Sipuleucel-T (sip-T) induced lytic CD8+ T cell responses to target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 162-162
Author(s):  
Emmanuel S. Antonarakis ◽  
David I. Quinn ◽  
Adam S. Kibel ◽  
Daniel Peter Petrylak ◽  
Tuyen Vu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Cell Responses ◽  
Hormone Sensitive ◽  
Ctl Activity

162 Background: Sip-T is an FDA-approved immunotherapy for patients (pts) with asymptomatic or minimally symptomatic metastatic CRPC. Sip-T is manufactured from autologous peripheral blood mononuclear cells cultured with the immunogen PA2024, a fusion antigen of prostatic acid phosphatase (PAP) conjugated to granulocyte macrophage colony-stimulating factor. After sip-T, antibody and T cell responses to PA2024 and/or PAP correlate with improved survival. To further elucidate the mechanism of sip-T–induced immune responses, we evaluated the proliferative and lytic ability of PA2024- and PAP-specific CD8+ T cells. Methods: Mononuclear blood cells were labeled with the membrane dye carboxyfluorescein succinimidyl ester (CFSE) and cultured with PA2024 or PAP. In vitro proliferative and lytic CD8+ (cytotoxic T lymphocyte [CTL]) T cell responses to these antigens were evaluated by flow cytometry. For proliferation, progressive dilution of CFSE was measured. For CTL activity, the loss of intracellular granzyme B (GzB), indicating exocytosis of this apoptosis-mediating enzyme, was assessed. Samples were from 2 sip-T clinical trials STAND (NCT01431391) and STRIDE (NCT01981122), hormone-sensitive and CRPC pts, respectively. Results: Six wk after sip-T administration, CD8+ PAP- and PA2024-specific responses were observed (n=14 pts assessed). The magnitude of PA2024-specific CD8+ proliferative responses was greater than that for PAP-specific responses. CD8+ T cells from a subset of pts who exhibited PA2024- and/or PAP-specific proliferative responses were assessed for lytic ability. After in vitro antigen stimulation, CTL activity in all evaluated samples (n=14, PA2024; n=13, PAP) was demonstrated by a significant decrease (p<0.05) in intracellular GzB relative to a no-antigen control. Conclusions: Sip-T induced CD8+ CTL proliferation against the target antigens PAP and PA2024. Moreover, antigen-specific CTL activity provides the first direct evidence that sip-T can induce tumor cell lysis. These antigen-specific CD8+ lytic abilities were observed within 6 wk following sip-T, suggesting rapidly generated immune responses. Clinical trial information: NCT01431391; NCT01981122.

Suppression of CTL Responses against AAV-Capsid Epitopes by Peptide-Induced Regulatory T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 377-377 ◽  
Author(s):  
Daniel J Hui ◽  
Gary C Pien ◽  
Etiena Basner-Tschakarjan ◽  
Federico Mingozzi ◽  
Jonathan D Finn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Transgene Expression ◽  
T Cell Responses ◽  
Effector Cells ◽  
Cell Responses ◽  
Ctl Activity

Abstract Abstract 377 Hemophilia B represents a promising model for the development of adeno-associated viral (AAV) vectors-based gene therapeutics. In the first clinical trial for AAV serotype 2 mediated gene transfer of Factor IX (F.IX) to the liver of severe hemophilia B subjects, transgene expression was short-lived with a gradual decline of F.IX levels. The loss of transgene expression was accompanied by a transient transaminitis, which we hypothesized to be the result of the reactivation of a pool of capsid-specific memory CD8+ T cells originated from a previous exposure to wild-type AAV. These results were unanticipated since previous work in small and large animal models showed that AAV administration is uneventful, allowing prolonged expression of F.IX transgene at therapeutic levels. We developed an in vitro cytotoxicity assay using a human hepatocyte cell line expressing HLA-B*0702, a common MHC class I allele for which the AAV capsid immunodominant epitope VPQYGYLTL was identified. Using this model, we demonstrated that HLA-matched AAV-specific effector CD8+ T cells were able to lyse target hepatocytes transduced with AAV-2. We now use this in vitro model of CTL killing of AAV-transduced hepatocytes to demonstrate the efficacy of a novel strategy to circumvent undesirable immune response through the engagement of regulatory T cells. A recently characterized MHC Class II-restricted T cell epitope (Tregitope) in the Fc fragment of IgG has been shown to induce regulatory T cells in vitro and in vivo (Blood, 2008; 112: 3303-3311). AAV-specific HLA-B*0702 effector cells expanded in the presence of a human Tregitope peptide resulted in 79% to 89% inhibition of cytotoxic activity against peptide-pulsed and AAV-transduced target cells, respectively. These results were confirmed using PBMCs from 5 different donors. A similar degree of inhibition of CTL activity was observed for the HLA allele A*0101, which binds to the AAV-derived epitope SADNNNSEY; co-culture of effector cells with the Tregitope inhibited CTL-mediated killing by 60%. Interestingly, the same Tregitope efficiently mediated suppression of CTL activity in subjects carrying different HLA alleles, indicating a high level of promiscuity of Tregitope binding. Staining for the regulatory T cell markers CD4, CD25, and FoxP3 supported the hypothesis that Tregitopes suppress T cell responses by expanding regulatory T cells; 62.2% of the CD4+ population stained positive for CD25 and FoxP3 in PBMCs expanded against AAV epitopes in the presence of Tregitope, compared with PBMCs expanded against an AAV epitope alone (3.63%), or against an AAV epitope and an irrelevant control peptide (1.94%). Polyfunctional analysis for markers for T cell activation showed that CD8+ T cells incubated in the presence of Tregitope had an approximately 5-fold decrease in production of IL-2 and IFN-γand a 2-fold reduction in TNF-α production, indicating levels of activation close to naïve CD8+ T cells. We further characterized the mechanism of action of Tregitopes by showing that Tregitopes are required at the time of CD8+ T cell priming, as CTL activity of AAV-expanded CD8+ T cells against transduced hepatocytes was not inhibited by the CD4+ T cell fraction of PBMC expanded separately in vitro with Tregitopes only. We conclude that the use of Tregitopes represents a promising strategy for antigen-specific, Treg-mediated modulation of capsid-specific T cell responses. Disclosures: Martin: EpiVax: Employment. De Groot:EpiVax, Inc.: Employment, Equity Ownership.

BK Polyomavirus–Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination

2020 ◽  
Author(s):  
Maud Wilhelm ◽  
Amandeep Kaur ◽  
Marion Wernli ◽  
Hans H Hirsch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Kidney Transplant ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Bk Polyomavirus

Abstract Background BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Methods Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. Results BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1–) and cytotoxic. Conclusions Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

Mutated Nucleophosmin 1 (NPM1) Is an Immunogenic Target and Patients with NPM1mut Acute Myeloid Leukemia (AML) Showed High Expression of Different Leukemia-Associated Antigens (LAAs)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3592-3592
Author(s):  
Susanne Hofmann ◽  
Vanessa Schneider ◽  
Lars Bullinger ◽  
Yoko Ono ◽  
Anita Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Microarray Analysis ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
High Expression ◽  
Chromium Release ◽  
Cell Responses ◽  
Favorable Prognosis

Abstract Abstract 3592 Nucleophosmin gene 1 mutations (NPM1mut) are one of the most frequent molecular alterations in AML and distinct immune responses might contribute to the favorable prognosis of AML patients with NPM1mut. Recently, we showed specific T cell responses of CD4+ and CD8+ T cells against epitopes derived from mutated regions of NPM1 (Greiner et al., Blood. 2012 May 16, Epub). In the present study, we investigated clinical parameters and the clinical outcome of NPM1mut AML patients in accordance to their immune responses against different NPM1 epitopes. Moreover, we examined the quantitative expression of different leukemia-associated antigens (LAAs) in NPM1mutAML patients. In ELISpot analysis of 33 healthy volunteers and 27 AML patients, we detected T cell responses of CD4+ and CD8+ T cells against epitopes derived from the mutated region of NPM1. We performed further tetramer assays against the most interesting epitopes and chromium release assays to show the cytotoxicity of peptide-specific T cells. Microarray analysis was performed to analyze the expression of different LAAs in NPM1mut and NPM1wtAML patients. Two epitopes (peptide #1 and #3) derived from NPM1mut induced CD8+ T cell responses. 33% of the NPM1mut AML patients showed immune responses against peptide #1 and 44% against peptide #3. NPM1mut AML patients showed a significantly higher frequency of T cell responses against peptide #3 in contrast to HVs (p=0.046), whereas for peptide #1 the frequency of T cell responses of AML NPM1mut patients and HVs was not significantly different. Specific lysis of pulsed T2 cells but also NPM1mut leukemic blasts was detected in chromium release assays. Therefore, overlapping peptides (OL) were analyzed in ELISpot assays and the peptide called OL8 showed favorable results to activate both CD8+ and CD4+ T cells. We performed survival analysis for these 33 NPM1mut patients analyzed by ELISpot comparing cases with or without specific T cell responses. Our data suggest a trend to a better overall survival (OS) for patients with specific T cell responses against peptide #1 or #3. However, the patient numbers are small and the data have to be interpreted carefully. Analyses with material from larger controlled clinical trials with a high number of patients with NPM1mut AML have to be performed. Our microarray analysis of 30 AML patients showed a high expression of different LAAs like RHAMM, WT-1 and BCL-2 in all subtypes of cells of NPM1mutAML patients, also in leukemic progenitor cells. This demonstrates that NPM1 is an AML subtype suitable for poly-targeted immunotherapeutic trials. Taken together, NPM1mut might constitute an interesting target structure for individualized immunotherapeutic approaches in NPM1mut AML patients. We hypothesize that immune responses to NPM1 mutation may contribute to the favorable prognosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TLR9 and Dendritic Cells Are Required for CD8+ T Cell Responses to the AAV Capsid

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 552-552 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Roland W Herzog
Keyword(s):  
Pattern Recognition ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Post Injection ◽  
Cell Responses

Abstract CD8+ T cell responses to the adeno-associated virus (AAV) capsid have posed a significant barrier to transduction in clinical trials of AAV-mediated gene therapy for hemophilia B, as reactivation of a memory CTL response to the capsid is capable of eliminating transduced hepatocytes in the absence of immunosuppression. Recently, it has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can influence the development of adaptive immune responses to AAV-mediated gene transfer. In particular, reports have implicated TLR2 (AAV capsid), TLR9 (AAV genome), and MyD88 (downstream signaling adaptor of both these TLRs). Herein, we have used a modified AAV2 with an insertion of the immunodominant MHC class I epitope of ovalbumin into the capsid (AAV2-SIINFEKL) to study the mechanism of CD8+ T cell responses to the AAV capsid. Using an H2-Kb-SIINFEKL tetramer reagent, we determined that anti-capsid CD8+ T cell responses depended on the TLR9-MyD88 pathway. While the frequency of circulating capsid-specific CD8+ T cells peaked around 7-10 days post-injection and subsided after about 21 days in wild type (WT) mice, tetramer-positive cells were not detected in TLR9-/- or MyD88-/- mice. The kinetics and magnitude of the response was unaltered in TLR2-/- mice. Mice deficient in STING, a downstream adaptor of multiple cytoplasmic DNA sensing pathways, also developed comparable capsid-specific CD8+ T cell frequencies to WT mice, suggesting that this is not a general effect of pattern recognition of DNA. Interestingly, the frequency of capsid-specific CD8+ T cells was not reduced in AP3-/- mice, which are deficient in type I IFN signaling downstream of TLR9. Adoptively transferred OVA-specific OT-1 T cells proliferated in WT but not TLR9-/- mice that received AAV2-SIINFEKL, confirming the importance of TLR9. The effect was antigen-specific, as OT-1 cells in WT mice that received AAV2 lacking SIINFEKL showed minimal proliferation comparable to TLR9-/- mice. In addition to pattern-recognition receptors, we also assessed the role of antigen-presenting cells in the CD8+ T cell response to capsid. The formation of capsid-specific CD8+ T cells was unaltered in mice that received gadolinium chloride to inactivate macrophages, or in B cell-deficient μMT mice. Depletion of B cells in WT mice prior to vector administration also failed to affect the anti-capsid CD8+ T cell response. However, transient depletion of dendritic cells (DCs) in CD11c-DTR mice resulted in a delayed development of capsid-specific CD8+ T cells. Seven days post-injection, DC-depleted mice had a significantly reduced frequency of tetramer-positive CD8+ T cells which recovered to normal by 10 days, likely due to the repopulation of DCs before the input capsid was completely cleared. Overall, our results show that TLR9 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses. Disclosures Herzog: Genzyme: AAV-FIX technology Patents & Royalties.

scholarly journals Enhanced Anti-HIV Functional Activity Associated with Gag-Specific CD8 T-Cell Responses

2010 ◽  
Vol 84 (11) ◽  
pp. 5540-5549 ◽  
Author(s):  
B. Julg ◽  
K. L. Williams ◽  
S. Reddy ◽  
K. Bishop ◽  
Y. Qi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Replication ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Cell Counts ◽  
Inhibit Virus Replication

ABSTRACT Effective HIV-specific T-cell immunity requires the ability to inhibit virus replication in the infected host, but the functional characteristics of cells able to mediate this effect are not well defined. Since Gag-specific CD8 T cells have repeatedly been associated with lower viremia, we examined the influence of Gag specificity on the ability of unstimulated CD8 T cells from chronically infected persons to inhibit virus replication in autologous CD4 T cells. Persons with broad (≥6; n = 13) or narrow (≤1; n = 13) Gag-specific responses, as assessed by gamma interferon enzyme-linked immunospot assay, were selected from 288 highly active antiretroviral therapy (HAART)-naive HIV-1 clade C-infected South Africans, matching groups for total magnitude of HIV-specific CD8 T-cell responses and CD4 T-cell counts. CD8 T cells from high Gag responders suppressed in vitro replication of a heterologous HIV strain in autologous CD4 cells more potently than did those from low Gag responders (P < 0.003) and were associated with lower viral loads in vivo (P < 0.002). As previously shown in subjects with low viremia, CD8 T cells from high Gag responders exhibited a more polyfunctional cytokine profile and a stronger ability to proliferate in response to HIV stimulation than did low Gag responders, which mainly exhibited monofunctional CD8 T-cell responses. Furthermore, increased polyfunctionality was significantly correlated with greater inhibition of viral replication in vitro. These data indicate that enhanced suppression of HIV replication is associated with broader targeting of Gag. We conclude that it is not the overall magnitude but rather the breadth, magnitude, and functional capacity of CD8 T-cell responses to certain conserved proteins, like Gag, which predict effective antiviral HIV-specific CD8 T-cell function.

T Cell Responses in Patients Vaccinated with Telomerase (hTERT)-mRNA Transfected Dendritic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 373-373
Author(s):  
Else Marit Inderberg Suso ◽  
Anne-Marie Rasmussen ◽  
Steinar Aamdal ◽  
Svein Dueland ◽  
Gustav Gaudernack ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cancer Patients ◽  
Cd8 T Cells ◽  
Stable Disease ◽  
T Cell Responses ◽  
Htert Mrna ◽  
Cell Responses

Abstract Abstract 373 Two cancer patients were vaccinated with dendritic cells (DC) loaded with telomerase (hTERT) mRNA to investigate the safety, tolerability and immunological response to vaccination prior to the start of a new phase I/II clinical trial. Following written informed consent one primary lung adenocarcinoma with metastasis and one patient with a relapsed pancreatic ductal type of adenocarcinoma, were treated with autologus monocyte-derived DC transfected with mRNA encoding hTERT. The patients first received four weekly injections administered intradermally followed by monthly booster injections. Peripheral blood mononuclear cells (PBMC) at each vaccination time point were tested in vitro with transfected DC and a panel of 24 overlapping hTERT peptides. In addition, hTERT-specific CD8+ T cells were monitored by pentamer staining. The treatment was well tolerated with minor side effects. Immune responses against telomerase-transfected DC and some of the overlapping hTERT peptides were detected in both patients. We also detected hTERT-specific CD8+ T cells in both patients by pentamer staining in post-vaccination samples. The lung cancer patients obtained a stable disease that lasted 18 months while the patient with pancreas cancer who started the DC vaccination in July 2007 following palliative chemotherapy, still is in stable disease by continuously boost vaccination. T-cell responses against telomerase epitopes have also been identified in both non-vaccinated cancer patients and cancer patients previously vaccinated with telomerase peptide. Since patients with these findings often show extraordinary clinical courses of their disease we hypothesize that it exists a high degree of immunogenicity and HLA promiscuity for some telomerase epitopes. In this study we have shown that vaccination with hTERT-mRNA transfected DC is safe and able to induce robust immune responses to several telomerase T-cell epitopes both in CD4+ and CD8+ T cells. This opens up the possibility for a broad clinical application of mRNA hTERT DC vaccines. Furthermore, responding T cells identified in these patients are strong candidates for T-cell receptor cloning and the receptors identified can thereafter be transferred into T cells creating the next generation of immuno-gene therapy with retargeted T cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of Adaptive Vγ2Vδ2+ T-Cell Responses during Active Mycobacterial Coinfection of Simian Immunodeficiency Virus SIVmac-Infected Monkeys

2003 ◽  
Vol 77 (5) ◽  
pp. 2998-3006 ◽  
Author(s):  
Dejiang Zhou ◽  
Xiaomin Lai ◽  
Yun Shen ◽  
Prabhat Sehgal ◽  
Ling Shen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Simian Immunodeficiency Virus ◽  
Γδ T Cells ◽  
Subsequent Development ◽  
T Cell Responses ◽  
Cell Responses ◽  
Immunodeficiency Virus

ABSTRACT Adaptive immune responses of γδ T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vγ2Vδ2+ T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vγ2Vδ2+ T cells after Mycobacterium bovis BCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vγ2Vδ2+ T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vγ2Vδ2+ T-cell responses was associated with profound CD4+ T-cell deficiency and subsequent development of SIVmac-related tuberculosis-like disease in the coinfected monkeys. Consistently, Vγ2Vδ2+ T cells from coinfected monkeys displayed a reduced capacity to expand in vitro following stimulation with phosphoantigen. The reduced ability of Vγ2Vδ2+ peripheral blood lymphocytes (PBL) to expand could be restored to some extent by coculture of these cells with CD4+ T cells purified from PBL of SIV-negative monkeys. Furthermore, naïve monkeys inoculated simultaneously with SIVmac and BCG were unable to sustain expansion of Vγ2Vδ2+ T cells at the time that the coinfected monkeys developed lymphoid depletion and a fatal tuberculosis-like disease. Nevertheless, no deletion in Vδ2 T-cell receptor repertoire was identified in SIVmac-BCG-coinfected macaques, implicating an SIVmac-induced down-regulation rather than a clonal exhaustion of these cells. Thus, an SIVmac-induced compromise of the adaptive Vγ2Vδ2+ T-cell responses may contribute to the immunopathogenesis of the SIV-related tuberculosis-like disease in macaques.

scholarly journals Selection and T-cell antigenicity of synthetic long peptides derived from SARS-CoV-2

2022 ◽  
Vol 103 (1) ◽  
Author(s):  
Katarzyna Piadel ◽  
Amin Haybatollahi ◽  
Angus George Dalgleish ◽  
Peter Lawrence Smith
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Hla Class I ◽  
Peripheral Blood Mononuclear ◽  
Cell Responses

The pandemic caused by SARS-CoV-2 has led to the successful development of effective vaccines however the prospect of variants of SARS-CoV-2 and future coronavirus outbreaks necessitates the investigation of other vaccine strategies capable of broadening vaccine mediated T-cell responses and potentially providing cross-immunity. In this study the SARS-CoV-2 proteome was assessed for clusters of immunogenic epitopes restricted to diverse human leucocyte antigen. These regions were then assessed for their conservation amongst other coronaviruses representative of different alpha and beta coronavirus genera. Sixteen highly conserved peptides containing numerous HLA class I and II restricted epitopes were synthesized from these regions and assessed in vitro for their antigenicity against T-cells from individuals with previous SARS-CoV-2 infection. Monocyte derived dendritic cells were generated from these peripheral blood mononuclear cells (PBMC), loaded with SARS-CoV-2 peptides, and used to induce autologous CD4+ and CD8+ T cell activation. The SARS-CoV-2 peptides demonstrated antigenicity against the T-cells from individuals with previous SARS-CoV-2 infection indicating that this approach holds promise as a method to activate anti-SAR-CoV-2 T-cell responses from conserved regions of the virus which are not included in vaccines utilising the Spike protein.

scholarly journals Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma

2021 ◽  
Vol 9 (9) ◽  
pp. e002754
Author(s):  
Eva Bräunlein ◽  
Gaia Lupoli ◽  
Franziska Füchsl ◽  
Esam T Abualrous ◽  
Niklas de Andrade Krätzig ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Immune Checkpoint ◽  
T Cell Responses ◽  
Functional Avidity ◽  
Cell Responses

BackgroundNeoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation.MethodsThree neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient’s immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient’s TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing.ResultsSelected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation.ConclusionsWe performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.

T Cell Responses Directed Against HLA A*0201-Restricted Epitopes Derived from WT1 Are Readily Identifiable in Patients with AML and CML, but Not in Patients with ALL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2526-2526
Author(s):  
Katayoun Rezvani ◽  
Jason Brenchley ◽  
David Price ◽  
Yasemin Kilical ◽  
Matthias Grube ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Healthy Donors ◽  
Myeloid Leukemias

Abstract The WT1 gene contributes to leukemogenesis and all adult ALL, AML and CML express WT1 RNA by quantitative real-time reverse transcription polymerase chain reaction (qPCR) techniques. WT1 may therefore be a useful antigenic target for immunotherapy. Four HLA-A*0201-restricted WT1 T cell epitopes have been identified: Db126 (RMFPNAPYL), WH187 (SLGEQQYSV), WT37-45 (VLDFAPPGA) and WT235 (CMTWNQMNL), but only Db126 has been extensively studied in myeloid leukemias. Here, we sought CD8+ T cells directed against these epitopes in 12 healthy SCT donors, 6 patients with AML, 8 with, CML and 6 with ALL prior to SCT. All patients tested with myeloid or lymphoid leukemias expressed MHC class I, B7.1 and WT1. To detect very low frequencies of WT1-specific CD8+ T cells, we used qPCR for interferon-g (IFN-g) mRNA. After isolation, 106 CD8+ T cells were stimulated with C1R-A2 cells (MHC class I-defective LCL cells transfected with HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM to determine their functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3 hr coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated that the IFN-g copy number was linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/104 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/104 CD8 copies in peptide pulsed/unpulsed cultures. Responses to at least one WT1 peptide were detected in 5/8 CML patients, 4/6 patients with AML and 7/12 healthy donors. Notably, a response was not elicited to WT1 in any of the 6 patients with ALL, despite evidence of immune competence as shown by a normal CMV response. Five of five CML responders and 3/4 AML responders recognized 2 or more WT1 epitopes, while the 7 healthy donors recognized only one WT1 epitope. The range of IFN-g mRNA copies/104 CD8 copies was 289–13584, 418–45891 and 160–2683 for CML, AML and healthy donors respectively. WT1-specific tetramer-positive CD8+ T cells displayed both central memory (CD45RO+CD27+CD57−) and terminally differentiated effector memory phenotypes (CD45RO-CD27−CD57+). As multiple WT1-derived epitopes can be targeted simultaneously, it is likely that T cell response to WT1 is polyclonal. These results are important because the presence of memory WT1 responses in patients with myeloid leukemias and healthy individuals should favor vaccination as a means to expand immune responses to leukemia in the autologous and allogeneic transplant setting. Furthermore, the presence of CD8+ T cell responses to multiple WT1 epitopes should favor robust polyclonal immune responses to leukemia. However, failure to detect CD8+ T cell responses to WT1 in ALL patients suggests that WT1 may not be a useful antigen to target for immunotherapeutic purposes in this patient group.

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