Generation and characterization of peptide-specific, MHC-restricted cytotoxic T lymphocyte (CTL) and helper T cell lines from unprimed T cells under microculture conditions

Abstract Neonatal alloimmune thrombocytopenia (NAIT) can cause severe complications such as intrauterine death or intracranial hemorrhage (ICH) in the newborn, and is caused by the transfer of platelet-depleting antibodies from the mother to the fetus during pregnancy. These antibodies react with allogeneic epitopes, most commonly human platelet antigen (HPA) 1a, when present on fetal platelets. Although these responses are thought to be a result of a T cell-dependent immune response, HPA 1a specific T cells have not yet been isolated. To examine whether HPA 1a specific T cells could be detected and isolated, we collected PBMC post delivery from an HPA 1a negative mother who gave birth to an HPA 1a positive neonate suffering from severe thrombocytopenia (platelet count <50×109/L). The cells were stimulated with HPA 1a peptides (20aa) in long term cultures supplemented with IL-7 and IL-2, and subsequently, IL-15. After 4 weeks in culture these cells were labeled with CFSE dye and restimulated with HPA 1a or control peptides. After additional 2 weeks in culture supplemented with IL-2 and IL-15, specific proliferative responses were detectable by CFSE dye dilution by flow cytometry. The cells were cloned by fluorescent-activated cell sorting (FACS) and expanded in numbers with anti-CD3 stimulation in the presence of irradiated allogeneic PBMC and IL-2. The resulting clonal T cell lines were characterized in proliferation assays, ELISPOT assays and phenotyped by flow cytometry. All clones were CD3+, CD4+ and CD19−, and the majority of the clones proliferated and secreted cytokines in response to stimulation with HPA 1a peptides, but not control peptides. In ELISPOT assays, peptide-pulsed antigen-presenting cells were required for T cell detection. These clonal HPA 1a specific CD4+ T cell lines represent formal evidence of the existence of HPA 1a specific T cell responses related to NAIT and will serve as important tools for further characterization of maternal immune responses associated with NAIT.

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T and B cells that recognize the same antigen do not transcribe similar heavy chain variable region gene segments.

Journal of Experimental Medicine ◽

10.1084/jem.158.1.192 ◽

1983 ◽

Vol 158 (1) ◽

pp. 192-209 ◽

Cited By ~ 33

Author(s):

E Kraig ◽

M Kronenberg ◽

J A Kapp ◽

C W Pierce ◽

A F Abruzzini ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Cell Lines ◽

Variable Region ◽

Antigenic Specificity ◽

Helper T Cell ◽

Region Gene ◽

Vh Gene ◽

T Cell Lines

We have attempted to determine whether T cells and B cells that have the same antigenic specificity and whose receptors share idiotypic determinants in fact express similar VH gene segments. To do this, we have obtained and characterized a cDNA clone containing the entire coding sequence for the VH gene from a glutamic acid60/alanine30/tyrosine10 (GAT)-binding immunoglobulin that carries the CGAT idiotype. The GAT-VH clone was hybridized to Northern blots of GAT-specific T cell RNAs; there was no evidence of a T cell transcript that hybridized to the GAT-VH probe. The T cells analyzed included: (a) 10 GAT-binding suppressor T cell hybridomas, 6 of which secreted factors with CGAT idiotypic determinants, (b) one GAT-specific helper T cell hybridoma, and (c) two GAT-specific helper T cell lines grown in the absence of feeder cells. The detection limit of the Northern blot analysis was 1-2 copies of a particular mRNA species per cell for the hybridomas and 5-10 copies per cell for the T cell lines. Therefore, we conclude that T and B lymphocytes responding to GAT do not utilize similar VH gene segments. Furthermore, the presence of idiotypic determinants on T lymphocytes does not necessarily imply close structural similarity between T and B cell antigen receptors.

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Rapid selection of antigen-specific T lymphocytes by retroviral transduction

Blood ◽

10.1182/blood.v96.1.109 ◽

2000 ◽

Vol 96 (1) ◽

pp. 109-117 ◽

Cited By ~ 51

Author(s):

Guenther Koehne ◽

Humilidad F. Gallardo ◽

Michel Sadelain ◽

Richard J. O'Reilly

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Cytotoxic T Lymphocyte ◽

Interleukin 2 ◽

Retroviral Vectors ◽

Retroviral Transduction ◽

Alloreactive T Cells ◽

T Cell Lines ◽

Selection Of

Abstract Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT+T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT+ fraction relative to the untransduced T-cell population. NIT+ fractions transduced on day 8 possessed more CD4+ T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

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Rapid selection of antigen-specific T lymphocytes by retroviral transduction

Blood ◽

10.1182/blood.v96.1.109.013k44_109_117 ◽

2000 ◽

Vol 96 (1) ◽

pp. 109-117 ◽

Cited By ~ 1

Author(s):

Guenther Koehne ◽

Humilidad F. Gallardo ◽

Michel Sadelain ◽

Richard J. O'Reilly

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Cytotoxic T Lymphocyte ◽

Interleukin 2 ◽

Retroviral Vectors ◽

Retroviral Transduction ◽

Alloreactive T Cells ◽

T Cell Lines ◽

Selection Of

Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT+T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT+ fraction relative to the untransduced T-cell population. NIT+ fractions transduced on day 8 possessed more CD4+ T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

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Analysis of T cell hybridomas. I. Characterization of H-2 and Igh-restricted monoclonal suppressor factors.

Journal of Experimental Medicine ◽

10.1084/jem.154.5.1390 ◽

1981 ◽

Vol 154 (5) ◽

pp. 1390-1402 ◽

Cited By ~ 37

Author(s):

M Minami ◽

K Okuda ◽

S Furusawa ◽

B Benacerraf ◽

M E Dorf

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Cell Lines ◽

Immune Suppression ◽

Cellular Interactions ◽

Suppressor T Cells ◽

Suppressor Activity ◽

T Cell Lines

Five hybridoma T cell lines were prepared by fusion of second order suppressor T cells (Ts2) with the BW5147 thymoma. The culture supernates from these T cell hybrids contained a factor, TsF2, which specifically suppressed 4-hydroxy-3-nitrophenyl acetyl hapten (NP)-induced cutaneous sensitivity responses. TsF2 activity was observed when the factor was administered during the effector phases of the immune response. TsF2 bears I-J determinants and has binding specificity for NPb idiotypic determinants. TsF2 suppressor activity could be absorbed on antigen-primed H-2-incompatible T cells but cannot suppress H-2-incompatible mice. In addition to this H-2 restriction, which maps to the I-J subregion, monoclonal TsF2 also has an Igh genetic restriction. The present results are combined with previous data to describe the cellular interactions leading to immune suppression.

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Characterization of cell surface molecules involved in the recognition of antigen-presenting cells by cloned helper T-cell lines

Cellular Immunology ◽

10.1016/0008-8749(88)90186-4 ◽

1988 ◽

Vol 115 (2) ◽

pp. 334-351 ◽

Cited By ~ 1

Author(s):

Kelly P. Kearse ◽

David R. Cassatt ◽

Alan M. Kaplan ◽

Donald A. Cohen

Keyword(s):

T Cell ◽

Cell Surface ◽

Cell Lines ◽

Antigen Presenting Cells ◽

Cell Surface Molecules ◽

Helper T Cell ◽

Surface Molecules ◽

T Cell Lines ◽

Antigen Presenting

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Characterization of collagen-specific T cells derived from pathogenic and nonpathogenic rat T cell lines

Cellular Immunology ◽

10.1016/0008-8749(90)90288-3 ◽

1990 ◽

Vol 130 (2) ◽

pp. 472-489 ◽

Cited By ~ 2

Author(s):

Grace Ku ◽

Ernest Brahn ◽

Mitchell Kronenberg

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

T Cell Lines

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Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells.

Journal of Experimental Medicine ◽

10.1084/jem.158.3.836 ◽

1983 ◽

Vol 158 (3) ◽

pp. 836-856 ◽

Cited By ~ 420

Author(s):

J Kaye ◽

S Porcelli ◽

J Tite ◽

B Jones ◽

C A Janeway

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cell Lines ◽

Cell Activation ◽

T Cell Proliferation ◽

Accessory Cell ◽

Helper T Cell ◽

T Cell Lines ◽

Antigen Presenting

Two antisera and a monoclonal antibody raised in BALB.K mice against cloned, major histocompatibility complex (MHC)-restricted, antigen-specific helper T cell lines are described. These antibodies are specific for individual cloned T cell lines and are potent inducers of T cell proliferation. The induction of T cell proliferation by these antibodies requires the presence of an adherent accessory cell. There is no H-2 restriction between this accessory cell and the cloned T cell, nor is this antibody-induced proliferation blocked by a monoclonal anti-Fc receptor antibody. The requirement for an accessory cell, however, is eliminated in the presence of an IL-1- or IL-2-rich supernatant. Thus this system allows the analysis of helper T cell activation with only a single cell type present. Anti-T cell sera also induce T cell-dependent B cell proliferation and immunoglobulin secretion. The induction of T cell-dependent B cell activation by these sera does not require H-2-matched T cells and B cells. The specificity of these antibodies and their ability to stimulate cloned helper T cells in the absence of antigen and antigen-presenting cells strongly suggest that these antibodies are directed against antigen and/or Ia recognition sites on the T cell.

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