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.

Therapeutic Anti-Tumor Immunity Mediated by Transiently Engrafting Allogeneic Lymphocytes: The “Allogeneic Effect” Revisited.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5255-5255
Author(s):  
Heather J. Symons ◽  
M. Yair Levy ◽  
Jie Wang ◽  
Xiaotao Zhou ◽  
Ephraim J. Fuchs
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Host Immune System ◽  
Tumoricidal Activity ◽  
Tumor Bearing ◽  
Anti Tumor Activity ◽  
Allogeneic Lymphocytes

Abstract The “allogeneic effect” refers to the induction of host B cell antibody synthesis or host T cell cytotoxicity, including tumoricidal activity, by an infusion of allogeneic lymphocytes. We have previously shown that treatment of mice with cyclophosphamide (Cy) followed by infusion of CD8+ T cell-depleted allogeneic spleen cells (Cy + CD8− DLI) induces anti-tumor activity in a model of minimal residual leukemia, even though the donor cells are eventually rejected by the host immune system. The purpose of the current investigation was to test the activity of Cy + CD8− DLI in the treatment of well-established cancer, and to characterize the mechanisms of the anti-tumor effect. BALB/c mice were inoculated intravenously (IV) with the syngeneic A20 lymphoma/leukemia or the RENCA renal cell carcinoma on day 0 and were then treated with nothing, Cy alone on day 14, or Cy + CD8− DLI from MHC-mismatched C57BL/6 donors on day 15. In both tumor models, the combination of Cy + CD8− DLI significantly prolonged survival compared to mice treated with nothing or with Cy alone. While depletion of CD4+ T cells from the DLI significantly diminished the beneficial effect of CD8− DLI, purified CD4+ T cells alone were inactive, demonstrating that donor CD4+ T cells and another population of cells were required for optimal anti-tumor activity. Several observations pointed to an active role for the host immune system in the anti-tumor activity of Cy + CD8− DLI. First, host T cells participated in the anti-tumor effect of treatment with Cy alone, since the drug’s activity was diminished in tumor-bearing scid mice or in normal BALB/c mice depleted of T cells. Second, while Cy + CD8− DLI caused no GVHD in tumor-bearing but immunocompetent BALB/c recipients, it caused fatal acute GVHD in either tumor-bearing scid or T-cell depleted BALB/c mice. Finally, the anti-tumor effect of Cy + CD8- DLI was also significantly inhibited in BALB/c mice that were depleted of CD8+ T cells. These results demonstrate that transiently engrafting T cells administered after Cy can induce significant anti-tumor effects against both solid and liquid tumors. We propose that upon recognition of alloantigen on host antigen-presenting cells (APCs), allogeneic donor CD4+ T cells deliver activating ligands to the APCs, thereby generating effective “help” to break tolerance in tumor-specific host CD8+ T cells. This mechanism may correspond to the “allogeneic effect” in the anti-tumor response described over three decades ago.

Donor Immunization with WT1 Peptide Augments Anti-Leukemic Activity After MHC-Matched Bone Marrow Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1896-1896
Author(s):  
Holbrook E Kohrt ◽  
Antonia MS Mueller ◽  
Jeanette B Baker ◽  
Matthew J Goldstein ◽  
Evan Newell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Antigen ◽  
Cd4 T Cell ◽  
Leukemia Cells ◽  
Peptide Vaccination ◽  
Tumor Bearing ◽  
Ifn Γ ◽  
Anti Tumor Activity

Abstract Abstract 1896 The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part due to immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilm's Tumor 1 (WT1) peptides, induces a T cell population that is tumor antigen specific. We determined whether BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent anti-tumor activity when combined with allotransplantation. WT1 peptide vaccinations of healthy syngeneic or allogeneic donor mice with a 9-mer WT1 peptide (amino acids 126–134, the WT1 9-mer which has the highest binding affinity for H-2Db) and Incomplete Freund's Adjuvant induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that compared to vaccination with IFA alone, four weekly WT1 vaccinations induced an increased percentage of WT1-tetramer+CD8 T-cells (0.15% vs. 1%) in the peripheral blood 28 days following the first vaccination (Figure A *p<.001). CD8 T-cells producing IFN-γ+ after co-culture with tumor cells were similarly increased (0.11% vs. 13.6%) at this timepoint (Figure B *p<.001). They were CD44hi suggesting a memory phenotype, specifically reactive to WT1-expressing tumor (FBL3 and not H11), and increased in a vaccination dose-dependent fashion (Figure A and B). Four weekly WT1 vaccinations prevented tumor growth in donors following intravenous leukemia challenge. In contrast, in tumor-bearing mice, WT1 vaccinations failed to induce WT1-tetramer+ or IFN-γ+ CD8 T-cells and were ineffective as a therapeutic vaccine based on intensity of bioluminescence from luciferase-labeled FBL3 leukemia and mortality. BMT from WT1 vaccinated MHC-matched donors including LP/J and C3H.SW, but not C57BL/6 syngeneic donors, into C57BL/6 recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of luciferase-labeled FBL3 leukemia and survival of 70–90% of mice. Interestingly, the transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells, likely as a result of alloreactive and tumor-antigen reactive T cells contained with the donor total CD8+ T cells. Total and tetramer+CD8+ T cells required CD4+ T cell help for maximal anti-tumor activity, which was equivalent in efficacy from immunized or unimmunized CD4+ T cell donors. Total CD4+ T cells, alone, from immunized donors provided no anti-tumor activity. The infused donor LP/J or C3H.SW CD8+ T cells collected from cured C57BL/6 recipients, were highly reactive against WT1-expressing FBL3 leukemia cells (14% IFN-γ+) compared to non-WT1-expressing H11 leukemia cells (5% IFN-γ+). The circulating, WT1-tetramer+CD8+ T cell population expanded in cured recipients, peaking at 3.5% on day 50 and contracting through day 100 post-BMT to 0.56%. These findings show that peptide vaccination of donor mice with a tumor antigen dramatically enhances GvT activity and is synergistic with allogeneic BMT. This novel and broadly applicable approach, using leukemia-associated antigen immunization to enhance GvT by creating an “educated” donor T cell graft for allogeneic transplantation of patients with acute myeloid leukemia and myelodysplastic syndrome, is currently being translated to a Phase 1 clinical trial at our institution. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 702 TJ-CD4B (ABL111), a Claudin18.2-targeted 4-1BB tumor engager induces potent tumor-dependent immune response without dose-limiting toxicity in preclinical studies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A730-A730
Author(s):  
Wenqing Jiang ◽  
Zhengyi Wang ◽  
Zhen Sheng ◽  
Jaeho Jung ◽  
Taylor Guo
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Gene Expression Analysis ◽  
Cell Activation ◽  
Clinical Development ◽  
Cynomolgus Monkeys ◽  
Anti Tumor Activity

Background4-1BB (CD137) is a co-stimulatory receptor that stimulates the function of multiple immune cells. Its ability to induce potent anti-tumor activity makes 4-1BB an attractive target for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic toxicities, we have developed a novel Claudin18.2 (CLDN18.2) x 4-1BB bispecific antibody, TJ-CD4B (ABL111) that stimulates 4-1BB pathway only when it engages with Claudin 18.2, a tumor-associated antigen specifically expressed in gastrointestinal cancers. TJ-CD4B (ABL111) is now being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04900818).MethodsTJ-CD4B (ABL111) was evaluated in vivo using the human 4-1BB knock-in mice bearing CLDN18.2 expressing MC38 tumor cells. Pharmacodynamic effects upon treatment were characterized in tumor tissue and blood. Immunophenotyping of the tumor microenvironment (TME) and peripheral blood was performed by flow cytometry. Soluble biomarkers were measured using Luminex-based multiplex assay. In-depth gene expression analysis was performed on primary human CD8+ T cells that were co-cultured with CLDN18.2 expressing cells in the presence of anti-CD3 using NanoString nCounter®. Pharmacokinetic (PK) and toxicity study were performed in cynomolgus monkeys.ResultsTJ-CD4B (ABL111) elicited complete tumor regression in 13 out of 18 MC38 tumor bearing mice given at a dose above 2 mg/kg. Dose-dependent anti-tumor activity was associated with enhanced T cell activation in TME and expansion of memory T cells in the peripheral blood. Increased CD8+ T cells number and proliferation were observed in both tumor nest and surrounding stroma while the level of soluble 4-1BB in the serum was also elevated in response to the treatment. In vitro gene expression analysis by Nanostring revealed TJ-CD4B(ABL111) effectively activated immune pathways characterized by IFN?-signaling and T cell inflammation. Preclinically, TJ-CD4B was well tolerated at the repeated doses up to 100 mg/kg/wk in cynomolgus monkeys without the adverse influence on the liver function which is generally affected by 4-1BB activation. Besides, no cytokine release or immune activation was observed in the periphery.ConclusionsTJ-CD4B (ABL111) is a novel CLDN18.2 dependent 4-1BB bispecific agonist antibody that induced T cell activation and memory response in tumor with CLDN18.2 expression, leading to a strong anti-tumor activity in vivo. TJ-CD4B did not induce systemic immune response nor hepatic toxicity due to the CLDN18.2 dependent 4-1BB stimulation. These data warrant the current clinical development in phase I trial to validate the safety properties and tumor specific responses.

Augmentation of Therapeutic Tumor Vaccine Efficacy by Genetic Engineering of CD4+ T Cell Help for Tumor Vaccine-Reactive CD8+ T Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3175-3175
Author(s):  
Sanju Jalla ◽  
Erin McCadden ◽  
Jie Wang ◽  
Ephraim J. Fuchs ◽  
Katharine A. Whartenby
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Autologous Tumor Cell ◽  
Autologous Tumor ◽  
Tumor Bearing ◽  
Cd4 T Cell Help ◽  
T Cell Help

Abstract Since CD4+ T cell help has been proposed to be required for maintaining the activity of tumor-specific CD8+ T cells, tolerance in tumor-specific CD4+ T cells may seriously impair the efficacy of therapeutic tumor vaccines. To overcome this problem, we devised a strategy to “engineer” CD4+ T cell help by treating tumor-bearing animals with nonmyeloablative conditioning and transplantation of autologous hematopoietic stem cells (HSCs) that have been genetically modified, via lentiviral transduction, to express an antigen containing “foreign” CD4+ T cell epitopes. After hematopoietic reconstitution, animals received the combination of an autologous tumor cell vaccine and an infusion of primed CD4+ T cells specific for the expressed epitopes. Using influenza hemagglutinin (HA) as the model antigen, we first confirmed that transplantation of HA-transduced HSCs led to efficient expression of HA by antigen-presenting cells, as demonstrated by the clonal expansion of adoptively transferred, HA-specific CD4+ transgenic T cells in mice receiving HA-transduced HSCs but not in mice receiving nerve growth factor receptor (NGFR) gene-transduced HSCs. Next, BALB/c mice harboring 13 day old, metastatic 4T1 mammary cancer were treated with removal of the primary, nonmyeloablative conditioning and transplantation of HA-transduced syngeneic HSCs, and following hematopoietic reconstitution, with concomitant autologous tumor cell vaccination and adoptive transfer of in vitro activated, HA-specific transgenic CD4+ T cells. This therapy was successful in curing the majority of tumor bearing mice, and was superior to the same therapy given to mice transplanted with NGFR-transduced stem cells. Finally, we found that the anti-tumor effect of vaccination plus exogenous T cell help was abolished by the adoptive transfer of either CD4+ or CD8+ T cells from tumor-bearing mice, suggesting that tumor-bearing mice contain both potential effectors and suppressors of anti-tumor immunity, the latter of which are abolished by the non-myeloablative conditioning. These results highlight the importance of CD4+ T cell help in the induction of therapeutic anti-tumor immunity.

Circulating T Cells Derived From Patients with Low Risk Myelodysplastic Syndromes Show Altered Chemokine Receptor Expression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4018-4018
Author(s):  
Kristoffer E Sand ◽  
Astrid Olsnes Kittang ◽  
Øystein Bruserud
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chemokine Receptor ◽  
Cd8 T Cells ◽  
Central Memory ◽  
Low Risk ◽  
Receptor Expression ◽  
Effector Memory ◽  
Circulating T Cells ◽  
Cx3cr1 Expression

Abstract Abstract 4018 Several T cell abnormalities have been described in myelodysplastic syndromes (MDS), and such abnormalities may become important for identification of patients who will benefit from T cell targeting immunosuppressive treatment. Chemokine receptor repertoires are important in the regulation of both T cell migration and function and may therefore be important in the development of MDS. Materials and Methods: The chemokine receptor expression by circulating T cells were investigated by multicolor flow cytometry for patients with newly diagnosed low risk MDS (N=16) and for healthy controls (N=18). CD3+CD8+ and CD3+CD8- cells were examined for expression of CCR2-7, CXCR3-4 and CX3CR1 (only 7 unselected patients examined for CX3CR1 expression). CCR6 and CXCR4 expression was also investigated for specific T cell subsets defined by CD62L and CD45RA expression (naïve, central memory, effector memory and terminal effector). CX3CR1 expression was stratified for CD8+ and CD8- subpopulations according to high, low and negative expression of CCR5. Results: Chemokine receptor profiles showed several differences between low risk MDS patients and controls. Total CD8+ T cells from MDS patients showed increased expression of CCR3 (p=0.005) and decreased expression of CCR7 and CCR4 (p=0.023 and p=0.036 respectively), whereas the CD8- T cells from MDS patients showed increased expression of CX3CR1 (p=0.043). In contrast, CCR6 expression was increased only by CD8+ central memory T cells (p=0.044). Finally, CD8+CCR5- and CD8-CCR5high cells from MDS patients showed increased expression of CX3CR1 compared with the controls (p=0.011 and p=0.049). Discussion: CCR7 is mainly expressed by central memory and naïve T cells whereas CX3CR1 is especially expressed by cytotoxic effector lymphocytes independent of the lymphocyte subclass (i.e. CD4, CD8, delta/gamma and NK cells). The observed changes are in line with a shift from naïve/central memory to effector/effector memory dominance, especially in the CD8+ population. A similar shift has been described previously using CD45RA and CD62L (Zou et al, Leukemia 2009). Our median values are in line with such a shift. CCR6 expression is associated with IL17 production both for CD8+ and CD4+cells, and increased levels of circulating CD4+ IL17 producing cells in low risk MDS have been described. Conclusions: The chemokine receptor profiles of circulating T cells differ between low risk MDS patients and healthy controls, especially for the CD8+ T cell subset. These differences may be important for T cell trafficking and disease development, and they may reflect a shift from naïve to effector/effector memory cell dominance in low risk MDS. Disclosures: No relevant conflicts of interest to declare.

TNFR2 Is Expressed Preferentially By Late Differentiated CD8 T-Cells and Can be Triggered By TNFR2-Specific Ligand to Induce Cell Death of Recently Activated Antigen-Specific T Cells: A Possible Role of TNFR2 in T-Cell Deflation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4352-4352
Author(s):  
Mohammad Raeiszadeh ◽  
Matthew Verney ◽  
Charles Craddock ◽  
Harald Wajant ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Receptor Expression ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Cell Transplant ◽  
Gamma Chain ◽  
Transplant Patients

Abstract Recent evidence suggests that Tumor Necrosis Factor (TNF) can selectively kill antigen-specific autoreactive CD8+ T-cells through engagement with TNF Receptor 2 (TNFR2) (1). Within the immune system, TNFR2 expression is restricted to subsets of T-cells, a profile which is in marked contrast to the ubiquitous pattern of expression of TNFR1. However, the spectrum and physiological significance of TNFR2 expression by CD8+ T-cell subpopulations is unknown. In this study we analysed the expression of TNFR2 by CD8 T-cell subsets isolated from normal healthy donors by flow cytometry. In addition, in order to understand the physiological significance of TNFR2 expression on recently activated T cells, we further studied expression on CMV-specific CD8 T-cells which expanded in stem cell transplant patients in response to episodes of CMV reactivation. The expression of TNFR2 was compared to that of other common gamma chain receptors including IL2R and IL7R, and to the expression of a receptor for inflammatory cytokine IL6. TNFR2 expression was found to increase during differentiation of CD8+ T cells. In particular, TNFR2 expression was seen on 6.5% of naïve, 14.6% of central memory, 37.9% of effector memory and 45.2% of CD45RA-revertant effector memory (TEMRA) CD8+ T cells. In contrast, common gamma chain cytokine receptor expression was skewed towards less differentiated T-cell subsets. For example, IL-7R was expressed by 63% of central memory populations but only 18.4% of the TEMRA subset. Comparable expression of IL2R was 12.1% on TCM and 2% on TEMRA. Of interest, IL-6 receptor expression was predominantly expressed by naïve CD8 T-cells (69.5%). In support of these results, we went on to show that expression of TNFR2 was inducible on primary T cells following activation with anti-CD3 and IL-2 in vitro. Healthy CMV seropositive donors had a larger median number of CD8+ T cells expressing TNFR2 (53%) in comparison to CMV seronegative donors (15%), (p<0.0001), consistent with the known accumulation of differentiated T-cells within CMV seropositive individuals.The expression of TNFR2 was then examined on CMV-specific CD8 T-cells which were undergoing acute expansion in response to viremia in six haemopoietic stem cell transplant patients. The expansion of CMV-specific CD8 T-cells was accompanied by an increase in the intensity of TNFR2 expression which later decreased during the retraction of antigen-specific T-cells during resolution of viremia. In order to explore the functional significance of TNFR2 expression, T-cells isolated from healthy donors were treated with recombinant TNFR2-specific ligand. This induced cell loss ranging from 13% to 60% of all CD8 T-cells in relation to untreated control cells, with selective depletion of the TNFR2+ population. A similar proportion of CMV-specific T-cells from transplant patients were eliminated by ex vivo stimulation of TNFR2. In conclusion our work shows that TNFR2 expression increases during differentiation of CD8+ T cells. In addition, we were able to utilize virus-specific T cells from SCT patients to show that expression is increased during the acute response to stimulation with antigen. We also provide evidence that TNFR2 activation can lead to the partial elimination of antigen-specific CMV-specific T-cells and it may thus play an important role in the ‘deflation’ of a pathogen-specific T-cell immune response following resolution of infection. These data suggest that TNFR2 expression may act as a ligand to signal activation-induced cell death in late differentiated populations of CD8+ T cells. Further investigations are required to assess the molecular pathways of TNFR2 signalling that are activated following receptor ligation in vivoand whether or not these are disrupted in disorders associated with chronic CD8+ T cell lymphproliferation. (1) L. Ban et al, PNAS 2008, 105: 3644 Disclosures No relevant conflicts of interest to declare.

scholarly journals Acute Myeloid Leukemia (AML) Blasts Influence the Gene Expression Signature and Co-Signaling Receptor Expression of CD8+ T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1700-1700
Author(s):  
Hanna A. Knaus ◽  
Sofia Berglund ◽  
Hubert Hackl ◽  
Raúl Montiel-Esparza ◽  
Mark J. Levis ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Induction Chemotherapy ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Receptor Expression ◽  
Inhibitory Receptor ◽  
Canonical Pathways

Abstract Background: T cell dysfunction in AML remains poorly understood. Our previous studies of AML-associated T cell dysfunction (Knaus, ASH 2015) have focused on expression of multiple inhibitory receptors by T cells in AML patients. Transcriptional signatures, however, remain relatively unexplored, as does the role of Blast/T cell interactions on T cell function. Deciphering those could be crucial for integration of future immunotherapies into clinical practice. Therefore, we aimed to characterize CD8+ T cell gene expression signatures in newly diagnosed AML patients before and after treatment, and to decipher the effects of AML blasts on the expression of co-signaling molecules by CD8+ T cells in co-culture experiments. Methods: Serial peripheral blood (PB) samples (at diagnosis and at the recovery after induction chemotherapy) were collected. To study transcriptional signatures, RNA isolated from FACS-purified PB CD8+ T cells from 6 patients [3 responders (R) and 3 non-responders (NR)] and 4 healthy controls (HC) was analyzed with the Human Prime View Gene Expression Array (Affymetrix). The data were normalized and log transformed. Expression fold change (FC), p values and false discovery rate were determined. Enrichment of canonical pathways was determined using Ingenuity Pathway Analysis (IPA, QIAGEN). To study AML blast-T cell interactions, we FACS-purified T cells and primary AML blasts at diagnosis (n=13) and T cells from HC (n=12). T cells were cultured in vitro for 3 days in the presence or absence of blasts (T cell:blast ratio 1:10) and analyzed by flow cytometry. Results: The transcriptional profile of CD8+ T cells at AML diagnosis significantly differed from that of HC. Genes were selected based on >2 FC between patient and HC, and p< 0.01. We identified a total of 453 dysregulated genes, of which 237 were up- and 216 down-regulated. Upregulated genes included immune inhibitory receptors LILRB1, 2B4, KLRG1, CD160, the transcription factors EOMES, TBET, TIGIT and cytokines (granzyme-A/B/K). In contrast, co-stimulatory receptor genes were downregulated, including CD40LG, CD28, CD30LG and CD28H. Canonical pathways analysis with IPA revealed that the NFAT pathway (involved in T cell differentiation and self-tolerance) was highly upregulated, while co-stimulatory CD28, ICOS and OX40 signaling pathways were downregulated in CD8+ T cells at AML diagnosis. Next, we compared R to NR after induction chemotherapy. There were a total of 351 dysregulated genes; 108/243 genes were up-/down-regulated, respectively. R patients upregulated immune stimulatory receptor genes like ICOS, whereas the top expressed genes for NR patients included the co-inhibitory receptor TIM3; several members of the inhibitory LIR receptor family; LST1 (involved in inhibition of lymphocyte proliferation); TWEAK-APRIL (associated with T cell apoptosis); and CD39 (terminally exhausted CD8+ T cells). In line with these findings, IPA showed that the co-stimulatory ICOS and OX40 signaling pathways were enriched in R patients. In contrast, the NFAT pathway, which had been highly upregulated at diagnosis, remained enriched in NR, but not in R patients. Results were confirmed by qPCR. The culture assay showed that the presence of primary AML blasts significantly reduced the viability of both AML and HC T cells (p <0.005 in both cases). The presence of AML blasts also significantly decreased the frequency of primary AML T cells expressing co-stimulatory receptors 41BB, ICOS and OX40, while it increased the frequency of HC T cells expressing co-inhibitory receptor 2B4 and the senescence/exhaustion marker CD57 compared to their counterparts cultured without blasts. Conclusions: Our study provides insight into the genomic CD8+ T cell signatures of AML patients at diagnosis and following chemotherapy. At diagnosis, T cells overexpressed genes that negatively regulate T cell immune responses, while genes that positively regulate immune responses were downregulated. Interestingly, after induction chemotherapy these changes persisted in NR only. Additionally, a pattern of decreased viability and co-stimulatory receptor expression was seen after in vitro co-culture of T cells with AML blasts, whereas immune inhibitory receptor expression was increased. Our data suggests that the blasts themselves influence the T cell phenotype and genotype in AML patients and that remission is associated with reversion to HC pattern. Disclosures Levis: Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Millennium: Consultancy, Research Funding.

scholarly journals Combination rhIL-15 and Anti-PD-L1 (Avelumab) Enhances HIVGag-Specific CD8 T-Cell Function

2020 ◽  
Vol 222 (9) ◽  
pp. 1540-1549
Author(s):  
Bruktawit A Goshu ◽  
Hui Chen ◽  
Maha Moussa ◽  
Jie Cheng ◽  
Marta Catalfamo
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Cd8 T Cell ◽  
Peripheral Tissues ◽  
T Cell Function ◽  
Interleukin 15 ◽  
Checkpoint Receptors

Abstract In chronic HIV infection, virus-specific cytotoxic CD8 T cells showed expression of checkpoint receptors and impaired function. Therefore, restoration of CD8 T-cell function is critical in cure strategies. Here, we show that in vitro blockade of programmed cell death ligand 1 (PD-L1) by an anti-PD-L1 antibody (avelumab) in combination with recombinant human interleukin-15 (rhIL-15) synergistically enhanced cytokine secretion by proliferating HIVGag-specific CD8 T cells. In addition, these CD8 T cells have a CXCR3+PD1−/low phenotype, suggesting a potential to traffic into peripheral tissues. In vitro, proliferating CD8 T cells express PD-L1 suggesting that anti-PD-L1 treatment also targets virus-specific CD8 T cells. Together, these data indicate that rhIL-15/avelumab combination therapy could be a useful strategy to enhance CD8 T-cell function in cure strategies.

IL-15 Plays a Critical Role in CD8+ T-Cell Recovery after Allogeneic Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1414-1414
Author(s):  
Frances T. Hakim ◽  
Kenton Allen ◽  
Jesse M. Carson ◽  
Michael Boyiadzis ◽  
Sarfraz A. Memon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Receptor Expression ◽  
Brdu Incorporation ◽  
Effector Memory ◽  
Cell Populations ◽  
Cell Recovery ◽  
Post Transplant

Abstract In severely lymphopenic hosts, CD4+ and CD8+ T cell populations increase rapidly through peripheral homeostatic expansion, a process in which IL-7 has been found to play a key role. Because of the marked differences in the kinetics of CD4+ and CD8+ T cell repopulation following hematopoietic stem cell transplants (HSCT), we have investigated the roles of additional cytokines in early repopulation. Interleukin-15 (IL-15) supports the proliferation, terminal differentiation, and survival of NK, NKT and memory CD8+ T-cell populations, all of which increase disproportionately in the early transplant period. We therefore investigated the role of IL-15 in post-transplant CD8+ T cell recovery by assessing plasma IL-15 levels, IL-15 receptor expression and IL-15-induced proliferation by BrdU incorporation. In patients undergoing non-myeloablative HLA-matched allogeneic HSCT for hematological and non-hematological malignancies, IL-15 levels in the plasma increased concurrent with the loss of lymphocytes during each cycle of inductive chemotherapy, and peaked at a 50-fold increase over pretreatment levels at day of transplant, a time when CD8+ T cell levels were usually less than 1 cell/μL. Plasma IL-15 levels fell rapidly in the first two weeks, during the rapid recovery of NK and CD8+ T cell populations, returning to pretransplant levels by 1–2 months. Overall, during the cytoreductive transplant and for the first year post transplant, the IL-15 levels were inversely proportional to the level of CD8 T cells (P &lt; 0.0001; r = −0.73). In the first weeks after transplant, CD8+T-cells expressed elevated levels of CD122, but had little or no expression of CD25, the IL-2Ralpha chain. Levels of CD122 remained elevated for several months, while expression of CD127, IL-7Ralpha, was reduced. In vitro BrdU incorporation assays demonstrated that CD8+ T-cells from both normal donors and transplant recipients responded primarily to IL-15, not IL-7 or IL-2. CD4+ T cells, in contrast, responded primarily to IL-7. A higher proportion of CD28+CD45RA− memory and CD28−CD45RA+/− effector-memory CD8+ T-cells incorporated BrdU than did naive CD28+CD45RA+ CD8+ T cells. Finally, IL-15, not IL-2 or IL-7, was found to maintain survival and support expansion in culture of the CD28−CD57+ terminal effector cells that dominate post transplant CD8+ T-cells populations. These data, describing an inverse relationship between the levels of free plasma IL-15 and CD8+ T cells, elevated expression of IL-15 receptor chain and strong responsiveness of post transplant CD8+ T cells to IL-15, suggest that IL-15 serves as a critical homeostatic cytokine post transplant, supporting the initial rapid generation CD8+ T cells and maintaining elevated levels of memory/effector CD8+ T-cell populations in the post transplant period.

First Generation Chimeric Antigen Receptor Display Functional Defects In Key Signal Pathways Upon Antigen Stimulation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2088-2088
Author(s):  
Johannes Duell ◽  
Sarah Lurati ◽  
Marcus Dittrich ◽  
Tanja Bedke ◽  
Martin Pule ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Target Cell ◽  
First Generation ◽  
Cytokine Production ◽  
Receptor Expression ◽  
Signal Pathways ◽  
Mhc I ◽  
Split Anergy

Abstract Abstract 2088 Introduction: A promising strategy for tumor therapy is the adoptive transfer of tumor specific T cells which are endowed with chimeric antigen receptors (CAR). First generation CARs are constructed by single chain antibodies and as signal domain the ζ chain of the CD3 complex. However, clinical trials are disappointing as adoptive transferred T-cells showed only modest persistence in patients resulting in limited clinical activity. We there for hypothesized that CAR expressing T-cells in comparison to unmodified T-cells display signaling defects when stimulated via their CARs. Methods: Cytomegalovirus(CMV)pp65 MHC I restricted CD8+ T-cells were generated, isolated by tetramer selection and modified with first generation CAR targeting CD19 and purified based on their receptor expression to more than > 95% purity. T-cell receptor (TCR) and CAR expression were quantified by Quantibright beads. Effector function of both T-cell populations were analyzed for specific lysis, cytokine production (IFN-g, TNF-a) and proliferation (CSFE) upon target cell stimulation. Phosphorylation of Erk, Jnk, p38 and PLC-γ was measured and analyzed with CBA Flexsets from BD. All statistical analyses have been performed using the statistical software package R. Signal peak intensities have been compared using the nonparametric wilcoxon rank sum test. Results: CMV-specific MHC-I restricted TCR as well as the CARs are expressed at same density levels and T-cells show equally lysis of targets either in the time of lysis onset as well the maximal lysis. In contrast, cytokine production (IFN, TNF-a) as well as antigen driven proliferation was reduced in CAR expressing T-cells when compared to CMV-specific CD8+ T-cells upon target exposure. PLC-γ was phosphorylated within minutes after target contact by CMV-specific CD8+ T-cells whereas CAR transduced CMV-specific CD8+ T-cells showed no significant phosphorylation of PLC-γ to target cell exposure. T-cell activated via CAR's demonstrated a statistically significant reduction of maximal phosphorylation in comparison to CMV-specific T-cells for ERK, for JNK and for p38. To exclude that CAR modification of CMV-specific CD8+ T-cells may impair signaling, CAR-CMV-specific CD8+ T-cells were exposed to CMVpp65 expressing targets. Killing, cytokine production and signal intensity were restored in comparison to parental CMV-specific CD8+ T-cells. Conclusion: CAR expressing T-cells show functionally signs of split anergy by efficient target elimination but fails to produce significant levels of cytokines and do not proliferate in response to target stimulation. Split anergy is not due to reduced expression of the CAR's but due to a complete lack of phosphorylation of PLC-γ as well as reduced phosphorylation of MAP-kinases ERK, p38 and JNK. These results potentially explain why primary CAR expressing T-cells fail to show significant clinical efficacy. Analysis of adequate phosphorylation, as proposed here, may be a powerful tool to identify the most promising second generation CARs for clinical studies. Disclosures: No relevant conflicts of interest to declare.

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