, and homing capacities in vivo. When cotransferred with naive CD4+CD25− effector T cells after thymectomy and T-cell depletion in CBA mice that received CBK (H2k+Kb) skin grafts, the expanded Treg cells preferentially accumulated in the graft-draining lymph nodes and within the graft while preventing CBK but not third-party B10.A (H2k+Dd) skin graft rejection. In wild-type CBA, these donor-specific Treg cells significantly delayed CBK skin graft rejection without any other immunosuppression. Taken together, these data suggest that in vitro–generated tailored Treg cells could be considered a therapeutic tool to promote donor-specific transplant tolerance.

scholarly journals Donor-type CD4+CD25+ Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation

2002 ◽  
Vol 196 (3) ◽  
pp. 389-399 ◽  
Author(s):  
Petra Hoffmann ◽  
Joerg Ermann ◽  
Matthias Edinger ◽  
C. Garrison Fathman ◽  
Samuel Strober
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Graft Versus Host Disease ◽  
Treg Cells ◽  
Allogeneic Bone ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor Type ◽  
Acute Graft

Acute graft-versus-host disease (aGVHD) is still a major obstacle in clinical allogeneic bone marrow (BM) transplantation. CD4+CD25+ regulatory T (Treg) cells have recently been shown to suppress proliferative responses of CD4+CD25− T cells to alloantigenic stimulation in vitro and are required for ex vivo tolerization of donor T cells, which results in their reduced potential to induce aGVHD. Here we show that CD4+CD25+ T cells isolated from the spleen or BM of donor C57BL/6 (H-2b) mice that have not been tolerized are still potent inhibitors of the alloresponse in vitro and of lethal aGVHD induced by C57BL/6 CD4+CD25− T cells in irradiated BALB/c (H-2d) hosts in vivo. The addition of the CD4+CD25+ Treg cells at a 1:1 ratio with responder/inducer CD4+CD25− T cells resulted in a >90% inhibition of the mixed leukocyte reaction and marked protection from lethal GVHD. This protective effect depended in part on the ability of the transferred CD4+CD25+ T cells to secrete interleukin 10 and occurred if the Treg cells were of donor, but not host, origin. Our results demonstrate that the balance of donor-type CD4+CD25+ Treg and conventional CD4+CD25− T cells can determine the outcome of aGVHD.

scholarly journals Cd4+Cd25+ Immune Regulatory Cells Are Required for Induction of Tolerance to Alloantigen via Costimulatory Blockade

2001 ◽  
Vol 193 (11) ◽  
pp. 1311-1318 ◽  
Author(s):  
Patricia A. Taylor ◽  
Randolph J. Noelle ◽  
Bruce R. Blazar
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
Tolerance Induction ◽  
Solid Organ ◽  
Induction Of Tolerance

Immune regulatory CD4+CD25+ cells play a vital role in the induction and maintenance of self-tolerance and are essential for T cell homeostasis and the prevention of autoimmunity. Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. To determine whether CD4+CD25+ cells regulate T cell responses to alloantigen and are critical for tolerance induction, murine CD4+ T cells were tolerized to alloantigen via ex vivo CD40 ligand (CD40L)/CD40 or CD28/cytotoxic T lymphocyte–associated antigen 4/B7 blockade resulting in secondary mixed leukocyte reaction hyporesponsiveness and tolerance to alloantigen in vivo. CD4+CD25+ T cells were found to be potent regulators of alloresponses. Depletion of CD4+CD25+ T cells from the CD4+ responder population completely abrogated ex vivo tolerance induction to alloantigen as measured by intact responses to alloantigen restimulation in vitro and in vivo. Addback of CD4+CD25+ T cells to CD4+CD25− cultures restored tolerance induction. These data are the first to indicate that CD4+CD25+ cells are essential for the induction of tolerance to alloantigen and have important implications for tolerance-inducing strategies targeted at T cell costimulatory pathways.

scholarly journals 764 Expansion with IL-15 increases cytotoxicity of Vγ9Vδ2 T cells and is associated with higher levels of cytotoxic molecules and T-bet

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A812-A812
Author(s):  
Pia Aehnlich ◽  
Per Thor Straten ◽  
Ana Micaela Carnaz Simoes ◽  
Signe Skadborg ◽  
Gitte Olofsson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Adoptive Cell Therapy ◽  
Hematological Cancers ◽  
Cytotoxic Molecules ◽  
Vγ9vδ2 T Cells ◽  
T Cell Expansion

BackgroundAdoptive cell therapy (ACT) is an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle.MethodsIn this study, we explored the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro.ResultsWe could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1000U/ml interleukin (IL)-2 and (b) 100U/ml IL-2+100U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in phenotype, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells harbor increased amounts of perforin, granzyme B and granulysin in a resting state and release more upon activation. IL-2/IL-15-expanded Vγ9Vδ2 T cells also showed higher levels of transcription factor T-bet, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity.ConclusionsThese results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

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