Efficient Induction and Isolation of CMV-Specific CD8+ T Cells from CMV Seronegative Donors for the Treatment of CMV Reactivation in CMV Seropositive Patients Transplanted with a CMV Seronegative Donor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1053-1053 ◽  
Author(s):  
Inge Jedema ◽  
Marian van de Meent ◽  
Pim L.J. van der Heiden ◽  
Erik W.A. Marijt ◽  
Pauline Meij ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Memory T Cells ◽  
Immunomagnetic Bead ◽  
Healthy Donors ◽  
Donor T Cells ◽  
The Impact ◽  
Cmv Reactivation

Abstract Cytomegalovirus (CMV) disease is a significant cause of morbidity and mortality after allogeneic stem cell transplantation (allo-SCT) in CMV seropositive (CMV+) patients. In a recent cohort of CMV+ patients, we investigated the impact of donor CMV serostatus on the severity of CMV reactivation after T cell depleted allo-SCT. A high incidence of CMV related mortality was seen in patients transplanted with a CMV- donor (5/20) whereas no CMV-related deaths occurred after transplantation with a CMV+ donor (0/20). In most CMV+ patients transplanted with a CMV+ donor reconstitution with CMV specific (memory) T cells was found. We recently performed a phase I/II clinical study using isolated CMV-specific CD8+ memory donor T cells for the treatment of patients with persistent CMV reactivation despite seropositivity of the donor. In this study we demonstrated the feasibility of isolating and selecting CMV-specific CD8+ memory T cells from CMV+ donors using the interferon-gamma (IFNg) capture assay and CliniMACS isolation after peptide stimulation of the CMV-specific donor T cells. We have illustrated the in-vivo potential of these T cells after adoptive transfer in 5 patients, resulting in clearance of the CMV load. However, no suitable method was available for the induction of primary immune responses against CMV for the treatment of persistent CMV reactivation in patients transplanted with a CMV- donor. In the current study we investigated the possibility to induce and isolate CMV-specific T cells from CMV- healthy donors by in-vitro priming and selection. We used as responder cells CD14- CD45RO- PBMC from HLA-A1, A2, A3, B7, or B8 positive CMV- healthy donors (n=10). By CD45RO depletion we removed the majority of regulatory T cells (Tregs) capable of inhibiting the initiation of the response. Mature monocyte-derived dendritic cells (DCs) were loaded with a cocktail containing 1μg of each relevant CMV pp65, pp50, or IE1 derived 9-mer peptide, depending on the HLA type of the donor. Naïve donor T cells were cocultured in a 10/1 ratio with peptide loaded DCs. From day 4 on 5 ng/mL IL-7 and IL-15 was added to the culture. At day 10, the responses were specifically restimulated with peptide loaded autologous PBMC. Cytokines were refreshed twice weekly. At day 20 CMV-specific CD8+ T cells were detected and isolated by specific tetramer staining and flowcytometric cell sorting, by specific pentamer staining and immunomagnetic bead isolation, or further enriched by another restimulation, followed by isolation of CD137 expressing T cells at day 21. In 10/10 CMV seronegative donors CMV specific T cells could be detected at day 20 of the immune response in frequencies ranging from 0.01–0.4%. These tetramer positive cells could be isolated and expanded both in bulk cultures and clonally. Functional CMV-specific T cells against all 3 major immunogenic CMV proteins pp65, pp50, and IE1 were detected and isolated with different dominant responses detected in different donors. In conclusion, we have developed a method for the in-vitro induction and isolation of functional CMV-specific CD8+ T cells from CMV- donors. This may allow the treatment of serious CMV-related complications in CMV+ patients transplanted with a CMV- donor.

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

Blood ◽  
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.

scholarly journals IL-2 Induces Selective Activation of Helios-Positive Regulatory T Cells and CD56bright NK Cells in Vitro and in Patients with Chronic Gvhd Receiving Low-Dose IL-2 Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 919-919 ◽  
Author(s):  
Masahiro Hirakawa ◽  
Tiago R Matos ◽  
John Koreth ◽  
Edouard Forcade ◽  
Jennifer Whangbo ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Advisory Committees ◽  
Low Concentrations ◽  
Healthy Donors

Abstract Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation (SCT). Treg constitutively express high-affinity interleukin-2 (IL-2) receptors and murine models have established that IL-2 is a critical homeostatic regulator of Treg in vivo. We previously reported that daily administration of low-dose IL-2 in patients with cGVHD induces selective expansion of Treg and NK cells and results in clinical improvement in approximately 50% of patients. However, the mechanisms responsible for these selective effects and the influence of IL-2 therapy on other lymphocytes have not been established due to the limited resolution of traditional cell analytic methods such as flow cytometry. Methods: Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct T, B and NK cell subsets and 11 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map. Results: In unstimulated lymphocytes from healthy donors, constitutive expression of CD25 (IL-2Ra) at high levels was restricted to Treg and CD56bright NK cells. Central memory (CM) and effector memory (EM) subsets of conventional CD4 T cells (Tcon) and CM CD8 T cells expressed low levels of CD25. Within the Treg population, the highest expression of CD25 was closely associated with expression of Helios transcription factor. Helios+ Treg also express higher levels of FoxP3, HLA-DR and CD95 and lower levels of BCL2 compared to Helios- Treg. To examine responses to IL-2, we stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with IL-2 for 15 min in vitro (Figure 1). At low IL-2 concentrations (1 to 10 IU/ml), pSTAT5 was preferentially activated in Treg. Notably, pSTAT5 activation was more robust in memory Treg than naïve Treg and in Helios+ Treg than Helios- Treg. In addition, we observed activation of pSTAT5 in CD56bright NK cells at low concentrations of IL-2 (10 IU/ml). Higher IL-2 concentrations (100-1000 IU/ml) were required to activate pSTAT5 in Tcon, CD8 T cells and CD56dim NK cells. At high IL-2 concentrations, pSTAT5 was activated in all Treg, NK, Tcon and CD8 subsets. To examine the response to IL-2 in vivo, we examined PBMC from 14 patients with chronic GVHD receiving daily low-dose IL-2 using the same CyTOF panel of markers. Without additional in vitro stimulation, pSTAT5 expression was increased preferentially in Helios+ Treg. Peak pSTAT5 expression occurred 1 week after starting IL-2 and decreased with continued IL-2 therapy. Similarly, increased expression of FoxP3, CD25, HLA-DR and Ki67 occurred primarily in Helios+ Treg with peak expression at 1 week. At later time points during IL-2 therapy, changes in Treg included increased expression of CD95, CTLA4, PD-1, BIM and BCL2. Although there was no activation of pSTAT5 in CD4 Tcon and CD8 T cells, expression of PD-1 increased in effector memory subsets of Tcon and CD8 T cells 1 week after starting IL-2 therapy. Selective expansion of CD56bright NK cells was also noted, with peak activation at 1 week. No other changes were noted in Tcon, CD8 T cells and B cells. All changes observed during IL-2 therapy returned to baseline levels 4 weeks after treatment was stopped. However, examination of PBMC from 8 patients who received continuous daily low-dose IL-2 therapy for approximately 1 year showed that all of the changes noted above persisted during extended therapy. Conclusion: Comprehensive analysis of T, B and NK cells from healthy donors revealed that low concentrations of IL-2 result in selective activation of Helios+ Treg and CD56bright NK cells. Higher concentrations of IL-2 are required for activation of CD4 Tcon, CD8 T cells and CD56dim NK cells. Identical populations are activated in patients with cGVHD receiving daily low-dose IL-2 and these functional effects persist during extended IL-2 therapy. Although the function of Helios transcription factor is not well defined, Helios expression identifies those Treg most primed to respond to low concentrations of IL-2 in vitro and in vivo. Disclosures Armand: Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

scholarly journals 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Thomas Stübig ◽  
Anita Badbaran ◽  
Tim Luetkens ◽  
York Hildebrandt ◽  
Djordje Atanackovic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Phenotype ◽  
Target Cells ◽  
T Helper 1 ◽  
Cell Immunity ◽  
The Impact

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigatedin vitroandin vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells.In vivodata confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

Requirements for Retroviral Targeting of a Suicide Gene to Alloreactive Memory Stem T Cells for Adoptive Immunotherapy of Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2176-2176
Author(s):  
Attilio Bondanza ◽  
Lothar Hambach ◽  
Shin Kaneko ◽  
Sara Mastaglio ◽  
Bart Nijmeijer ◽  
...  
Keyword(s):  
T Cells ◽  
Memory T Cells ◽  
Suicide Gene ◽  
Effector T Cells ◽  
Donor T Cells ◽  
Specific Effector ◽  
Ifn Γ ◽  
Homeostatic Cytokines

Abstract In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.

The Pan- JAK Inhibitor Ruxolitinib Impairs T-Cell Activation, Cytokine Production and Proliferation In Vivo and In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2001-2001 ◽  
Author(s):  
Thomas Stuebig ◽  
Christine Wolschke ◽  
Haefaa Alchalby ◽  
Francis Ayuk ◽  
Marion Heinzelmann ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Post Transplantation ◽  
Effector Cells ◽  
Proliferation Capacity ◽  
Healthy Donors ◽  
The Impact

Abstract Myelofibrosis (MF) is a clonal myeloproliferative neoplasm, in which the JAK2-V617F mutation is frequently observed. The appearance in up to 50% of the cases makes the JAK2 mutation attractive as therapeutical target. In 2012 Ruxolitinib (Ruxo) a pan-JAK inhibitor was approved for the treatment of MF and showed efficacy in disease treatment, irrespectively of the JAK2V617 mutation status. Currently allogeneic stem cell transplantation (allo SCT) remains the only curative treatment option for MF. To further improve transplant outcome in MF reduction of spleen size and constitutional symptoms prior transplantation is a reasonable target. Harnessing graft versus myelofibrosis post transplantation by immune-modulating drugs may help to reduce the risk of relapse. Ruxolitinib may be used as pre- and post-transplantation drug to improve transplant outcome. However the impact of Ruxolitinib on the immune system, especially on T-cells, is poorly understood. Here we investigated the effects of Ruxolitinib on T-cells in vivo and in vitro. T-cells from healthy donors were isolated by magnetic cell sorting to pan CD3+, CD4+ and CD8+ fraction. All three different cell subsets were cultured with different dosages of Ruxolitinib (100, 250, 500, 750nM and 1µM) for additional 48h. Thereafter cells were analysed for cell growth, cell death, RNA expression, immune phenotype. Additionally, immune profiles of 9 patients were analysed for the changes of the T-cell compartment during the treatment with Ruxolitinib over a period of 3 weeks T –cells from healthy donors showed a dosage dependent impairment in the proliferation capacity compared to non-treated control cells (4.1x106 CD3 cells /ml vs. 1.9x106 CD3 cells /ml, p<0.05), additionally KI67 expression was reduced from 48% in control cells to 12% in 100nM treated CD3 cells and 9% in 500nM treated CD3 cells, p<0.05. Strikingly apoptotic cell death increased from 11% in control cells to 43% and 48% in 100nM and 500nM Ruxo treated cells, p<0.03. Analysing the immune phenotype of Ruxo treated CD3, CD4 and CD8 cells we found a significant reduction in the expression of activation marker like CD25 and HLA-DR (38% vs. 6% and 4.5% respectively, p<0.05 and 63% vs. 47% and 40% respectively, p<0.05). Furthermore, we found that the effector cells, marked by CCR7/CD45RA expression, decreased in the CD8 compartment from 22% to 10.5% and 7.8% respectively, p<0.05. When analysing regulatory T-cells we also observed a decrease in a dose dependent manner (4% vs. 1.2% and 0.8%, p=0.05). While control Treg showed a KI67 expression of >60%, Ruxo (100nM) treated T-reg did not expressed KI67. Likewise to CD8 effector cells and Tregs we found a decrease in pro-inflammatory TH1 and TH17 cells in vitro (27% vs. 14% and 12% for TH1 cells and 6% vs. 4% and 4% for TH17 cells). Next, we analysed mRNA expression and found that pro-inflammatory cytokines like IL23, IL18, IL7 were down regulated after Ruxo treatment. To in contrast to pro- inflammatory cytokines, p53 and cell cycle inhibitor of the cip/waf locus showed to be up regulated in CD3 and CD4 cells suggesting that the observed increase in apoptosis in T-cells is mediated by p53. We next investigated the impact of Ruxolitinib on T-cells in patients. Therefore we analysed the blood of patients treated with Ruxolitinib in weekly intervals. Likewise to in vitro CD3 cells showed a decrease which turned to be significant after two and three weeks of treatment (1560/µl vs. 688/µl and 410/µl, p<0.05), this was mainly through the reduction of CD8+ T-cells (630/µl before treatment vs. 250/µl at week 2 and 200/µl at week 3, p <0.05). We also observed a decrease of CD3+/ HLA-DR+ (as activation marker) from 355/µl before to 130/µl and 70/µl however this did not reached statistical significance. The same was found for Tregs in vivo (5.6% vs. 2.3% and 1.9%, respectively). These data argue that treatment of T-cells by Ruxolitinib impairs their proliferation capacity by inducing apoptosis through an up regulation of p53. This increase of cell death applies all analysed T-cell compartments, and thereby may explains why Ruxo treated T-cells were less able to show a pro-inflammatory as well as regulatory phenotype. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Absence of NLRP6 in Donor T Cells Exacerbates Gvhd

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2766-2766
Author(s):  
Masahiro Suto ◽  
Eri Matsuki ◽  
Masahiro Miyata ◽  
Erika Sekiguchi ◽  
Hiroya Tamaki ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Responses ◽  
Donor T Cells ◽  
Induced Apoptosis

Abstract The Nlrp6 (NOD-like receptor family pyrin domain containing 6) inflammasome is important for intestinal epithelial cell innate immune responses and for maintaining gut homeostasis by preventing microbial dysbiosis. Contrary to its role in epithelial cell inflammasome-mediated responses, we recently showed that Nlrp6 in gut epithelial cells exacerbates GVHD in a manner independent of the inflammasome or gut microbiota. However, donor allogeneic T cells are also critical for GVHD development, yet, the function of Nlrp6 in allogeneic T cells is unknown. We hypothesized that Nlrp6 deficient donor T cells would ameliorate experimental GVHD. To test our hypothesis, WT-BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x10 6 bone marrow and 1.0x10 6 splenic CD90 +T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Nlrp6 -/- donors. Contrary to our hypothesis, the survival of allogeneic recipients of Nlrp6 -/- donor T cells was significantly worse than those receiving WT-B6 T cells (p&lt;0.05). Nlrp6 -/- donor T cells also caused greater GVHD mortality and morbidity in an MHC mismatched haploidentical B6 into B6D2F1 model (p&lt;0.05) and an MHC mismatched B10.BR into B6 model. Similar results were obtained using B6 into BALB/c and B6 into B6D2F1 models performed at the University of Michigan, suggesting our results were not unique to local environmental factors. By contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. Because the B6 into C3H.sw model is largely driven by CD8+ T cells whereas the previous models are mediated by both CD4+ and CD8+ T cells, we examined whether Nlrp6 separately regulates CD4+ and CD8+ T cell-mediated GVHD. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x10 6 CD4+ or CD8+ T cells from B6-WT or Nlrp6 -/- animals. GVHD severity and mortality (P&lt;0.05) were enhanced only when Nlrp6 -/- CD4+ T cells were transplanted. These data suggested that Nlrp6 regulates allogeneic T cell responses in a subset-specific manner. To explore how Nlrp6 regulates intrinsic responses in donor T cell subsets, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation. Consistent with the lack of increased GVHD induced by CD8+ Nlrp6 -/- donor T cells in the B6 into C3H.sw model, Nlrp6 -/- CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, when stimulated with either anti-CD3/CD28 antibodies or lethally irradiated allogeneic antigen presenting cells in a mixed lymphocyte reaction. In addition, activation-induced apoptosis was decreased in Nlrp6 -/- CD4+ T cells compared to WT T cells. Importantly, Treg suppressive function was not altered in Nlrp6 -/- T cells. Therefore, increased proliferative responses and resistance to activation-induced apoptosis may have contributed to the enhanced GVHD caused by Nlrp6 -/- donor T cells. Increased Th1 and Th17 polarization is associated with worse GVHD. Because only CD4+ Nlrp6 -/- T cells enhanced GVHD, we tested whether Nlrp6 influenced T helper cell differentiation into Th1, Th17, and Th2 subsets. Consistent with our in vivo data, Th1 in vitro differentiation was enhanced in Nlrp6 -/- CD4+ T cells. To determine the molecular signaling events altered by Nlrp6 deficiency, we tested various T cell activation signaling pathways and found that phosphorylation of ZAP-70 was increased in Nlrp6 -/- T cells. These data suggested that Nlrp6 in donor T cells may regulate allo-immune responses via ZAP-70 pathway. GVH and graft-versus-tumor (GVT) responses are intricately linked. Because CD8+ responses were not affected by Nlrp6 deficiency, we hypothesized that GVT responses would be unaltered in Nlrp6 -/- donor T cells. Indeed, Nlrp6 -/- T cells showed equivalent in vivo GVL responses to MLL-AF4 leukemia cells as WT-T cells. Hence Nlrp6in donor T cells is not required for GVT responses. Altogether our data suggested that Nlrp6 negatively-regulates allogeneic donor CD4+ T cell responses, possibly via negative regulation of ZAP-70 signaling, resulting in mitigation of GVHD and maintenance of robust GVT responses. Disclosures Ishizawa: AbbVie: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; Bristol Myers Squibb: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Consultancy; SymBio: Honoraria, Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; IQVIA: Research Funding.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

scholarly journals The critical immunosuppressive effect of MDSC-derived exosomes in the tumor microenvironment

2020 ◽  
Author(s):  
Mohammad H. Rashid ◽  
Thaiz F. Borin ◽  
Roxan Ara ◽  
Raziye Piranlioglu ◽  
Bhagelu R. Achyut ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
Cell Types ◽  
Biological Macromolecules

AbstractMyeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME), and our perception regarding the role of MDSCs in tumor promotion is attaining extra layer of intricacy in every study. In conjunction with MDSC’s immunosuppressive and anti-tumor immunity, they candidly facilitate tumor growth, differentiation, and metastasis in several ways that yet to be explored. Alike any other cell types, MDSCs also release a tremendous amount of exosomes or nanovesicles of endosomal origin and partake in intercellular communications by dispatching biological macromolecules. There has not been any experimental study done to characterize the role of MDSCs derived exosomes (MDSC exo) in the modulation of TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant amount of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those are in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cells in vivo by treating the mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that immunosuppressive and tumor-promoting functions of MDSC are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

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