Direct Genotyping of Coding Non-Synonymous SNPs for Identification of Novel Minor Histocompatibility Antigens.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3237-3237
Author(s):  
Brad Stone ◽  
Scott Graves ◽  
Arnold Kas ◽  
Alexander Ford ◽  
Nathan Standifer ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Granzyme B ◽  
Epitope Prediction ◽  
Hla Class I ◽  
Cd8 T Cell ◽  
Histocompatibility Antigens ◽  
Synonymous Snps ◽  
Minor Histocompatibility Antigens ◽  
T Cell Lines

Abstract Traditional methods for identifying minor histocompatibility antigens (mHags) are technically challenging and biased against discovery of mHags not expressed in the peripheral blood. In this work, we propose a rapid, unbiased, genetic approach for identification minor antigens resulting from disparities in coding non-synonymous SNPs (“C SNPS”). This approach is capable of testing for responses to candidate minor antigens expressed in virtually any tissue, including those expressed exclusively in tissues targeted by GVHD. The first step in our approach begins with comparison of donor and recipient C SNP genotypes generated using C SNP microarrays. These arrays interrogate approximately 80% of human C SNPs predicted to occur in greater than 5% of the population. Comparison of C SNP genotypes directly identifies protein-altering alleles present in the recipient but not the donor (hereafter referred to as “recipient-restricted” alleles), thereby identifying a transplant-specific set of candidate minor antigens. The second step utilizes conventional HLA-class I epitope prediction performed on all linear peptides that include amino-acid residues defined by a recipient-restricted allele. This two step filtering process identifies a small, “testable” number candidate minor peptide epitopes for an individual expressed HLA-class I allele. Candidate epitopes can then be synthesized, pooled and tested at diagnosis of GVHD using a commercial Granzyme B ELISPOT assay. As proof-of-principal for a direct genotyping approach, we have analyzed C SNP genotypes, performed epitope prediction and generated T-cell lines specific for candidate minor antigens using DNA and PBL from a pair of disease-free HLA-identical siblings. Analysis of 10,000 C SNPs shows that approximately 2,000 C SNP alleles are restricted to one sibling within the pair. BIMAS epitope prediction of short unique peptide sequences determined by each sibling-restricted allele identifies approximately 100 candidate minor epitopes predicted to bind HLA-A*0201. These candidate minor epitopes were ranked using expression microarrays performed on EBV-transformed LCL derived from each sibling, with candidates derived from highly expressed genes ranked above those from genes with lower expression levels. A pool of 12 candidate minor epitopes that were both unique to sibling “A” and derived from genes highly expressed in LCL were synthesized and used to generate CD8+ T-cell lines from sibling “B”. Stimulation utilized autologous (sibling “B”-derived) mature dendritic cells loaded with candidate minor epitopes. After several rounds of in-vitro stimulation, each T-cell line was tested for responses to EBV-LCL from sibling “B” and sibling “A” using a Granzyme B ELISPOT kit. Five out of sixteen lines responded to LCL from sibling A while no line responded to autologous LCL. Thus we show that this approach frequently generates CD8+ T-cell lines specific for sibling-derived target cells, suggesting that this approach efficiently identifies genuine, novel, endogenously processed and presented minor epitopes. Deconvolution of the peptide pool suggests that at least two out of the twelve candidate minor epitopes are naturally processed and presented.

Development of Chronic Lymphocytic Leukemia (CLL) Reactive Cytotoxic T Lymphocytes after Non-Myeloablative Hematopoietic Stem Cell Transplant Correlates with Anti-Leukemia Response.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 413-413
Author(s):  
Tetsuya Nishida ◽  
Ana Kostic ◽  
David G. Maloney ◽  
Rainer F. Storb ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Histocompatibility Antigens ◽  
Antitumor Response ◽  
Hematopoietic Stem ◽  
Minor Histocompatibility Antigens ◽  
Cell Responses ◽  
T Cell Lines

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) following non-myeloablative (NM) conditioning is a promising approach for treating patients with advanced fludarabine refractory CLL. In this setting, a graft versus leukemia (GVL) effect mediated by donor T cells is critical for tumor eradication. We have evaluated the development of alloreactive and CLL-reactive cytotoxic T lymphocyte (CTL) responses in patients after NM-HSCT to determine if the generation of detectable T cell responses was associated with an antitumor response. Seven patients with fludarabine refractory CLL were conditioned with fludarabine (30mg/m2 x 3 doses) and total body irradiation (2 Gy) prior to receiving G-CSF mobilized peripheral blood stem cells from an HLA matched donor. Peripheral blood mononuclear cells (PBMC) were obtained from the recipient pretransplant and at intervals after NM-HSCT. When chimerism showed a major proportion of donor CD3+ T cells, the postransplant PBMC were stimulated in vitro with recipient CLL cells from the pretransplant collections. CLL cells lack or express low levels of co-stimulatory and adhesion molecules, and are poor stimulators of T cells in vitro. Thus, prior to their use as stimulators and targets, the CLL cells were activated with CD40 ligand (CD40L), which upregulates costimulatory, adhesion, and MHC molecule expression, and turns CLL cells into effective antigen presenting cells. The cultures were stimulated weekly and supplemented with IL2 and IL7. After two stimulations, the T cell lines were tested for cytotoxicity against donor and recipient target cells including recipient CLL. T cell lines generated from four patients with a good antitumor response after NM-HSCT exhibited cytotoxicity against recipient CLL and EBV transformed B cells (B-LCL), but not against donor B-LCL. By contrast, T cell lines generated from three patients with persistent or progressive disease after NM-HSCT did not have cytotoxicity against recipient CLL, despite the development of GVHD in all patients. Multiparameter flow cytometry and IFN-g secretion assay of T cell lines from patients with an antitumor response showed that both CD8+ and CD4+ T cells produced INF-g in response to recipient CLL. We sorted and expanded CD8+ INF-g+ and CD4+ IFN-g+ T cells and both subsets were able to lyse CLL cells. The cytotoxicity of CD4+ and CD8+ T cells was inhibited completely by concanamycin A, suggesting perforin is the major mechanism for leukemia cell lysis. Twenty-one CD8+ T cell clones specific for distinct minor histocompatibility antigens expressed on CLL were isolated from T cell lines of the four responding patients. Multiple specificities were recognized in three of the four patients. Screening a cDNA expression library has identified the genes encoding two minor histocompatibility antigens recognized by CD8+ T cells, and their characterization is in progress. These findings suggest that the development after NM-HSCT of early, diverse, alloreactive T cell responses specific for antigens expressed by CLL may be an important predictor of outcome. The identification of the antigens recognized may facilitate the development of strategies to evoke an effective antitumor response in a larger fraction of patients.

scholarly journals Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

PLoS ONE ◽  
2010 ◽  
Vol 5 (8) ◽  
pp. e12120 ◽  
Author(s):  
Alexis Morice ◽  
Béatrice Charreau ◽  
Bérangère Neveu ◽  
Sophie Brouard ◽  
Jean-Paul Soulillou ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Cross Reactivity ◽  
Class I ◽  
Class I Mhc ◽  
Mhc Molecules ◽  
T Cell Lines

Establishment and characterization of αs1-casein–specific T-cell lines from patients allergic to cow’s milk: Unexpected higher frequency of CD8+ T-cell lines

1996 ◽  
Vol 97 (6) ◽  
pp. 1342-1349 ◽  
Author(s):  
Haruyo Nakajima ◽  
Satoshi Hachimuraa ◽  
Shinya Nishiwakia ◽  
Toshiyuki Katsuki ◽  
Naoki Shimojo ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Cow’S Milk ◽  
Cow's Milk ◽  
T Cell Lines

Generation of Cytomegalovirus-Specific T Lymphocytes Using Protein-Spanning Pools of pp65-Derived Over-Lapping Pentadecapeptides for Adoptive Immunotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2236-2236
Author(s):  
Guenther Koehne ◽  
Deepa Trivedi ◽  
Roxanne Y. Williams ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Alleles ◽  
Hla Genotypes ◽  
T Cell Lines

Abstract Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We utilized a pool of 138 synthetic overlapping pentadecapeptides over-spanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T-cells and in analyses of T-cell lines from 5 separate HLA-A*0201+ individuals demonstrate that this pp65-derived pentadecapeptide-pool selectively induced T-cells specifically reactive against sub-pools of pentadecapeptides which contained the HLA-A*0201 binding epitope NLVPMVATV. The specificity of these T-cells for this immunodominant nonapeptide was confirmed by MHC-tetramer staining and intracellular interferon-γ production, demonstrating that 38 – 60% of the CD8+ cell population were specific for this A*2-restricted peptide after 3 weeks of culture. These T cells also killed both nonapeptide-pulsed and CMV-infected target cells. In subsequent experiments using auotlogous monocyte-derived DC’s pulsed with the pentadecapeptide pool for the stimulation of CMV-specific T-cell lines in individuals other than HLA-A*2, the generated T cells selectively recognized 1–3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide loaded targets sharing single HLA class I or II alleles permitted the identification the restricting HLA alleles. Those T-cell lines from HLA-A*2 neg. donors contained high frequencies of CD4 and/or CD8 T-cells selectively reactive against peptides presented by other HLA alleles including known epitopes such as aa 341–350QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as aa 267–275HERNGFTVL (HLA-B*4001 and B* 4002). In some donors, the peptide-specific IFN-g+ T-cells generated have been predominantly CD4+ T-cells. Like the peptide-specific CD8+ T-cells, we could determine both epitope and HLA-class II restricting element, e.g. aa513–523 FFWDANDIYRI (HLA-DRB1* 1301). These CD4+ T-cells also consistently exhibited cytotoxic activity against infected targets as well as peptide-loaded cells expressing the restricting HLA class II allele. Thus, synthetic overlapping pentadecapeptides spanning the sequence of the immunodominant protein of CMV-pp65, when loaded on DCs can consistently stimulate the in vitro generation of CD8+ and CD4+ T-cell lines from seropositive donors of diverse HLA genotypes. These cell lines are selectively enriched for T-cells specific for a limited number of immunodominant epitopes each presented by a single HLA class I or class II allele. This approach fosters expansion and selection of HLA-restricted CMV-pp65-reactive T-cell lines of high specificity which also lyse CMV-infected targets and may have advantages for generating virus-specific T-cells for adoptive immunotherapy.

Generation of Cytomegalovirus (CMV)-Specific CD4 and CD8 T Cell Lines Using Protein-Spanning Pools of pp65 and IE1 Derived Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 477-477
Author(s):  
Erica Dander ◽  
Giuseppina Li Pira ◽  
Ettore Biagi ◽  
Fabrizio Manca ◽  
Andrea Biondi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
Target Cells ◽  
Pulsed Dc ◽  
Ifn Γ ◽  
T Cell Lines

Abstract BACKGROUND: Reactivation of latent CMV in immunocompromised recipients of allogeneic stem cell transplantation remains a major cause of morbidity and mortality. Reconstitution of immunity by CMV specific immunotherapy is an attractive alternative to drugs currently used, which show high toxicity and are sometimes ineffective. It has been demonstrated that CD4 helper T-cell function is crucial for the persistence of in vivo transferred CD8 CMV-specific CTL. Based on this finding, we have explored the feasibility of generating both anti-CMV CD4 and anti-CMV CD8 T-cell lines. METHODS: Dendritic Cells (DC) were generated from donor peripheral blood (PB) monocytes after a 7-day culture in the presence of GM-CSF plus IL-4 and matured with TNF-α, IFN-α, IFN-γ, IL1-β, POLI I:C. Matured-DC were then pulsed with a pool of 50 peptides spanning pp65 and IE1 proteins which are recognised by both CD4 and CD8 T lymphocytes. Donor T cells were stimulated three times at a T cell/DC ratio of 1:6 on day 0, +7 and +14 with mature peptide pulsed-DC. At the end of the culture the specificity of generated T cells was determined as percentage of pentamer-positive cells and intracellular IFN-γ production after incubation with peptide pulsed-DC. Cultured T cells were also analysed for their ability to proliferate in response to peptide pulsed-target cells, to kill them in a standard citotoxicity assay and to migrate in response to inflammatory (CXCL9, CCL3 and CCL5) and constitutive (CXCL12) chemokines. RESULTS: CMV-specific T cell lines were generated from five CMV seropositive donors. In four cases CD4 and CD8 CMV-specific T cell lines were expanded successfully. Cultured T cells expressed CD8 (mean= 70%, range 60–81%) and CD4 (mean= 20%, range 15–28%) and showed a CD45RA- CCR7- Effector Memory phenothype (mean=26%, range 19–30%) or a CD45RA+ CCR7- T Effector Memory RA-Positive phenothype (mean=67%, range 59–77%). An enriched CMV-specific T cell population was observed after staining with pentamers (7–45% pentamer-positive T cells). Furthermore, 90% of CD8+ and 40% of CD4+ T cells expressed high levels of intracytoplasmatic perforin and granzyme. In 4/5 cases tested, cutured T cells showed a cytolitic activity against CD8-peptide pulsed target cells (average lysis=50%, range 40–55%) and to a lesser extent against CD4-peptide pulsed target cells (average lysis=35%, range 30–40%). In addition, cultured T lymphocytes were able to proliferate and to produce intracytoplasmic IFN-γ (average production=50%, range 35–60%) after exposure to peptide-pulsed DC. Finally, Cultured T cells strongly migrated in response to chemokines (CXCL9, CCL3 and CCL5) involved in the recruitment of effector cells during viral infection. DISCUSSION: In conclusion, a great advantage of this method is represented by the possibility to generate anti-CMV CD4+ T cells, which could support in vivo the persistence of re-infused CMV-specific CTL. Moreover, the possibility of generating peptides under GMP conditions would facilitate the translation of this approach into clinical intervention.

Generation of Acute Myeloid Leukemia (AML)-Specific Autologous CD4 and CD8 T Cell Lines by Limiting Dilution AML Dendritic Cell Culture.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2018-2018
Author(s):  
Rui-kun Zhong ◽  
Thomas A. Lane ◽  
Edward D. Ball
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Cd8 T Cell ◽  
Ifn Gamma ◽  
T Cell Lines ◽  
Limiting Dilution ◽  
Acute Myeloid

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure

Universal CD137 Expression upon Activation Allows Efficient Isolation of a Broad Repertoire of Virus-Specific CD8+ and CD4+ T Cells for Adoptive Immunotherapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2222-2222
Author(s):  
Maarten L. Zandvliet ◽  
J.H. Frederik Falkenburg ◽  
Inge Jedema ◽  
Roelof Willemze ◽  
Henk-Jan Guchelaar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Cell Populations ◽  
T Cell Lines ◽  
Kinetics Of ◽  
Ifng Production

Abstract Reactivation of adenovirus (ADV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can cause serious morbidity and mortality during the prolonged period of immune deficiency following allogeneic stem cell transplantation. It has been shown that adoptive transfer of donor-derived virus-specific T cells can be a successful strategy to control viral reactivation. To provide safe and effective anti-viral immunotherapy, we aimed to generate combined CD8+ and CD4+ T cell lines with high specificity for a broad range of viral epitopes. Isolation by the IFNg capture assay of virus-specific T cells that produce IFNg upon activation allows the generation of highly specific T cell lines without the need for extensive culture. However, it has been recently shown that specific upregulation of the co-stimulatory molecule CD137 upon antigen-specific activation of CD8+ and CD4+ T cells can also be used for isolation. We therefore analyzed IFNg production and CD137 expression by CD8+ and CD4+ T cells upon incubation of peripheral blood mononuclear cells (PBMC) from seropositive donors with peptides corresponding to 17 defined MHC class I restricted minimal epitopes from 10 different ADV, CMV, EBV and influenza (FLU) proteins, and 15-mer or 30-mer peptides containing MHC class II restricted epitopes from CMV pp65 or ADV hexon. Using tetramer and intracellular IFNg staining we first determined the fraction of CD8+ T cells that produced IFNg upon activation with the minimal epitopes. Specific IFNg production was observed for 58–100% of tetramer+ CD8+ T cells specific for CMV pp65 (n=6), and 83% for FLU (n=1), but only 18–58% for CMV pp50 (n=3) or IE-1 (n=3), 4–91% for EBV latent (n=3) and lytic (n=3) epitopes, and 41–63% for ADV hexon (n=2). In contrast to the variation in the fraction of IFNg-producing cells, we observed homogeneous upregulation of CD137 by the virus-specific tetramer+ T cell populations upon activation. In 2 cases where no CD137 expression by tetramer+ T cells could be detected, no IFNg production was observed either. These data suggest that the majority of CD8+ T cells specific for CMV pp65 or FLU can be isolated on basis of IFNg production, but only part of CD8+ T cell populations specific for other viral proteins, while complete virus-specific CD8+ T cell populations may be isolated on basis of CD137 expression. Activation of CD4+ T cells specific for CMV pp65 or ADV hexon with 15-mer or 30-mer peptides induced both specific IFNg production and CD137 expression. To investigate whether multiple virus-specific T cell populations could be isolated simultaneously, we next determined the kinetics of IFNg production after activation with defined MHC class I epitopes or peptides containing MHC class II epitopes. CMV- and EBV-specific CD8+ T cells and CMV-specific CD4+ T cells showed a rapid induction of IFNg production, which peaked after 4 hours and decreased thereafter. In contrast, ADV- and FLU-specific CD8+ T cells and ADV-specific CD4+ T cells, predominantly having a more early differentiation phenotype (CD27+CD28+) compared to CMV- and EBV-specific T cells, showed peak IFNg production after 8 hours that continued for more than 48 hours. This difference in phenotype and IFNg kinetics may suggest that the persistent and frequent presentation of CMV and EBV epitopes in vivo, in contrast to an intermittent exposure to ADV and FLU epitopes, drives differentiation and shapes the kinetics of the IFNg response of specific T cells. Kinetic analysis of CD137 expression showed uniform upregulation by virus-specific CD8+ T cell populations from day 1 to day 4 after activation, which peaked at day 2, suggesting that this may be the optimal time point for CD137-based isolation. In a limited number of experiments, virus-specific CD8+ and CD4+ T cells could be isolated based on CD137 expression within the same timeframe. These data indicate that virus-specific T cell populations can be more efficiently isolated at one time point on basis of CD137 expression than on basis of IFNg production, due to differences in IFNg kinetics. In conclusion, this study shows that T cell lines generated by CD137 isolation may comprise a significant number of virus-specific T cells which do not produce IFNg, but may have other effector functions. Furthermore, CD137-based enrichment may be more robust and allows the efficient simultaneous isolation of multiple virus-specific T cell populations due to uniform kinetics of CD137 expression.

Derivation ofHerpesvirus saimiri-Transformed CD8+T Cell Lines with Noncytotoxic Anti-HIV Activity

1997 ◽  
Vol 82 (3) ◽  
pp. 274-281 ◽  
Author(s):  
Carl E. Mackewicz ◽  
Roland Orque ◽  
Jae Jung ◽  
Jay A. Levy
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
T Cell Lines ◽  
Anti Hiv
Sign in / Sign up

Export Citation Format

Share Document