The Expression and Mechanism of Tim3 and PD-1 in Patients with Acute Myeloid Leukemia

Abstract Objective: To investigatethe expression and the role of Tim3 and PD-1 on T cells of peripheral blood in patients with acute myeloid leukemia(AML). Consistently discuss the clinical significance of Tim3 and PD-1 co-expression on these cells and the mechanism of Tim3-mediated immune escape. Methods: Collectedclinical data of 36 patients who had been diagnosed with AML by bone marrow MICM classification, and then gathered their heparin anticoagulation peripheral blood before any treatment. The heparin anticoagulation peripheral blood from 20 healthy volunteers was gathered as normal control. We used the methods of immune fluorescence labeling and flow cytometry to detect expression of Tim3 on T cells. Then detected co-expression of Tim3 and PD-1 on T cells, consistently with IFN-γ secreting level on T cells. Results: 1.The proportions of CD4+ T cells and CD8+ T cells on lymphocytes in AML patients were (15.28±10.99)% and (9.19±7.54)% respectively, which were significantly lower than the proportion of normal controls [(31.12±2.22)% and (21.59±4.22)% respectively] (P<0.05). 2.The levels of Tim3 expression on CD4+ T cells and CD8+ T cells in AML patients were (4.77±3.560)% and (5.90±4.91)% respectively, which were significantly higher than the levels of normal controls [(0.73±0.62)% and (0.96±0.54)% respectively] (P<0.05). 3.Tim3 and PD-1 were co-expression on CD4+ T cells and CD8+ T cells in AML patients. 4.The IFN-γ secreting level in Tim3+ CD8+ T cells was significantly decreased than Tim3- CD8+ T cells (P<0.05). Conclusion: 1.The high expression of Tim3 on peripheral blood T cells in AML patients mediate T cell exhaustion/dysfunction, which can be an important mechanism of immune escape in leukemia. 2.PD-1 and Tim3 co-expression On CD4+ T cells and CD8+ T cells in AML patients which were dificient in there ability to produce IFN-γ. Further investigations are needed to explore the correlation of co-expression PD1 and Tim3 with process of AML, thus probably making Tim3 become another new immunotherapy target. Disclosures No relevant conflicts of interest to declare.

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Indoleamine 2,3-Dioxygenase-1 (IDO1) Is Expressed by a Subset of Childhood Acute Myeloid Leukemias and Restrains IFN-γ Production by T Cells

Blood ◽

10.1182/blood.v120.21.1430.1430 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1430-1430

Author(s):

Valentina Folgiero ◽

Daniela Natale ◽

Daria Pagliara ◽

Roberta Caruso ◽

Luciana Vinti ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Leukemia ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Myeloid Leukemia ◽

Ido1 Expression ◽

Blast Cells ◽

Ifn Γ ◽

Acute Myeloid

Abstract Abstract 1430 Background. Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan (TRP) into kynurenine (KYN) and other immune suppressive molecules. The IDO1-driven production of KYN promotes the development, stabilization and activation of regulatory T cells (Treg), while suppressing effector T cells, all of which may contribute to immune system impairment in cancer-bearing individuals. It has been previously shown that IDO1 mRNA and protein are detectable in blast cells from 52% of adult patients with newly diagnosed acute myeloid leukemia (AML), in correlation with expanded Treg cells. Importantly, high copy numbers of IDO mRNA may be a negative independent predicting variable for overall and relapse-free survival in adult AML. Patients and methods. We investigated IDO1 expression and function in 21 children with acute leukemia [10 AML (median age 13 years, range 6–16), 9 B-cell precursor (BCP)-ALL, 1 infant acute leukemia with MLL rearrangement and 1 T-cell ALL] and in 1 patient with Ph+ chronic myeloid leukemia (CML). Amongst patients with AML, 3 children had secondary AML, 3 patients had core-binding factor (CBF)+ AML, 3 patients had FLT3-ITD+AML and 1 patient had AML with t(6;9). TRP and KYN levels were measured with reverse phase (RP)-HPLC. Results. Cells from either BCP-ALL or T-cell ALL expressed IDO1 neither constitutively nor after challenge with 100 ng/ml interferon (IFN)-γ, a prototypical inducer of IDO, whereas they up-regulated both phosphorylated STAT-3 and surface programmed death ligand 1 (PD-L1), an IFN-γ-inducible co-inhibitory receptor also implicated in tumor-induced immune evasion. By contrast, leukemia blast cells from 5 out of 10 AML and from Ph+ CML up-regulated IDO1 protein expression after in vitro challenge with IFN-γ (median 20-fold increase, range 11.9–120, compared with unstimulated AML cells). KYN levels significantly increased in supernatants of AML cells treated with IFN-γ for 72h (10.8 μM/L, range 8.15–20.5) compared with unstimulated cultures (1.4 μM/L, range 1.1–2.2), in parallel with TRP consumption (6.3 μM/L, range 3.0–14.1, after challenge with IFN-γ compared with 18.2 μM, range 13.7–20.9, in unstimulated cultures). The IFN-γ-induced increase of IDO expression was significantly inhibited by pre-treatment of leukemia cells with STAT3 inhibitors (median 1.95-fold compared with unstimulated AML cells, range 0.9–27.7), but not with STAT5 inhibitors. In line with these results, STAT3 inhibition prevented the IFN-γ-induced release of KYN in culture supernatants. Western blot runs of immuno-precipitated proteins with specific antibodies suggested a physical interaction between IDO and STAT3 in IFN-γ-challenged leukemia blasts, but not in unstimulated samples. In a mixed tumor cell lymphocyte culture (MTLC), AML blasts primed with IFN-γ significantly inhibited Th1 cytokine production by allogeneic CD8+ T cells, while enhancing IL-4 release by CD4+ T cells. These effects were potentiated by the supplementation of MTLCs with exogenous KYN. The intracellular levels of IL-17A were unaffected by the exposure of allogeneic T cells to AML blasts. The addition of D,L-1-methyl-tryptophan (1MT), an IDO inhibitor, to the co-cultures of T cells and AML blasts incompletely restored IFN-γ production by CD8+ T cells. By contrast, STAT3 inhibitors fully reverted the AML-induced skewing of IFN-γ/IL-4 secretion. Conclusions. Blast cells from a subset of childhood AML, but not those from BCP-ALL or T-cell ALL, express functional IDO1 and restrain IFN-γ secretion by CD8+ T cells, while enhancing IL-4 production by CD4+ T cells. From a therapeutic standpoint, STAT3 inhibitors may effectively interfere with IDO1 expression by AML cells, thus tipping the Th1/Th2 balance in favor of anti-leukemia immune responses. Disclosures: No relevant conflicts of interest to declare.

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Identification and characterization of epitopes of the receptor for hyaluronic acid–mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2–positive patients with acute myeloid leukemia

Blood ◽

10.1182/blood-2004-12-4787 ◽

2005 ◽

Vol 106 (3) ◽

pp. 938-945 ◽

Cited By ~ 78

Author(s):

Jochen Greiner ◽

Li Li ◽

Mark Ringhoffer ◽

Thomas F. E. Barth ◽

Krzysztof Giannopoulos ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Hyaluronic Acid ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

T Cell Epitopes ◽

Computer Algorithms ◽

Cd8 Cells ◽

Acute Myeloid

AbstractThe receptor for hyaluronic acid–mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen. To define T-cell epitopes of RHAMM/CD168 toward specific immunotherapies for acute myeloid leukemia (AML), 10 potential HLA-A2–binding RHAMM/CD168 peptides (R1 to R10) were synthesized based on computer algorithms and screened by enzyme-linked immunospot (ELISPOT) analysis using CD8+ T cells isolated from peripheral blood (PB) of patients with AML and healthy donors. We found that CD8+ cells from 7 of 13 (54%) patients with AML presensitized with peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) specifically recognized T2 cells pulsed with R3 (39%) or R5 (15%) peptide. In contrast, only 4 of 21 (19%) healthy volunteers had CD8+ cells reactive with R3- or R5-pulsed T2 cells after presensitization. The presence of R3 peptide–specific effector T cells in the peripheral blood of patients with AML could be confirmed by staining as HLA-A2/R3 peptide tetramer+ CCR7-CD45RA+ cells. In chromium-51 release assays, peptide-primed CD8+ T cells from patients with AML were able to lyse RHAMM/CD168 peptide–pulsed T2 cells, AML blasts, and dendritic cells generated thereof (AML DCs). Transfection of COS7 cells with RHAMM/CD168 cDNA revealed that peptides R3 and R5 are naturally processed epitopes of RHAMM/CD168 that are presented in an HLA-A2–restricted manner. In summary, RHAMM/CD168 is a promising target for immunotherapies in patients with AML, and we have therefore initiated a clinical vaccination trial with R3 peptide. Because RHAMM/CD168 is also expressed in various other hematologic malignancies and solid tumors, vaccines targeting this antigen may have even wider application.

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HLA-G Expression on Blasts and Tolerogenic Cells in Patients Affected by Acute Myeloid Leukemia

Journal of Immunology Research ◽

10.1155/2014/636292 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 15

Author(s):

Grazia Locafaro ◽

Giada Amodio ◽

Daniela Tomasoni ◽

Cristina Tresoldi ◽

Fabio Ciceri ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Immune Escape ◽

Human Leukocyte ◽

Treg Cells ◽

Leukemic Cells ◽

Tolerogenic Dendritic Cells ◽

Acute Myeloid

Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4+T cells in the peripheral blood of AML patients at diagnosis. Moreover, we explored the possible influence of the 3′ untranslated region (3′UTR) ofHLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4+T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4+T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the host’s immune system. Further studies on larger populations are required to verify our findings.

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Vaccination of Acute Myeloid Leukemia Patients with Dendritic Cells Electroporated with mRNA Encoding the Wilms’ Tumor Protein WT1: A Phase I/II Trial.

Blood ◽

10.1182/blood.v110.11.158.158 ◽

2007 ◽

Vol 110 (11) ◽

pp. 158-158 ◽

Cited By ~ 2

Author(s):

Zwi N. Berneman ◽

Ann Van Driessche ◽

Ann Van de Velde ◽

Griet Nijs ◽

Tessa Braeckman ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Dendritic Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Wilms Tumor ◽

Rna Expression ◽

Acute Myeloid

Abstract To date, Wilms’ tumor protein (WT1) is acknowledged as a valuable target for active specific immunotherapy in several solid and hematological malignancies, such as leukemia. Preclinical data from our laboratory and that of Hans Stauss have already shown that WT1 RNA-electroporated dendritic cells (DC) stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia2005;19:1863–1871). Therefore, we started a phase I/II dose-escalation trial in which patients with acute myeloid leukemia (AML) in remission received intradermal injections with WT1 RNA-loaded DC. Feasibility, safety and immunogenicity of the vaccine were investigated. Seven patients received four biweekly DC vaccines. A delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients underwent an apheresis and monocytes were isolated using CD14-labeled magnetic beads by CliniMACS. DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, GM-CSF and IL-4 and maturated with PGE2 and TNF-a. Keyhole limpet hemocyanin (KLH) was added during maturation as a CD4+ helper antigen. Mature DC were harvested, electroporated with WT1 mRNA and used as vaccines. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by qRT-PCR. When the patient was HLA-A2+, tetramer staining was performed to detect WT1-specific CD8+ T cells. Before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. There was successful DC generation and vaccine production in all patients selected. No serious adverse events or toxicity was seen and all vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 3/5 patients who had an increased WT1 mRNA level in peripheral blood at the start of DC vaccination. A vaccine-specific immune response was demonstrated in 7/7 patients by an in vivo DTH reaction both to KLH as well as to WT1. By tetramer analysis, detectable levels of WT1-specific CD8+ T cells could be demonstrated during the course of the vaccination both in the peripheral blood as well as in the expanded DTH-infiltrating T cells from the skin biopsies. Preliminary data from immunomonitoring in pre- and post-vaccination T cell samples from 3 patients show a mixed T helper (Th)1/Th2 response towards the KLH and the WT1 protein following vaccination. We conclude that vaccination of AML remission patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the vaccine elicits anti-vaccine T-cell responses in vivo and a decrease in WT1 RNA expression levels was observed during MRD monitoring in some vaccinated patients.

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Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.

Blood ◽

10.1182/blood.v110.11.2973.2973 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2973-2973

Author(s):

Il-Kang Na ◽

Anne Letsch ◽

Ines Noack ◽

Sandra Bauer ◽

Jens Geginat ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Human Bone ◽

Ifn Γ

Abstract Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion. Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype. Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).

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Immune Cell Phenotype and Function After Treatment With Blinatumomab For Childhood Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

Blood ◽

10.1182/blood.v122.21.2668.2668 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2668-2668 ◽

Cited By ~ 4

Author(s):

Alice Bertaina ◽

Perla Filippini ◽

Valentina Bertaina ◽

Barbarella Lucarelli ◽

Aurelie Bauquet ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Lymphoblastic Leukemia ◽

Γδ T Cells ◽

Cell Phenotype ◽

Ifn Γ

Abstract Background Blinatumomab is a bi-specific monoclonal antibody designed to engage and tether cytotoxic T-cells (CTL) to CD19-expressing target B cells. An ongoing phase I multicenter study in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has shown that blinatumomab induces morphological and molecular remissions, defined as minimal residual disease (MRD) levels <10-4, in 47% of patients [Gore L, et al. J Clin Oncol 31, 2013 (suppl; abstr 10007)]. It is presently unknown whether and to what extent blinatumomab affects T-cell phenotype and function in pediatric patients with BCP-ALL. Patients and Methods Eight children diagnosed with relapsed/refractory BCP-ALL at the Bambino Gesù Children’s Hospital in Rome (median age at diagnosis 5.8 years, range 0.5-14.6) received blinatumomab as continuous intravenous infusion for 28 consecutive days, followed by a 2-week drug-free period. Four out of 8 patients were given repeated treatment courses. Peripheral blood samples were collected before treatment (day 0) and weekly thereafter, for 4 consecutive weeks. Bone marrow (BM) aspirates were available on days 0 and +29 of each drug course. Peripheral blood mononuclear cells (PBMC) were labeled with appropriate combinations of fluorochrome-conjugated monoclonal antibodies to quantitate naïve/memory T cells, αβ/γδ-expressing T cells and other immune effectors with potential anti-leukemia activity, such as CD3+CD56+ natural killer (NK) T cells and CD3-CD56+ NK cells. T-cell production of interferon (IFN)-γ, interleukin (IL)-4 and IL-17 was measured at the single-cell level, after short-term (4-hour) stimulation with phorbol myristate acetate (PMA) and ionomycin. The TCR-Vβ Repertoire Kit® (Beckman Coulter, Milan, Italy) allowed the flow cytometry analysis of 24 different Vβ specificities on T cells, thus covering approximately 70% of the normal human TCR-Vβ repertoire. Results Peripheral blood lymphocytes reached their nadir on day +1 (median 300/µL of blood [inter-quartile range 40-380] compared with 1,080/µL of blood at baseline [inter-quartile range 360-2,310]; p=0.0037 by Mann-Whitney U test for paired data), expanded within 7 days up to 3.5-fold above baseline, and included both CD4+ and CD8+ T cells. By contrast, the frequency of both CD3+CD56+ NK T cells and CD3-CD56+ NK cells remained unchanged compared to baseline. IFN-γ production by patient-derived CD4+ T cells exceeded that observed in CD4+ T cells from healthy controls by 2-fold, indicating robust T helper type 1 (Th1) polarization. The frequency of Th2/Th17 cells, defined as CD4+IL-4+ and CD4+IL-17+ cells, respectively, was not different after treatment compared to baseline. CD31 expression on recovering CD45RA+ naïve T cells, a surrogate phenotypic feature for recent thymic emigrants (RTEs), suggested that thymic output may contribute to T-cell expansion after blinatumomab administration. Non-significant changes in the relative proportion of TCR-αβ and TCR-γδ-expressing CD3+ T cells were detected after treatment (median 79.5% TCR-αβ+ T cells and 19.3% TCR-γδ+ T cells among total CD3+ cells) compared with baseline (median 87.4% TCR-αβ+ T cells and 12.2% TCR-γδ+ T cells among total CD3+ cells). Importantly, both CD3+CD8bright T cells and NK cells expressed lytic granule proteins, such as perforin and granzyme-B, at levels that increased during treatment. The analysis of Vβ TCR repertoire revealed a restricted usage of single Vβ domains by BM-resident CD8+ T cells, but not by CD4+ T cells. Specifically, the sum of Vβ within CD8+ T cells in the BM averaged 56.7±6.2% after blinatumomab, compared with 78±5.1% in healthy controls (p=0.04; Mann-Whitney U test for unpaired data). Conclusions Blinatumomab expands both CD31+CD45RA+ thymic-naïve and memory T cells with heightened IFN-γ production and is highly effective at clearing MRD in children with BCP-ALL. Skewing of the Vβ repertoire within BM-resident CD8+ T cells may be consistent with clonal expansions. Disclosures: Zugmaier: Amgen: Employment.

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The effect of Kefir on The Immune Response of Healthy Volunteers In Vitro

Journal of Agromedicine and Medical Sciences ◽

10.19184/ams.v3i2.5067 ◽

2017 ◽

Vol 3 (2) ◽

pp. 28

Author(s):

Desie Dwi Wisudanti

Keyword(s):

Immune Response ◽

T Cells ◽

Immune Responses ◽

Healthy Volunteers ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Fermented Milk ◽

Bioactive Components ◽

Ifn Γ

Kefir is a functional foodstuff of probiotics, made from fermented milk with kefir grains containing various types of beneficial bacteria and yeast. There have been many studies on the effects of oral kefir on the immune system, but few studies have shown the effect of bioactive components from kefir (peptides and exopolysaccharides/ kefiran), on immune responses. The purpose of this study was to prove the effect of kefir supernatant from milk goat on healthy immune volunteer response in vitro. The study was conducted on 15 healthy volunteers, then isolated PBMC from whole blood, then divided into 5 groups (K-, P1, P2, P3 and P4) before culture was done for 4 days. The harvested cells from culture were examined for the percentage of CD4+ T cells, CD8+ T cells, IFN-γ, IL-4 using flowsitometry and IL-2 levels, IL-10 using the ELISA method. The results obtained that kefir do not affect the percentage of CD4+ T cells and CD8+ T cells. The higher the concentration of kefir given, the higher levels of secreted IFN- γ and IL-4, but a decrease in IL-2 levels. Significant enhancement occurred at levels of IL-10 culture PBMC given kefir with various concentrations (p <0.01), especially at concentrations of 1%. These results also show the important effects of kefir bioactive components on immune responses. The conclusion of this study is that kefir can improve the immune response, through stimulation of IL-10 secretion in vitro.

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Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy

Cancers ◽

10.3390/cancers13194785 ◽

2021 ◽

Vol 13 (19) ◽

pp. 4785

Author(s):

Enrico Kittel-Boselli ◽

Karla Elizabeth González Soto ◽

Liliana Rodrigues Loureiro ◽

Anja Hoffmann ◽

Ralf Bergmann ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Immune Escape ◽

Early Stage ◽

Logic Gates ◽

Boolean Logic ◽

In Vivo Models ◽

Peptide Epitope ◽

Acute Myeloid

Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

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677 Reverse abscopal effect: intertumoral heterogeneity suppresses systemic CD8 T cell-mediated antitumor immunity and confers PD-1 inhibitor resistance in synchronous melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.677 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A705-A705

Author(s):

Shuyang Qin ◽

Booyeon Han ◽

Alexander Chacon ◽

Alexa Melucci ◽

Alyssa Williams ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Cd8 T Cells ◽

Antitumor Immunity ◽

Immune Escape ◽

Cd8 T Cell ◽

Abscopal Effect ◽

Immunogenic Tumor ◽

Ifn Γ

BackgroundDespite recent advancements in systemic therapy, only a minority of metastatic patients develop meaningful clinical responses to immune checkpoint inhibitors. Inherent genetic instability of melanoma generates genomically and microenvironmentally distinct metastases. These different tumor microenvironments (TMEs) contain numerous T cell suppression mechanisms, such as upregulation of the PD-1/PD-L1 exhaustion pathway. However, as synchronous metastases share one host immune system, intertumoral heterogeneity may result in increasing cross-talk between metastases that impairs systemic antitumor immunity and promotes PD-1 immunotherapy resistance.MethodsYUMM 1.7 (less immunogenic) and YUMMER 1.7 (more immunogenic cell line derived from YUMM following UVB irradiation) melanoma cell lines were simultaneously injected into opposite flanks of the same mice as a model of synchronous melanoma. We assessed tumor growth in wildtype, interferon-gamma (IFN-γ) knockout, and CD8-depleted mice as well as in response to PD-1 inhibitor. We characterized the TME with flow cytometry and performed TCR sequencing on tumor-infiltrating CD8 T cells.ResultsDistinct TMEs were observed for YUMM and YUMMER tumors simultaneously grown in the same mouse. The presence of the less immunogenic YUMM tumor allows the more immunogenic YUMMER tumors to escape IFN-γ and CD8 T cell-mediated rejection, despite abundant tumor-infiltrating, clonally expanded CD8 T cells. Identical immunodominant CD8 T cell clones were found in both YUMM and YUMMER tumors within the same mouse. Synchronous YUMMER-infiltrating CD8 T cells exhibit suppressed phenotypes, including increased persistence of surface PD-1 and decreased surface CD107a expressions. Simultaneously, these synchronous YUMMER tumors additionally upregulate macrophage surface PD-L1 expression, which potentially contributes to tumor immune escape. Lastly, synchronous YUMMER tumors become resistant to PD-1 inhibition, in direct contrast to control YUMMER tumors.ConclusionsIn a host with multiple melanoma lesions, immunogenicity of all tumors contribute to the systemic antitumor immune response. We show that two synchronous tumors with synonymous mutations (<40%), as is the case with metastatic patients, lead to skewed CD8 T cell expansion of the same clones in both tumors. The presence of a less immunogenic tumor prevents CD8 and IFN-γ mediated rejection of the more immunogenic tumor. Furthermore, CD8 T cells in the more immunogenic tumor exhibit decreased effector function and increased resistance to PD-1 blockade, as tumor-infiltrating macrophages concurrently become more immunosuppressive. These results are highly suggestive of a “reverse abscopal effect,” by which immunologically “cold” tumors generate systemic immunosuppression that facilitate PD-1 immunotherapy resistance and immune escape of all other tumors in synchronous metastatic melanoma patients.AcknowledgementsWe would like to thank Dr. Marcus Bosenberg from the Department of Dermatology at Yale University for kindly gifting us with the YUMMER 1.7 murine melanoma cell line.Ethics ApprovalAnimal experiments were approved by the University Committee on Animal Resources and performed in accordance with University of Rochester approved guidelines.

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