Negative effect of CTLA-4 on induction of T-cell immunity in vivo to B7-1+, but not B7-2+, murine myelogenous leukemia

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2146-2153 ◽  
Author(s):  
James L. LaBelle ◽  
Carrie A. Hanke ◽  
Bruce R. Blazar ◽  
Robert L. Truitt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Immunity ◽  
Myelogenous Leukemia ◽  
Cell Immunity ◽  
B7 Molecules ◽  
Negative Effect

Abstract B7 molecules provide important costimulatory signals to T cells, and B7 genes have been introduced into B7-negative tumor cells to enhance their immunogenicity. However, the role of B7 molecules in inducing tumor immunity is controversial because of conflicting results and reports of differential signaling through the B7 molecules and their ligands CD28 and CTLA-4. In this study, we compared the effect of B7-1 (CD80) and B7-2 (CD86) on the induction of T-cell immunity to C1498, a murine myelogenous leukemia. When cultured with exogenous cytokines in vitro, C1498/B7-1 and C1498/B7-2 induced syngeneic CD8+ T cells to kill parental C1498. In vivo, C1498/B7-1 grew progressively after subcutaneous injection, whereas C1498/B7-2 completely regressed after transient growth in naive mice. Spontaneous rejection of C1498/B7-2 resulted in immunity to challenge doses of C1498 and C1498/B7-1. Antibody-depletion studies in vivo showed that CD8+ T cells rejected C1498/B7-2, whereas only natural killer cells affected the growth of C1498/B7-1. Two approaches were used to determine whether preferential interaction of B7-1 with CTLA-4 contributed to the failure of C1498/B7-1 to activate CD8+ T cells in vivo. First, CTLA-4 specific monoclonal antibody was used to block B7-1–CTLA-4 interaction. Second, CTLA-4 deletional mutant (−/−) bone marrow chimeras were used as tumor hosts. In both systems, there was a significant increase in the rate of rejection of C1498/B7-1 tumors. Resistance to C1498/B7-1 in CTLA-4−/− hosts was mediated by CD8+ T cells. Blocking or deletion of CTLA-4 did not affect the growth of parental C1498, indicating that B7-1 was important for the induction of CD8+ T-cell immunity in the absence of CTLA-4.

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

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

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

Antibody Targeting of Activated Dendritic Cells Prevents AGVHD and Preserves T Cell Immunity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3240-3240
Author(s):  
Hannah Cullup ◽  
John Wilson ◽  
Alison M. Rice ◽  
Anne M. Dickinson ◽  
David J. Munster ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Immunity ◽  
Mouse Serum ◽  
Human Pbmc ◽  
Cell Immunity ◽  
Life Threatening

Abstract AGVHD is a life threatening complication of allogeneic HSCT, initiated by host and donor DC stimulation of donor T lymphocytes. Current AGVHD prophylaxis targets T lymphocytes, compromising anti-viral and therapeutic anti-leukemic responses. The presence of activated blood DC predicts for clinical AGVHD and we predict that a new strategy targeting therapy to activated DC should prevent alloimmune induction of AGVHD but preserve protective T cell immune responses. CD83 is a cell surface molecule expressed by activated DC. Having shown that a rabbit polyclonal antibody to human CD83 (RA83) depletes activated DC and suppresses alloimmune reactions in vitro, we tested the effect of RA83 treatment on T cell immunity in vitro and its ability to prevent human PBMC induced xenogeneic AGVHD in vivo. The effect of RA83 treatment on T cell numbers, proliferation and cytokine secretion in allogeneic MLR was compared with appropriate controls, including Campath-1H. Allogeneic responses in vitro were mirrored in a DC dependent in vivo model of xenogeneic AGVHD, in which 50×106 human PBMC were injected into an irradiated SCID mouse. Human cytokine levels were measured in MLR tissue culture supernatant (TCSN) and mouse serum at the time of sacrifice. Cytotoxic T cell responses to viral antigens (CMV and FMP) were analysed by specific pentamer analysis and Cr51 release assays, prior to and following HLA restricted peptide antigen specific expansion. RA83 was shown to have NK-mediated ADCC capacity. Cellular proliferation in the allogeneic MLR was reduced by both RA83 and Campath-1H treatment (p= 0.004 and 0.01 respectively vs controls) and both antibodies improved mouse survival in the human xenogenic AGVHD model (RA83: 93%, Campath-1H: 100%, p<0.0001). IFN-g was significantly reduced in the TCSN from MLR treated with RA83 (p=0.0391) and in sera taken from RA83 (p=0.0002) and Campath-1H (p=0.0051) treated mice. Serum IL-4 levels were maintained in RA83 and Campath-1H treated mice. The serum levels of IL-5, IL-8 and TNF in mice treated with RA83 were markedly reduced compared to controls (p=0.0256, 0.0025, 0.025, respectively), and the reductions were similar to those seen in Campath-1H treated mice. Similar numbers of T cells were recovered from RA83 treated and control MLR, and both CMV and FMP specific CD8+ T cells were retained. These cells were readily expanded by peptide pulsing and autologous restimulation and had specific cytotoxic activity comparable to control cultures (see figure). In contrast, Campath-1H treatment removed specific anti-viral responses (vs controls: CMV: p<0.00001 and FMP: p=0.0051). Specific antibody to CD83 depletes activated DC in vitro and prevents xenogeneic human DC dependent AGVHD in vivo. This was accompanied by a Th1 to Th2 skewing of the cytokine response. RA83, but not Campath-1H treatment retained normal numbers of T cells and maintained normal cytotoxic responses to common post-transplant viral infections. Depletion of activated DC may be an effective means of AGVHD control, which maintains T cell immunity to life threatening infections and potentially anti-leukaemia responses. RA83 treatment of allo-MLR preserves T cell anti-CMV immunity RA83 treatment of allo-MLR preserves T cell anti-CMV immunity

Targeting Therapeutic T Cells to Tumour Niches.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3009-3009
Author(s):  
Ben Carpenter ◽  
Sara Ghorashian ◽  
Emma Nicholson ◽  
James Edward Griffin ◽  
Maryam Ahmadi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Host Cells ◽  
Brdu Incorporation ◽  
Control Vector ◽  
Multiphoton Imaging ◽  
Cell Immunity

Abstract Abstract 3009 Background: Interactions between tumour cells and host cells within the microenvironment are important in promoting the development of cancer. Tumor niches provide crucial anti-apoptotic and anti-proliferative signals that drive tumor chemoresistance. The CXCR4-CXCL12 chemokine axis forms a critical component of this niche. CXCL12 produced by stromal cells has direct pro-survival effects upon tumor cells, promotes metastasis and recruits CXCR4-expressing regulatory T cell populations that block anti-tumour immunity. In this study, we have tested the hypothesis that targeting therapeutic T cells to CXCR4-dependent niches will improve eradication of tumours in mice. Methods: The murine CXCR4 gene was inserted into retroviral vector, pMP71. Murine T cells were transduced with CXCR4 or control vector and tested for homing in vitro to CXCL12 through chemotaxis assays. In vivo imaging of the putative endosteal bone marrow (BM) niche was performed by multiphoton imaging through cranial frontal bones in osteoblast (collagen 1-α-GFP) reporter mice. In vivo trafficking, competitive transfer and memory recall experiments were performed following transfer of transduced T cells to syngeneic, sub-lethally irradiated mice. Anti-tumour reactivity of CXCR4-transduced T cells was tested in models of allogeneic BM transplantation (BMT). Results: CXCR4-transduced T cells demonstrated enhanced migration towards CXCL12 in vitro. No differences in viability, phenotype or function were observed in CXCR4-transduced versus control T cells in the presence or absence of CXCL12. In competitive assays, CXCR4-transduced CD8 T cells demonstrated a 2-fold greater capacity than controls to home to the BM by 24h after transfer to sub-lethally-irradiated recipients. Multiphoton imaging through cranial frontal bones indicated that fluorescently labelled CXCR4-transduced T cells were closer than control cells to the endosteum (13 μm versus 17 μm, p<0.01). By 14 days, the numbers of CXCR4-transduced CD8 T cells in the BM were 15-fold greater than controls. To test immunity to model antigen, CXCR4 or control vector-transduced OT-1 TCR-transgenic CD8 cells were transferred to sub-lethally irradiated mice before challenge with OVA peptide-loaded dendritic cells. Pre-vaccination, CXCR4-transduced OT-1 cells demonstrated greater engraftment than controls in the BM and spleen. Seven days following vaccination, CXCR4 OT-1 cells demonstrated a greater capacity than control cells to generate IFN-γ to OVA-peptide. Four weeks following vaccination, CXCR4-transduced CD8 T cells showed increased frequencies of cells with a CD44highIL-7Rαhigh memory phenotype than controls, with a greater proportion of cells undergoing proliferation as evaluated by BrdU incorporation. To test T cell immunity against a tumor that exploits the CXCR4-CXCL12 axis to recruit regulatory T cells, B6 BM and CXCR4- or control transduced B6 T cells were transferred to irradiated BALB/c recipients given A20 tumor. Tumor growth was delayed to a greater extent following transfer of CXCR4-compared to control-transduced donor T cells. Conclusion: Over-expression of CXCR4 in CD8 T cells potentiates engraftment, initial effector function and generation of memory cells. Disclosures: Stauss: Cell Medica: Scientific Advisor Other.

Blocking VIP Signaling Abrogates Upregulation Of PD1 and Increases Proliferation Of Allo-Reactive T-Cells In a Mixed Lymphocyte Reaction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1041-1041
Author(s):  
Emily R Summerbell ◽  
Cynthia R. Giver ◽  
Sravanti Rangaraju ◽  
Katarzyna Anna Darlak ◽  
Edmund K. Waller
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Immunity ◽  
T Cell Proliferation ◽  
Cell Immunity

Abstract Introduction Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1 immunity and inhibits antiviral immunity. Decreased Th1 immunity is problematic for allogeneic bone marrow transplant (allo-BMT) patients requiring T-cell immunity against blood cancers (Graft-versus-Tumor) and against secondary infections such as CMV. VIPhyb, a modified VIP peptide, is a VIP receptor antagonist that decreases VIP signaling. VIP-knockout mice and mice treated with VIPhyb after allo-BMT are known to have better antiviral immunity and survival after CMV infection without increasing GvHD (Li et al. PLoS One. 2013 May 27;8(5):e63381) (Li et al. Blood. 2013 Mar 21;121(12):2347-51.), thus making VIPhyb of interest for pharmacological use in humans to improve the efficacy of allo-BMT The effects of VIPhyb on T-cell immunity are not yet fully profiled. This study aimed to analyze the effects of VIPhyb on CD4+ and CD8+ T-cell proliferation and activation in order to better understand the mechanistic implications of VIP inhibition on T-cell adaptive immunity. This study also aimed to show that mixed lymphocyte reactions (MLRs), an in vitro allo-BMT model, could be used to provide rapid and reliable results that are consistent with in vivo data. It was hypothesized that VIPhyb would increase T-cell immunity as profiled by: increased T-cell proliferation, CD69 and PD1 co-upregulation in early T-cell activation, and PD1 downregulation in T-cells after initial activation. Methods Splenocytes from two histoincompatible mice were cultured together at 37°C in a 1:1 ratio in a one-way MLR. BALB/c splenocytes (stimulators) were irradiated at 20Gy, and Pepboy splenocytes (responders) were labeled with CFSE to trace proliferation. VIPhyb was added daily to the cell cultures in doses of 0.1μM, 0.3μM, 1μM, or 3μM. Treatment groups were compared to a PBS control. Proliferation, CD69, and PD1 were assessed by flow cytometry on the BD FACSAria. All results are shown as mean ± SEM (n=3). One-way ANOVA tests with Dunnett post-tests were calculated using Prism software. *p < 0.05; **p < 0.01; ***p < 0.001 Results VIPhyb increased CD4+ and CD8+ T-cell proliferation: 3, 5, and 7 days after initiating a one-way MLR, CFSE expression of Pepboy responder T-cells was assessed using flow cytometry (Figure 1). As the VIPhyb dose increased, the percentage of initial splenocytes that underwent proliferation increased in both CD4+ and CD8+ T-cells. VIPhyb increased early T-cell CD69 expression and abrogated later PD1 upregulation in CD8+ T-cells: 3, 5, and 7 days after initiating a one-way MLR, expression levels of CD69 and PD1 on Pepboy responder T-cells were assessed by flow cytometry. Significant upregulation of CD69 on CD4+ and CD8+ T-cells on day 3 occurred with increasing VIPhyb doses (Figures 2A and 2B). PD1 was co-upregulated with CD69 during early activation, and VIPhyb significantly decreased PD1 expression on CD8+ T-cells on days 5 and 7 (Figures 2C and 2D). Conclusions VIPhyb increased T-cell proliferation; CD8+ T-cells were affected more significantly. VIPhyb increased early co-upregulation of CD69 and PD1 in all T-cells and significantly decreased later CD8+ T-cell PD1 expression, indicating that VIPhyb increases T-cell activation. We hypothesize that the decreased PD1 expression will be critical for understanding the pathways involved in VIP inhibition. Importantly, since it has been shown in vivo that VIPhyb does not increase GvHD, then it can be assumed that the VIPhyb-induced T-cell proliferation and activation will increase GvL and adaptive immunity without increasing alloreactivity. Notably, these results are consistent with published in vivo data, which demonstrates that the MLR can be used as a faster method of analyzing pharmacological compounds than in vivo experiments. Given these results, VIPhyb is still of interest as a potential therapy for allo-BMT patients. Disclosures: No relevant conflicts of interest to declare.

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

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

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

scholarly journals HER2-LAMP vaccines effectively traffic to endolysosomal compartments and generate enhanced polyfunctional T cell responses that induce complete tumor regression

2020 ◽  
Vol 8 (1) ◽  
pp. e000258 ◽  
Author(s):  
Alan Chen Chen ◽  
Renhuan Xu ◽  
Tao Wang ◽  
Junping Wei ◽  
Xiao-Yi Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Regression ◽  
T Cell Responses ◽  
Immune Checkpoint Blockade ◽  
Cell Responses ◽  
Cell Immunity

BackgroundThe advent of immune checkpoint blockade antibodies has demonstrated that effective mobilization of T cell responses can cause tumor regression of metastatic cancers, although these responses are heterogeneous and restricted to certain histologic types of cancer. To enhance these responses, there has been renewed emphasis in developing effective cancer-specific vaccines to stimulate and direct T cell immunity to important oncologic targets, such as the oncogene human epidermal growth factor receptor 2 (HER2), expressed in ~20% of breast cancers (BCs).MethodsIn our study, we explored the use of alternative antigen trafficking through use of a lysosome-associated membrane protein 1 (LAMP) domain to enhance vaccine efficacy against HER2 and other model antigens in bothin vitroandin vivostudies.ResultsWe found that inclusion of this domain in plasmid vaccines effectively trafficked antigens to endolysosomal compartments, resulting in enhanced major histocompatibility complex (MHC) class I and II presentation. Additionally, this augmented the expansion/activation of antigen-specific CD4+ and CD8+ T cells and also led to elevated levels of antigen-specific polyfunctional CD8+ T cells. Significantly, vaccination with HER2-LAMP produced tumor regression in ~30% of vaccinated mice with established tumors in an endogenous model of metastatic HER2+ BC, compared with 0% of HER2-WT vaccinated mice. This therapeutic benefit is associated with enhanced tumor infiltration of activated CD4+ and CD8+ T cells.ConclusionsThese data demonstrate the potential of using LAMP-based endolysosomal trafficking as a means to augment the generation of polyfunctional, antigen-specific T cells in order to improve antitumor therapeutic responses using cancer antigen vaccines.

scholarly journals IL-17RA-signaling modulates CD8+ T cell survival and exhaustion during Trypanosoma cruzi infection

2018 ◽  
Author(s):  
Jimena Tosello Boari ◽  
Cintia L. Araujo Furlan ◽  
Facundo Fiocca Vernengo ◽  
Constanza Rodriguez ◽  
María C. Ramello ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Trypanosoma Cruzi ◽  
Cell Survival ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Immunity ◽  
Cell Immunity ◽  
T Cell Survival

AbstractThe IL-17 family contributes to host defense against many intracellular pathogens by mechanisms not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes and their survival and appropriate response is orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. Absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells while recombinant IL-17 in vitro down-regulated the pro-apoptotic protein BAD and promoted activated CD8+ T cell survival. Phenotypic, functional and trancriptomic profiling showed that T. cruzi-specific CD8+ T cells arising in IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.

Peptides Derived From Mutated BCR-ABL Elicit T Cell Immunity In CML Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 887-887
Author(s):  
Ann Cai ◽  
Derin Keskin ◽  
Anselmo Alonso ◽  
David DeLuca ◽  
Wandi Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
T Cell Immunity ◽  
Myelogenous Leukemia ◽  
Class I ◽  
Immunogenic Tumor ◽  
Cell Responses ◽  
Cell Immunity

Abstract Abstract 887 Over 20 BCR-ABL mutations have been identified that result in imatinib resistance and relapse of chronic myelogenous leukemia (CML). CML is highly responsive to immunological manipulations and we therefore hypothesized that mutated BCR-ABL-derived peptides could serve as immunogenic tumor-specific targets. Herein, we present a multi-step strategy for identifying tumor-specific T cell epitopes generated from gene mutation. We first investigated whether peptides derived from 24 frequent BCR-ABL mutations could potentially bind 8 common class I MHC molecules by applying the well-validated prediction servers IEDB and NetMHC to tiled 9- and 10-mers around each mutation. More than 60 peptides were predicted to bind to one or more of the following alleles with IC50<1000: A*0201, A*0301, A*1101, B*0702, B*0801, B*1501, A*0101 or A*2402. From NetMHC, 24 of 84 (29%) were predicted as high (IC50<50), 42 (50%) as intermediate (IC50=50-500), and 18 (21%) as weak binders (1000> IC50>500). From IEDB, 9 of 61 (15%) were predicted as high, 35 (57%) as intermediate and 17 (38%) as weak binders. 24 of 84 mutated peptides (29%) and 24 of 61 mutated peptides (39%) were predicted as binding with at least two-fold higher affinity compared to parental peptides, using NetMHC and IEDB, respectively. These predictions indicated that cells from 7 of 9 imatinib-resistant CML patients had the potential to present at least one mutated BCR-ABL derived peptide by binding autologous HLA alleles (with IC50<1000). CML cells from 3 of the 5 patients had an E255K mutation and expressed HLA-A3, and were predicted to generate two promising candidate peptides: E255K-A247-255 (KLGGGQYGK, IEDB IC50=113; NetMHC IC50=192) and E255K-B255-263 (KVYEGVWKK, IEDB IC50=29; NetMHC IC50=28). Both peptides were predicted to bind HLA-A*0301 at least ten-fold more tightly than parental peptides. Using a competitive MHC binding assay, we confirmed that E255K-A and –B were high binders with IC50 scores of 208nM and 17nM, respectively and that they both had at least ten-fold fold greater affinity than parental peptides. In addition, E255K-B also bound to the other HLA-A3 superfamily members HLA-A*1101, HLA-A*3001, HLA-A*3101, HLA-A*6801 (IC50: 39–603nM). We next successfully generated T cell lines against E255K-B but not E255 K-A from a normal HLA-A3+ donor that demonstrated greater specificity against the mutated peptide (2330±325 SFC/million cells, by IFNγ-ELISPOT) than the parental peptide (1270±42 SFC/million cells). E255K-B is endogenously processed and presented since E255K-B reactive T cells also responded to HLA-A3+ antigen-presenting cells (APCs) that were transfected with a minigene encompassing 227 base pairs surrounding the E255K mutation (1900±85 SFC/million cells). Finally, we assessed the potential for E255K-B to stimulate T cell responses in CML patients. E255K-B elicits T cell immunity in vivo in that we could detect antigen-specific CD8+ T cell reactivity from two HLA-A3+ CML patients bearing the E255K mutation by IFNγ-ELISPOT and by antigen-specific tetramer staining. T cell responses could be abrogated in the presence of class I blocking antibody w6/32. For both patients, reactive T cells were polyfunctional, expressing GM-CSF, IP10 and TNFα in response to APCs pulsed with mutated peptide or expressing the E255K minigene. For Patient 2, E255K reactivity developed only in the setting of donor-derived engraftment following curative allogeneic stem cell transplantation. Further analysis to explore the kinetics of mutated peptide-specific immunity in relationship to burden of mutation-expressing leukemia cells is in progress. Our studies have demonstrated that leukemia-driven genetic alterations can provide immunogenic tumor-specific antigen targets associated with clinical response in vivo. Importantly, even though BCR-ABL mutations generate resistance to imatinib, they also create novel epitopes that can be effectively recognized by cytotoxic CD8+ T cells. Our studies support the development of specific, nontoxic and personalized vaccination strategies for eradication of drug-resistant CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reduced immune-regulatory molecule expression on human colonic memory CD4 T cells in older adults

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Stephanie M. Dillon ◽  
Tezha A. Thompson ◽  
Allison J. Christians ◽  
Martin D. McCarter ◽  
Cara C. Wilson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Older Persons ◽  
Age Groups ◽  
T Cell Immunity ◽  
Cd4 T Cell ◽  
Cell Immunity ◽  
Memory Cd4 T Cells

Abstract Background The etiology of the low-level chronic inflammatory state associated with aging is likely multifactorial, but a number of animal and human studies have implicated a functional decline of the gastrointestinal immune system as a potential driver. Gut tissue-resident memory T cells play critical roles in mediating protective immunity and in maintaining gut homeostasis, yet few studies have investigated the effect of aging on human gut T cell immunity. To determine if aging impacted CD4 T cell immunity in the human large intestine, we utilized multi-color flow cytometry to measure colonic lamina propria (LP) CD4 T cell frequencies and immune-modulatory marker expression in younger (mean ± SEM: 38 ± 1.5 yrs) and older (77 ± 1.6 yrs) adults. To determine cellular specificity, we evaluated colon LP CD8 T cell frequency and phenotype in the same donors. To probe tissue specificity, we evaluated the same panel of markers in peripheral blood (PB) CD4 T cells in a separate cohort of similarly aged persons. Results Frequencies of colonic CD4 T cells as a fraction of total LP mononuclear cells were higher in older persons whereas absolute numbers of colonic LP CD4 T cells per gram of tissue were similar in both age groups. LP CD4 T cells from older versus younger persons exhibited reduced CTLA-4, PD-1 and Ki67 expression. Levels of Bcl-2, CD57, CD25 and percentages of activated CD38+HLA-DR+ CD4 T cells were similar in both age groups. In memory PB CD4 T cells, older age was only associated with increased CD57 expression. Significant age effects for LP CD8 T cells were only observed for CTLA-4 expression, with lower levels of expression observed on cells from older adults. Conclusions Greater age was associated with reduced expression of the co-inhibitory receptors CTLA-4 and PD-1 on LP CD4 T cells. Colonic LP CD8 T cells from older persons also displayed reduced CTLA-4 expression. These age-associated profiles were not observed in older PB memory CD4 T cells. The decline in co-inhibitory receptor expression on colonic LP T cells may contribute to local and systemic inflammation via a reduced ability to limit ongoing T cell responses to enteric microbial challenge.

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

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

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

Sign in / Sign up

Export Citation Format

Share Document