Flow Cytometric Analysis of the Cytotoxic T-Cell Recall Response to Theileria parva in Cattle Following Vaccination by the Infection and Treatment Method

The apicomplexan hemoparasite, Theileria parva, causes East Coast fever (ECF), a frequently fatal disease of African cattle. Vaccine development has been impeded by incomplete understanding of protective immunity following natural exposure or the infection and treatment method (ITM) of immunization. This is attributable to a paucity of methods to characterize the memory T-cell repertoire following infection. To overcome this impediment, assays developed to study the immune response to other intracellular pathogens were adapted for use in studies with T. parva to enable definition of the phenotype and function of effector T cells in T. parva-immune cattle, facilitating vaccine development. As reported herein, stimulation of peripheral blood mononuclear cells (PBMC) from ITM-immunized steers with irradiated, autologous, T. parva-infected cell lines elicited a proliferative recall response comprised of CD45R0+/CCR7− CD4+ and CD8+ T cells. Subsequent co-incubation of stimulated cultures with infected cells demonstrated the presence of cytotoxic T cells (CTLs) with the ability to kill infected cells. Comparison of CTL activity in cultures depleted of CD4+ or CD8+ T cells demonstrated CTL activity was primarily attributed to CD8+ T cells. Importantly, stimulation of PBMC from vaccinated steers always elicited proliferation of CD4+ and CD8+ T cells. This was the first important observation obtained from the use of the assay described herein.

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Faculty Opinions recommendation of Specific recognition of the viral protein UL18 by CD85j/LIR-1/ILT2 on CD8+ T cells mediates the non-MHC-restricted lysis of human cytomegalovirus-infected cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018903.215491 ◽

2004 ◽

Author(s):

Eric Long

Keyword(s):

T Cells ◽

Human Cytomegalovirus ◽

Cd8 T Cells ◽

Viral Protein ◽

Specific Recognition ◽

Infected Cells

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Immunodominant Cytomegalovirus Epitopes Suppress Subdominant Epitopes in the Generation of High-Avidity CD8 T Cells

Pathogens ◽

10.3390/pathogens10080956 ◽

2021 ◽

Vol 10 (8) ◽

pp. 956

Author(s):

Kirsten Freitag ◽

Sara Hamdan ◽

Matthias J. Reddehase ◽

Rafaela Holtappels

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Escape ◽

Cd8 T Cell ◽

Antigenic Drift ◽

Murine Cytomegalovirus ◽

High Avidity ◽

Infected Cells ◽

Cd8 T Cell Memory

CD8+ T-cell responses to pathogens are directed against infected cells that present pathogen-encoded peptides on MHC class-I molecules. Although natural responses are polyclonal, the spectrum of peptides that qualify for epitopes is remarkably small even for pathogens with high coding capacity. Among those few that are successful at all, a hierarchy exists in the magnitude of the response that they elicit in terms of numbers of CD8+ T cells generated. This led to a classification into immunodominant and non-immunodominant or subordinate epitopes, IDEs and non-IDEs, respectively. IDEs are favored in the design of vaccines and are chosen for CD8+ T-cell immunotherapy. Using murine cytomegalovirus as a model, we provide evidence to conclude that epitope hierarchy reflects competition on the level of antigen recognition. Notably, high-avidity cells specific for non-IDEs were found to expand only when IDEs were deleted. This may be a host’s back-up strategy to avoid viral immune escape through antigenic drift caused by IDE mutations. Importantly, our results are relevant for the design of vaccines based on cytomegaloviruses as vectors to generate high-avidity CD8+ T-cell memory specific for unrelated pathogens or tumors. We propose the deletion of vector-encoded IDEs to avoid the suppression of epitopes of the vaccine target.

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148 Identification of prostate-restricted epithelial antigens for transgenic T cell adoptive therapy against prostate cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0148 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A161-A161

Author(s):

Diana DeLucia ◽

Tiffany Pariva ◽

Roland Strong ◽

Owen Witte ◽

John Lee

Keyword(s):

Prostate Cancer ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Cd8 T Cells ◽

Adoptive Therapy ◽

Mhc I ◽

Epithelial Antigens ◽

Ifn Γ ◽

Stimulation Of

BackgroundIn advanced prostate cancer (PCa), progression to castration-resistant PCa (CRPC) is inevitable and novel therapies for CRPC are needed. Adoptive transfer of T cells targeting tumor antigens is a promising approach in the cancer field. Unfortunately, identifying antigens expressed exclusively in prostate tumor cells has been challenging. Since the prostate is not an essential organ, we alternatively selected prostate-restricted epithelial antigens (PREAs) expressed in both malignant and normal prostate tissue for transgenic T cell studies.MethodsRNA-seq data sets identifying genes enriched in PCa were cross-referenced with the NIH Genotype-Expression database to identify PREAs. Using a novel molecular immunology approach, select PREAs and major histocompatibility complex class I (MHC-I) molecules were co-expressed in HEK293F cells, from which MHC–peptide complexes were efficiently isolated. Peptides were eluted and sequenced by mass spectrometry. Peptide–MHC binding was validated with a T2 stabilization assay and peptide immunodominance was determined using an interferon-γ (IFN-γ) ELISpot assay following stimulation of healthy HLA-A2+ peripheral blood mononuclear cells (PBMC) with peptide pools. Following peptide stimulation, CD8+ T cells with peptide-specific T cell receptors (TCR) were enriched by peptide–MHC-I dextramer labeling and fluorescence activated cell sorting for single cell TCR α/β chain sequencing.ResultsWe identified 11 A2+ peptides (8 previously unpublished) from prostatic acid phosphatase (ACPP), solute carrier family 45 member 3 (SLC45A3), and NK3 homeobox 1 (NKX3.1) that bound to HLA-A2 with varying affinities. Extended culture stimulation of PBMC with peptide pools from each PREA, compared to the standard overnight culture, revealed a greater number of IFN-γ producing cells overall and a greater breadth of response across all the peptides. Antigen specific CD8+ T cells were detectable at low frequencies in both male and female healthy PBMC for 7 of the 11 peptides. Dextramer-sorted antigen-specific cells were used for single-cell paired TCR αβ sequencing and transgenic T cell development.ConclusionsThrough this work we identified HLA-A2-presented antigenic peptides from the PREAs ACPP, SLC45A3, and NKX3.1 that can induce the expansion of IFN-γ producing CD8+ T cells. Through peptide–MHC-I dextramer labeling, we isolated PREA-specific CD8+ T cells and characterized TCR αβ sequences with potential anti-tumor functionality. Our results highlight a rapid and directed platform for the development of MHC-I-restricted transgenic CD8+ T cells targeting lineage-specific proteins expressed in prostate epithelia for adoptive therapy of advanced PCa.

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Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100588118 ◽

2021 ◽

Vol 118 (23) ◽

pp. e2100588118

Author(s):

Pengcheng Wei ◽

Kimberly R. Jordan ◽

Jonathan D. Buhrman ◽

Jun Lei ◽

Hexiang Deng ◽

...

Keyword(s):

T Cells ◽

Amino Acid ◽

T Cell ◽

Amino Acid Substitution ◽

Cd8 T Cells ◽

Tumor Antigens ◽

Vaccine Development ◽

Single Amino Acid ◽

Cell Repertoire ◽

Subtle Change

Tumors frequently express unmutated self-tumor–associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA–specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I–TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA–specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

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Infection of CD8+CD45RO+ Memory T-Cells by HIV-1 and Their Proliferative Response

The Open AIDS Journal ◽

10.2174/1874613600802010043 ◽

2008 ◽

Vol 2 (1) ◽

pp. 43-57 ◽

Cited By ~ 7

Author(s):

Naveed Gulzar ◽

Sowyma Balasubramanian ◽

Greg Harris ◽

Jaime Sanchez-Dardon ◽

Karen F.T. Copeland

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Proliferative Response ◽

Cell Infection ◽

Cellular Target ◽

Potential Reservoir ◽

Infected Cells ◽

Hiv 1

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis.

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Ex Vivo Characterization of Multiepitopic Tumor-Specific CD8 T Cells in Patients with Chronic Myeloid Leukemia: Implications for Vaccine Development and Adoptive Cellular Immunotherapy

The Journal of Immunology ◽

10.4049/jimmunol.174.12.8210 ◽

2005 ◽

Vol 174 (12) ◽

pp. 8210-8218 ◽

Cited By ~ 81

Author(s):

Monique Gannagé ◽

Michal Abel ◽

Anne-Sophie Michallet ◽

Stéphanie Delluc ◽

Marion Lambert ◽

...

Keyword(s):

T Cells ◽

Chronic Myeloid Leukemia ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Vaccine Development ◽

Ex Vivo ◽

Cellular Immunotherapy ◽

Adoptive Cellular Immunotherapy

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Enumeration of human antigen–specific naive CD8+ T cells reveals conserved precursor frequencies

Blood ◽

10.1182/blood-2009-10-251124 ◽

2010 ◽

Vol 115 (18) ◽

pp. 3718-3725 ◽

Cited By ~ 114

Author(s):

Cécile Alanio ◽

Fabrice Lemaitre ◽

Helen K. W. Law ◽

Milena Hasan ◽

Matthew L. Albert

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Vaccine Development ◽

Direct Detection ◽

Magnetic Bead ◽

Cell Precursor ◽

Adaptive Immune ◽

Precursor Frequency ◽

Magnetic Bead Enrichment ◽

Hiv 1

Abstract The number of antigen-specific naive CD8+ T cells is believed to be important in the shaping of adaptive immune responses, and is predictive for the magnitude of priming responses in mouse models. Because of extremely low precursor frequencies, knowledge about these cells comes from indirect techniques and estimations. Here, we present a strategy based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric cytometry, which permitted direct detection and analysis of CD8+ T cells reactive for 6 different naive epitopes (MART-126-35, HIV-1 Gag p1777-85, hepatitis C virus [HCV] NS31406-1415, HCV Core132-140, NY-ESO-1157-165, and cytomegalovirus [CMV] pp65495-503). Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6 × 10−6 to 1.3 × 10−4), but conserved frequencies among humans. Development of a procedure for direct assessment of T-cell precursor frequency in humans has important implications, with particular relevance to vaccine development and monitoring of tumor and self-reactive T cells.

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