Blockade of VIP-Signaling Enhanced Anti-Leukemic Activity in Murine Allogeneic BMT without Significantly Increased GvHD.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3001-3001
Author(s):  
Jian-Ming Li ◽  
Hyun Don Yun ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
T Cell ◽  
Small Molecule ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Transplant Recipients ◽  
Tumor Bearing ◽  
Allogeneic Bmt ◽  
Donor T Cells ◽  
Clinical Scoring

Abstract Abstract 3001 Background and Objective: Vasoactive intestinal peptide (VIP) has potent immune-suppressive activity and can generate tolerogenic dendritic cells (DC) in vitro that block graft versus host disease (GvHD) in mouse model of BMT. We have previously published that absence of VIP signaling dramatically decreases PD-1 expression on activated CD8 T-cells and increases cellular antiviral immunity (JI 2011, 187:1057). To determine whether blockade of VIP-signaling enhances the graft-versus-leukemia (GvL) activity of donor T-cells in an allogeneic BMT model, we treated tumor bearing B6B̂10BR allogeneic transplant recipients with a short course of daily s.c. injections of a small molecule VIP antagonist - VIPhyb or used VIP-knockout (VIP-KO) mice as BM donors. Methods: Recipient mice were inoculated with luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) by i.v. injection one day after lethal total body irradiation, then transplanted with the combination of 5 × 106 T cell-depleted BM (TCD-BM) plus splenocytes from either VIP-KO mice or wild-type (WT) littermates two days after irradiation. One group transplanted with WT BM and splenocytes received daily injections of 10 μg VIPhyb for one week; another group received saline injections. Survival, GvHD clinical sores (body weight, activity, posture, fur texture and skin), and bioluminescence imaging (BLI) were collected daily, twice a week, and weekly, respectively. Results: Transplantation of low dose (0.5 × 106) splenocytes from VIP-KO donors or low dose WT splenocytes in conjunction with VIPhyb-treatment dramatically improved tumor-free survival in the B6B̂10BR allogeneic BMT model compared with PBS-treated recipients of WT grafts (Figure 1). The best overall survival (70%) and lowest number of mice with detectable tumor (10%) were seen in the VIPhyb-treated group. VIPhyb-treated mice did not have increased GvHD as assessed by clinical scoring. Recipients of VIP-KO grafts had 40% survival with no detectable tumors by BLI, and were without significant GvHD by clinical scoring. In contrast, the recipients transplanted with TCD-BM alone (without added splenocytes) and recipients that received 0.5 × 106 splenocytes and TCD-BM from WT donors and treated with PBS had increased tumor growth detected by BLI following BMT, and all of the mice died by 2 months post-BMT. Moreover, in non-tumor bearing mice transplanted with an intermediate dose (1 × 106) of splenocytes, survival was not different among recipients engrafted with VIP-KO BM and T-cells (84 ± 6 %), WT BM and T-cells treated with VIPhyb (89 ± 8 %) and WT BM and T-cells treated with PBS (94 ± 5 %). A similar enhancement of the GvL effect and a corresponding survival advantage for VIP-signaling blockade was seen in tumor-bearing transplant recipients of TCD-BM plus 1 × 106 splenocytes, with significantly better survival among recipients of VIP-KO donor cells (50%), recipients of WT cells treated with VIPhyb (60%) compared with recipients of WT cells treated with PBS (20%; p=0.04). Furthermore, in non-tumor bearing mice that received a higher dose (3 × 106) of splenocytes, recipients of VIP-KO BM and recipients of WT BM treated with VIPhyb had no significant increase in GvHD compared with recipients of WT BM treated with PBS (66 ± 9 %, 71 ± 8 % and 71 ± 8 % survival at 80 days, respectively). The mechanism by which administration of a VIP antagonist enhanced anti-tumor immunity includes the effect of blocking VIP-signaling induction of cAMP, leading to fewer Treg and fewer tolerogenic DC. Of note, blocking VIP-signaling led to significant decreases in expression of PD-1 and PD-L1 on CD8+ T-cells and DCs, respectively. Conclusion: Treatment with a small molecule antagonist of VIP-signaling, VIPhyb, dramatically increased anti-leukemic activity of donor T-cells without significantly increased GvHD. Disclosures: No relevant conflicts of interest to declare.

Combining Early Heat Shock Protein Vaccination with Directed IL-2 Leads to Effective Anti-Tumor Immunity in Autologous Hematopoietic Cell Transplantation Recipients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 998-998
Author(s):  
Robert G. Newman ◽  
Eckhard R. Podack ◽  
Robert B. Levy
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Hematopoietic Cell Transplantation ◽  
Cd8 T Cell ◽  
Tumor Bearing ◽  
Donor T Cells ◽  
Vaccine Site ◽  
Autologous Hematopoietic Cell Transplantation

Abstract Abstract 998 Tumor relapse is still the major cause of morbidity and mortality in patients with hematologic cancers that undergo aggressive chemo-radiotherapy followed by autologous hematopoietic cell transplantation (auto-HCT). Hence, there is a critical need for new anti-tumor therapies. Heat shock protein (HSP) based vaccines elicit innate and adaptive immune responses in murine studies and have shown promise in clinical trials. The pre-clinical studies here investigated the efficacy of vaccination with tumor cells secreting the HSP fusion gp96-Ig together with directed IL-2 in tumor bearing auto-HCT recipients. To mimic clinical T cell replete auto-HCT, transplanted donor T cells were obtained from congenic tumor bearing mice (C57BL/6 CD45.2+ CD90.1+) that had been previously inoculated intraperitoneally (ip) with 4×106 OVA expressing lymphoma cells (E.G7). Some of these donor mice received 0.5×106 CD8 T cells specific for OVA257–264 (OT-I) to allow for tumor antigen specific T cell monitoring. Three weeks later, T cells were harvested from these animals bearing progressively growing tumor for use in T cell replete auto-HCT. Recipient mice (C57BL/6 CD45.2+ CD90.2+) received 9.5 Gy TBI with subsequent infusion of 5×106 congenic T cell depleted bone marrow cells (C57BL/6 CD45.1+ CD90.2+) supplemented with 2×106 enriched T cells from the tumor bearing donors. The following day, recipients were inoculated ip with 1×105 viable E.G7 lymphoma cells. Based on our prior findings, a multiple vaccination protocol was employed utilizing 1×107 irradiated E.G7 cells transfected to secrete the HSP fusion gp96-Ig (E.G7-gp96-Ig). Some recipients were administered IL-2 via specific antibody-cytokine complexes comprised of IL-2 and αIL-2 mAb clone S4B6 (IL-2/αIL-2CD122). This specific IL-2 complex has been shown to interact with cells expressing the β chain (CD122) of the IL-2 receptor, such as memory CD8 T cells and NK cells, but not with cells expressing the α chain (CD25). Compared to recipients of T cell replete auto-HCT vaccinated with parental E.G7 tumor cells who exhibited virtually no increase in antigen-specific CD8 T cells, marked expansion was detected in the blood after 2 vaccinations with E.G7-gp96-Ig, i.e. within 1 week of auto-HCT. This response reached a plateau after 3 vaccinations, and persisted throughout the 5 vaccine protocol. To quantitate this vaccine induced CD8 T cell expansion, analysis of the vaccine site, splenic and lymph node compartments was performed following 3 vaccinations, i.e. 2 weeks post-HCT. In contrast to the modest 25× increase observed after vaccination with parental E.G7 cells, a 175× expansion was detected following E.G7-gp96-Ig vaccination (6.8×106 vs. 3.8×104 input). Moreover, 75% of these gp96-Ig expanded CD8 T cells at the vaccine site were bifunctional, expressing IFN-γ and TNF-α following antigen specific stimulation ex vivo. Strikingly, combined treatment with vaccine cells secreting gp96-Ig together with IL-2/αIL-2CD122 complex resulted in a 1000× enhancement of antigen specific CD8 T cell numbers in all compartments analyzed. Tumor bearing auto-HCT recipients exhibited a median survival time (MST) of 1 month if not vaccinated or if vaccinated with parental E.G7 cells (Figure). However, vaccination with E.G7-gp96-Ig extended the MST by more than 2 weeks and ∼20% of recipients survived long term (>100 days). This effect was dependent on T cells since gp96-Ig vaccination alone without donor T cells resulted in no MST extension. Combination therapy with tumor cells secreting gp96-Ig and IL-2/αIL-2CD122 complex markedly elevated total CD8 T cells as well as NK cells at the vaccine site and in secondary lymphoid tissues, two populations that have been shown to facilitate HSP based vaccines. Notably, this strategy resulted in a MST >100 days with ∼60% of mice surviving indefinitely. We propose that 3 components are required together with auto-HCT to avoid relapse related mortality: (1) transplanted autologous T cells, (2) a pan-antigen vaccination approach that induces potent antigen presentation and activation of multiple antigen specific T cells, i.e. tumor cells secreting gp96-Ig, and (3) an adjuvant that potentiates this vaccine induced response, i.e. IL-2 delivered in the form of an antibody-cytokine complex. In total, this combinatorial protocol represents a promising regimen that could be translated into the clinic for patients with hematologic cancers. Disclosures: Podack: Heat Biologics, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3270-3270
Author(s):  
Mohammad S. Hossain ◽  
Ned Waller
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Lymph Nodes ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Post Transplant ◽  
Allogeneic Bmt ◽  
Donor T Cells ◽  
Organ Specific

Background: Allo-reactive donor T cells are primarily responsible for GvHD in allogeneic BMT. A number of studies have shown that increased allo-reactivity is found among the CD62L+ subset of donor T-cells, but the mechanisms for organ specific allo-reactivity are poorly defined. Our hypothesis is that rapid proliferation and migration of CD62L+ naive donor CD4+ and CD8+ T cells to specific organs leads to acute GvHD. Methods: We used a parent (C57BL/6) to (C57BL/6 × BALB/c) CB6F1 allogeneic BMT model with a combination of T cell depleted BM (TCD BM) and splenocytes. 30 × 106 congeneic donor splenocytes labeled with CFSE were transplanted with 5 × 106 TCD congeneic BM into lethally irradiated (11Gy) CB6F1 mice. Recipients were sacrificed within 3.5 days of transplant and FACS was used to measure proliferation of CFSE-labeled donor T-cells isolated from blood, spleen, liver, lungs, thymus, BM, lymph nodes, and peritoneal exudates cells (PEC). Syngeneic C57BL/6 recipients served as controls. At least 5 mice per group were used in each experiment. Results: There was increased homing of CFSE-labeled donor T-cells to most organs in allogeneic compared to syngeneic BMT recipients. CD45.1+ donor cells were 4-fold higher in spleen, p=0.01; 9-fold higher in liver, p=0.002; 14-fold higher in PEC, p=0.017; 136-fold higher in lung, p=0.0006; 126-fold higher in BM, P=0.002, 1482-fold higher in thymus p=0.002 compared to syngeneic recipients. Allogeneic and syngeneic recipients had equivalent numbers of donor CFSE-labeled lymphocytes in PBMC and lymph nodes. The tissue specific homing of CD4+ and CD8+ donor T-cells was also found significantly higher in most organs except the PBMC and LNs. Donor splenocytes were 80% CD62L+ before transplant, but the frequency of CD62L+ donor T-cells had declined to 15–16% in BM, 4–10% in liver, 17–30% in spleen and 10 to 25% in the thymus within 3.5 days post-transplant. In syngeneic recipients, 80% of donor T-cells remained CD62L+ within 3.5 days post-transplant. Most donor T-cells that divided rapidly lost expression of CD62L, while non-replicating donor CD4+ and CD8+ T cells remained predominately CD62L+. The expression of CD44 on donor T-cells were the opposite, with CD44+ cells undergoing less, and CD44− cells dividing more in allogeneic transplant recipients. In syngeneic BMT, donor CD4+ and CD8+ T-cells underwent minimal proliferation within the first 3.5 days post-transplant. Intracellular cytokine staining showed that high levels of IFN-g and TNF-a synthesis was seen among CD62L+ CD4+ and CD8+ T cells that had yet to divide (and had un-diluted CFSE staining). Conclusion: Migration of allogeneic donor T cells to tissues and local proliferation occurs rapidly after allogeneic BMT compared to recipients of syngeneic transplants. The dissociation of CD62L expression from lymph node homing suggests lack of the CD62L-receptor expression in lymph node HEV following irradiation, or a dominant effect of other chemokine receptors in directing donor T-cell preferentially to other organs. The marked and preferential homing of donor T-cells to the recipient thymus and bone marrow may play a role in achieving donor hematopoietic and T-cell chimerism in recipients of allogeneic BMT. Tissue specific homing of naive CD62L+ donor T-cells, with a high proliferative capacity, is likely responsible for the initiation of acute GvHD at these sites.

Enrichment of Donor Plasmacytoid Dendritic Cells in Bone Marrow Allografts Enhances Graft Vs. Leukemia Activity and Th1 Polarization of Donor T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4701-4701
Author(s):  
Kataryna Darlak ◽  
Ying Wang ◽  
Jian-Ming Li ◽  
Edmund K Waller
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Proliferation ◽  
Transplant Recipients ◽  
Post Transplant ◽  
Tumor Bearing ◽  
Donor T Cells ◽  
Selective Depletion ◽  
Th1 Polarization

Abstract Abstract 4701 Background: Allogeneic hematopoietic stem cell (HSC) transplant can cure patients with high risk and relapsed acute leukemia via a graft versus leukemia (GvL) effect. In our previous studies, depletion of CD11b+ cells (containing CD11b+ dendritic cells, CD11b+ NK cells, and myeloid suppressor progenitor cells) from donor bone marrow (BM) improved immune reconstitution and enhanced GvL effects without increased rates of GvHD in mice (Li, et al. BBMT 2004). We also have transplanted combinations of FACS purified HSC, donor T cells, and CD11b- dendritic cells (DC) and found similar improvement in GvL activity associated with donor T cells polarized towards a Th1 phenotype (Li, et al. JI 2009). In contrast, grafts containing FACS purified HSC, donor spleen T cells, and CD11b+ DC did not have significant GvL activity and donor T cells were polarized towards a Th2 phenotype after transplant. The objective of this study was to determine if enriching the relative numbers of donor plasmacytoid dendritic cells by selectively depleting CD11b+ DC from BM would yield similar enhancement of the GvL activity of donor T-cells as transplants of purified populations of HSC, CD11b-DC, and T cells in tumor-bearing mice. Methods: Selective depletion of CD11b+ DC from the BM allograft was achieved by FACS sorting and selectively removing the CD11b+ DC that comprised ∼1% of the BM. The CD11b+ DC-depleted graft thus contained ∼99% of all nucleated cells in the BM. Control undepleted BM grafts were also stained and sorted using only light scatter gates. To study the immunological effects of specific CD11b+ DC depletion, lethally irradiated B10.BR or BA.B10 recipients were transplanted with 3 × 10E6 CD11b+ DC FACS-depleted or undepleted BM cells and 1×10E6 spleen T cells from C57BL/6J or BA donors. Mice received 500,000 luciferase-positive LBRM cells (a T cell lymphoma) i.v. 1 day prior to transplant. Recipient mice were monitored for survival, weight change, and GvHD score (based on weight change, activity, posture, fur texture, and skin condition) throughout the duration of the experiment and for donor cell engraftment at days 30, 60, and 100 post transplant. Donor T cells were recovered from transplant recipients on days 3 and 10 post transplant and were examined for proliferation, Th1/Th2 polarization by flow cytometry, and ELISA measured serum cytokines. Results: BMT recipients of CD11b+ DC-depleted BM had higher survival than recipients of undepleted BM in tumor-bearing mice (p<0.05) (Figure 1). T cell chimerism at day 100 was > 95% for all mice, regardless of CD11b+ DC-depletion from the BM allograft. The level of GvHD in recipient mice did not differ significantly between groups. Donor T cell proliferation was increased on day 3 post transplant in recipients of CD11b+ DC-depleted BM (p< 0.05). On day 10 post transplant, serum IFN-g levels were increased in recipients of CD11b+ DC-depleted BM compared with undepleted BM(p<0.05). On day 10 post transplant, recipients of CD11b+ DC-depleted BM had significantly higher numbers of TNF-alpha producing donor CD8 T cells compared with donor CD8 T cells from recipients of undepleted BM (p<0.05) (Figure 2). Conclusions: Transplantation of BM allografts enriched for plasmacytoid DC by selective depletion of CD11b+ DC increased GvL activity of donor T cells without increasing GvHD. These data suggest that donor BM CD11b+ DC inhibit donor T-cell proliferation, Th1 polarization, and limit GvL activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation

Blood ◽  
2011 ◽  
Vol 118 (19) ◽  
pp. 5319-5329 ◽  
Author(s):  
Holbrook E. Kohrt ◽  
Antonia Müller ◽  
Jeanette Baker ◽  
Matthew J. Goldstein ◽  
Evan Newell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Antitumor Activity ◽  
Cd8 T Cells ◽  
Marrow Transplantation ◽  
Peptide Vaccination ◽  
Tumor Bearing ◽  
Allogeneic Bmt

Abstract The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part because of immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilms Tumor 1 (WT1) peptides, induces a T-cell population that is tumor antigen specific. We determined whether allogeneic BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent antitumor activity than either therapy alone. WT1 peptide vaccinations of healthy donor mice induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that peptide immunization was effective as a prophylactic vaccination before tumor challenge, yet was ineffective as a therapeutic vaccination in tumor-bearing mice. BMT from vaccinated healthy MHC-matched donors, but not syngeneic donors, into recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of FBL3 leukemia. The transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells and both required CD4+ T-cell help for maximal antitumor activity. These findings show that WT1 peptide vaccination of donor mice can dramatically enhance GvT activity after MHC-matched allogeneic BMT.

scholarly journals 578 CD8-targeted IL-2 drives potent anti-tumor efficacy and promotes action of tumor specific vaccines

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A607-A607
Author(s):  
Hussein Sultan ◽  
Kelly Moynihan ◽  
Yuang Song ◽  
Samuel Ameh ◽  
Ton Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Body Weight Loss ◽  
Tumor Rejection ◽  
High Dose ◽  
Therapeutic Effects ◽  
Toxicity Profile ◽  
Tumor Bearing

BackgroundIL-2 and currently available engineered variants are of interest for solid tumor treatment, but their efficacy and toxicity profiles remain suboptimal. These results reflect the pleiotropic signaling via IL-2 receptors on different cell types that may simultaneously drive desired and undesired responses. We hypothesized that restricting IL-2’s activity to CD8+ T cells would improve efficacy while also lowering its toxicity profile.MethodsWe developed a cis-targeted IL-2 that selectively acts on CD8+ T cells (CD8-IL2) and assessed its activity using the T3 progressor MCA sarcoma model, which was selected because (a) it is sensitive to anti-PD-1 therapy when tumors are small but develops insensitivity as tumor size increase, (b) rejection requires both CD4+ and CD8+ T cells and (c) rejection is dependent on tumor expression of two neoantigens: mItgb1 (MHC-II) and mLama4 (MHC-I).ResultsWhereas mice bearing 8-day T3 tumors had become insensitive to anti-PD-1 mediated tumor rejection, 90% of mice treated with single dose CD8-IL2 monotherapy rejected their tumors, while high dose IL-2 produced minimal efficacy. Efficacy occurred without body weight loss. These results suggest that CD8-IL2 can induce therapeutic effects at a time when tumors became insensitive to anti-PD-1. To assess this possibility in a more controlled manner, we used a tumor neoantigen vaccine model that depends on CD4+ T cell help for development of functional CD8+ T cells at both the priming stage in the lymph node as well as the effector stage at the tumor site. Mice bearing T3 tumors were vaccinated with a synthetic long peptide (SLP) containing the mLama4 neoepitope and either a high or low dose of an SLP containing the mItgb1 neoepitope. Whereas 85% of tumor bearing mice that received the vaccine containing mLama4 plus low dose mItgb1 SLP rejected their tumors, surprisingly none of the mice receiving high dose mItgb1 underwent tumor rejection. This high dose inhibition was reversed when CD8-IL2 was administered after high dose vaccination and at concentrations that had only modest activity in tumor bearing, non-vaccinated mice. With CD8-IL2 treatment, antigen specific T cells were expanded and displayed increased expression of activation-associated markers and reduced expression of exhaustion-associated markers.ConclusionsCD8-IL2 outperformed other forms of engineered IL-2 in anti-tumor efficacy, showed a significantly improved toxicity profile, and rescued deficient CD8 T cell responses resulting from poor CD4 help. In sum, we demonstrate high level antitumor efficacy and tolerability with a new form of targeted IL-2.Ethics ApprovalMice used in this study were between 8 and 12 weeks of age and were maintained in accordance with procedures approved by the Association for Assessment and Accreditation of Laboratory Animal Care and Accredited Animal Studies Committee of Washington University in St. Louis

scholarly journals 767 Activation of CD8+ T cells in the presence of multiple TLR agonists affects the expression of T-cell checkpoint receptors via IL-12 and type-1 interferon

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A802-A802
Author(s):  
Donghwan Jeon ◽  
Douglas McNeel
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cancer Immunology ◽  
Receptor Expression ◽  
Tlr Agonists ◽  
Tumor Bearing ◽  
Pathway Activation ◽  
Checkpoint Receptors ◽  
Anti Tumor Activity

BackgroundT-cell checkpoint receptors are expressed when T-cell are activated, and activation of these receptors can impair the function of T-cells and their anti-tumor efficacy.1 We previously found that T-cells activated with cognate antigen increase the expression of PD-1, while this can be attenuated by the presence of specific Toll-like receptor (TLR) agonists.2 3 This effect was mediated by IL-12 secretion from professional antigen presenting cells and resulted in CD8+ T cells with greater anti-tumor activity. In the current report, we sought to determine whether combination of TLR agonists can further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity.MethodsOT-1 CD8+ T cells were stimulated with peptide (SIINFEKL) and dendritic cells (DC) in the presence of two different TLR agonists. The cells were collected and evaluated for the expression of T-cell checkpoint receptors (PD-1, CTLA-4, CD160, CD244, LAG-3, TIM-3, TIGIT and VISTA) by flow cytometry, and for transcriptional changes by RNA-seq. Purified DC were stimulated with TLR combinations and evaluated for cytokine release by ELISA. The anti-tumor efficacy of vaccination using peptide and TLR agonist combinations was evaluated in EG7-OVA tumor-bearing mice.ResultsActivation of CD8+ T cells in the presence of specific TLR ligands resulted in decreases in expression of PD-1 and/or CD160. These changes in T-cell checkpoint receptor expression were modestly affected when TLR ligands were used in combination, and notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of tumor-bearing mice, co-administered with combinations of these agonists, showed greater anti-tumor effects. However, while the effect of TLR1/2 and/or TLR9 was abrogated in IL12KO mice, TLR3 demonstrated anti-tumor activity when co-administered with peptide vaccine. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and IFNß pathway activation following TLR3 stimulation. Stimulation of DC with TLR3 agonist, alone or in combination with other TLR agonists, resulted in increased IL-12 and IFNß secretion. Co-incubation of OT-1 splenocytes with rIL12 and/or rIFNß during peptide activation led to reduced expression of PD-1, and this could be reversed with antibodies blocking IL12R or IFNAR-1.ConclusionsMultiple TLR agonists can modulate the expression of T-cell checkpoint receptors, notably PD-1, by upregulating the secretion of IL-12 and IFNß. These data provide the mechanistic rationale for choosing optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.ReferencesJin H-T, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proceedings of the National Academy of Sciences 2010;107(33):14733–14738.Zahm CD, Colluru VT, McNeel DG. Vaccination with high-affinity epitopes impairs antitumor efficacy by increasing PD-1 expression on CD8+ T cells. Cancer Immunology Research 2017;5(8):630–641.Zahm CD, et al. TLR stimulation during T-cell activation lowers PD-1 expression on CD8+ T Cells. Cancer Immunology Research 2018;6(11):1364–1374.

scholarly journals Cortisol and epinephrine control opposing circadian rhythms in T cell subsets

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5134-5143 ◽  
Author(s):  
Stoyan Dimitrov ◽  
Christian Benedict ◽  
Dennis Heutling ◽  
Jürgen Westermann ◽  
Jan Born ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Circadian Rhythms ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Cell Counts

Abstract Pronounced circadian rhythms in numbers of circulating T cells reflect a systemic control of adaptive immunity whose mechanisms are obscure. Here, we show that circadian variations in T cell subpopulations in human blood are differentially regulated via release of cortisol and catecholamines. Within the CD4+ and CD8+ T cell subsets, naive cells show pronounced circadian rhythms with a daytime nadir, whereas (terminally differentiated) effector CD8+ T cell counts peak during daytime. Naive T cells were negatively correlated with cortisol rhythms, decreased after low-dose cortisol infusion, and showed highest expression of CXCR4, which was up-regulated by cortisol. Effector CD8+ T cells were positively correlated with epinephrine rhythms, increased after low-dose epinephrine infusion, and showed highest expression of β-adrenergic and fractalkine receptors (CX3CR1). Daytime increases in cortisol via CXCR4 probably act to redistribute naive T cells to bone marrow, whereas daytime increases in catecholamines via β-adrenoceptors and, possibly, a suppression of fractalkine signaling promote mobilization of effector CD8+ T cells from the marginal pool. Thus, activation of the major stress hormones during daytime favor immediate effector defense but diminish capabilities for initiating adaptive immune responses.

scholarly journals P01.13 MERTK signaling is critical for T cell proliferation and memory

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A14.2-A15
Author(s):  
RM Powell ◽  
MJW Peeters ◽  
A Rachbech ◽  
PT Straten
Keyword(s):  
T Cells ◽  
Cell Division ◽  
T Cell ◽  
Cd8 T Cells ◽  
Central Memory ◽  
T Cell Proliferation ◽  
Flow Cytometric ◽  
Donor T Cells ◽  
Memory Differentiation

BackgroundOverexpression of TAM receptors, including MERTK, in some cancers are integral for chemoresistance, proliferation and metastasis.1 Our group has previously demonstrated that T cells also express MERTK and engagement of MERTK signaling is responsible for increased proliferation, functional capacity and metabolic fitness.2 It is therefore important to further study the effect of MERTK inhibition on T cell function in the context of cancer treatments where MERTK inhibitors may play a role. Here we provide evidence that MERTK inhibition impacts greatly on T cell proliferation, specifically reducing phosphorylated mTOR. We have also demonstrated that MERTK expression is increased on CD8 central memory subsets during longterm expansion providing evidence that this signaling pathway may be important for sustaining T memory responses.Materials and MethodsFlow cytometric analysis was used to investigate the effect of titration of MERTK small molecule inhibitor UNC2025 on healthy donor T cells activated with CD3/CD28 dynabeads. Cell trace dye was used to track proliferation of CD4 and CD8 T cells along with markers of memory differentiation (CCR7 and CD45RO), activation (CD137) and function (IFNy, Tnfa and IL-2). MERTK signaling was assessed using phospho flow cytometric methodology of phosphorylated mTOR, AKT, ERK1/2, p38-MAPK and STAT5. Long term cultures of donor T cells of up to 28 days were investigated for MERTK expression alongside memory differentiation.ResultsWe demonstrated that moderate concentrations of MERTK inhibitor reduced proliferation of activated T cells. Despite inhibition of cell division, cell size still increased 2 fold compared to resting cells and cell viability remained unchanged. Additionally, the proportion of central memory to effector memory populations and intracellular cytokine production was not impacted. Analysis of molecules involved in MERTK signaling revealed that phosphorylated mTOR was significantly modulated following the addition of MERTK inhibitor. Long term culture of CD8 T cells demonstrated MERTK was significantly increased following early and late re-stimulation, and expression of MERTK was strongly associated with central memory subsets.ConclusionsOur results demonstrate that inhibition of MERTK signaling on T cells reduces cell division where mTOR is significantly impacted. Despite this, other functional aspects, such as intracellular cytokine production remain unchanged. Therefore, interruption of MERTK signaling on T cells has a specific effect on cell division rather than cytotoxic function on a cell by cell basis. This has potential ramifications on the use of MERTK inhibitors to treat tumors where the ability to form substantial cytotoxic T cell populations might be reduced. In addition, increased MERTK expression on central memory subsets during long term culture suggests this signaling pathway could be critical for generating memory pools of T cells and provide new avenues for the improvement of adoptive T cell therapy protocols.ReferencesCummings CT, Deryckere D, Earp HS, Graham DK. Molecular pathways: MERTK signaling in cancer. Clin Cancer Res 2013;19(19):5275–5280.Peeters MJW, Dulkeviciute D, Draghi A, et al. MERTK Acts as a Costimulatory Receptor on Human CD8+T Cells. Cancer Immunol Res 2019;7(9):1472–1484.Disclosure InformationR.M. Powell: None. M.J.W. Peeters: None. A. Rachbech: None. P.T. Straten: None.

scholarly journals Retarded recovery of functional T cell frequencies in T cell-depleted bone marrow transplant recipients

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 960-964 ◽  
Author(s):  
JP Daley ◽  
MK Rozans ◽  
BR Smith ◽  
SJ Burakoff ◽  
JM Rappeport ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
T Lymphocyte ◽  
Marrow Transplant ◽  
Transplant Recipients ◽  
Cell Compartment ◽  
Donor T Cells ◽  
Limiting Dilution

Abstract We have studied the effect of removing donor T cells by treatment with the monoclonal antibody Leu-1 and complement before marrow transplantation on the regeneration of functionally competent T lymphocytes in the blood at selected times after transplant. Using sensitive limiting-dilution methods that allow us to enumerate helper, cytotoxic, and proliferating T lymphocyte precursors, we report that regeneration of a functional T cell compartment is more severely impaired for the first 180 days after transplantation in those patients given T cell-depleted bone marrow than in recipients of untreated marrow. After this first 6 months, however, patients given T cell- depleted bone marrow had blood T cell frequencies comparable to those observed in patients given untreated marrow. Diminished frequencies of reactive T cells in recipients of depleted marrow could leave them more susceptible to infection or to the recurrence of neoplastic cells.

scholarly journals Functional analysis of clinical response to low-dose IL-2 in patients with refractory chronic graft-versus-host disease

2019 ◽  
Vol 3 (7) ◽  
pp. 984-994 ◽  
Author(s):  
Jennifer S. Whangbo ◽  
Haesook T. Kim ◽  
Sarah Nikiforow ◽  
John Koreth ◽  
Ana C. Alho ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Low Dose ◽  
B Cell Lymphoma ◽  
Host Disease ◽  
Graft Versus Host ◽  
Response Groups

Abstract Patients with chronic graft-versus-host disease (cGVHD) have a paucity of regulatory CD4 T cells (CD4Tregs) that mediate peripheral tolerance. In clinical trials, daily low-dose interleukin-2 (IL-2) has been administered safely for prolonged periods in patients with steroid-refractory cGVHD. Peripheral CD4Tregs expand dramatically in all patients during IL-2 therapy but clinical improvement was observed in ∼50% of patients. Here, we examined the impact of low-dose IL-2 therapy on functional T-cell markers and the T-cell repertoire within CD4Tregs, conventional CD4 T cells (CD4Tcons), and CD8+ T cells. IL-2 had profound effects on CD4Tregs homeostasis in both response groups including selective expansion of the naive subset, improved thymic output, and increased expression of Ki67, FOXP3, and B-cell lymphoma 2 within CD4Tregs. Similar changes were not seen in CD4Tcons or CD8 T cells. Functionally, low-dose IL-2 enhanced, in vitro, CD4Treg-suppressive activity in both response groups, and all patient CD4Tcons were similarly suppressed by healthy donor CD4Tregs. High-throughput sequencing of the T-cell receptor β (TCRβ) locus demonstrated that low-dose IL-2 therapy increased TCR repertoire diversity and decreased evenness within CD4Tregs without affecting CD4Tcons or CD8 T cells. Using clone-tracking analysis, we observed rapid turnover of highly prevalent clones in CD4Tregs as well as the conversion of CD4Tcons to CD4Tregs. After 12 weeks of daily IL-2, clinical responders had a greater influx of novel clones within the CD4Treg compartment compared with nonresponders. Further studies to define the function and specificity of these novel CD4Treg clones may help establish the mechanisms whereby low-dose IL-2 therapy promotes immune tolerance.

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