Delayed-type hypersensitivity (DTH) responses are a model of CD4+ T cell recruitment into the tissues in immune resistance and in allergic and autoimmune diseases. The presence of sensitized CD4+, DTH effector T cells in the circulation is not sufficient for recruitment into a tissue site challenged with antigen (Ag). Another Ag-specific cell called DTH- initiating cells is required. These cells elaborate Ag-specific factors that are analogous to IgE antibody and mediate DTH-initiation by leading to local release of the vasoactive amine serotonin. Using a panel of monoclonal antibodies to deplete DTH-initiating activity, an unusual phenotype for an Ag-specific cell was found: Thy-1+, CD5+, CD4-, CD8-, CD3epsilon-, sIg-, B220+, I1-2R-, and IL-3R+. Furthermore, DTH-initiating cells were induced in athymic nude mice but not in SCID mice, leading to the conclusion that DTH-initiating cells were primitive, relatively thymic independent, Ag-specific cells that employed rearranging genes to encode the Ag-specific factors that mediate DTH-initiation. Nude mice were used to immunize and boost DTH-initiating cells in the absence of contaminating CD4+ DTH-effector T cells, or CD8+ suppressor T cells that down-regulate DTH-initiating cells, and in vitro lines and clones of DTH-initiating cells were generated. the phenotype of the DTH- clones determined by FACS and molecular analysis confirmed that DTH-initiating cells are primitive Ag- specific cells of a unique mixed phenotype: for T-like markers: Thy-1+, CD5+, CD4-, CD8-, surface and mRNA CD3-, surface and mRNA alphabeta TCR- and delta TCR mRNA+; for B-like markers: surface and mRNA Ig-, B220+, CD23-; and for Mphi markers FcgammaR+, Mac1+, Class II+. The current specific aims are to: 1) Develop DTH-initiating clones of another Ag specificity to confirm Ag-specificity of DTH-initiation at the clonal level. 2) To clone, sequence, and express the product of the TCR- delta hybridizing gene that is transcribed in the DTH-initiating clones. 3) To clone the gene(s) that encode the DTH-initiating factors. An unamplified cDNA library will be constructed from the DTH-initiating clone in lambdalZAP and will be screened by several means to attempt to isolate cDNA(s) encoding the Ag specific DTH-initiating factor. Probing will be attempted with a polyclonal antibody for DTH-initiating factors, and also with a ligand probe consisting of a conjugate of the relevant hapten coupled to albumin to which is also linked alkaline phosphatase. Attempts will continue to biochemically purify the DTH-initiating factor to obtain amino acid sequence information to construct an oligonucleotide probe. Finally, hamster monoclonal antibodies will be developed to DTH-initiating factor to provide monospecific reagents to screen the library. It is thus hoped that knowledge will be advanced of the important in vivo phenomena of DTH-initiation to the level of molecular cloning of the biologically relevant molecule.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026689-05
Application #
3140568
Study Section
Immunological Sciences Study Section (IMS)
Project Start
1989-04-01
Project End
1997-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Szczepanik, M; Lewis, J; Geba, G P et al. (1998) Positive regulatory gamma delta T cells in contact sensitivity: augmented responses by in vivo treatment with anti-gamma delta monoclonal antibody, or anti-V gamma 5 or V delta 4. Immunol Invest 27:1-15
Ushio, H; Tsuji, R F; Szczepanik, M et al. (1998) IL-12 reverses established antigen-specific tolerance of contact sensitivity by affecting costimulatory molecules B7-1 (CD80) and B7-2 (CD86). J Immunol 160:2080-8
Ptak, W; Paliwal, V; Bryniarski, K et al. (1998) Aggregated immunoglobulin protects immune T cells from suppression: dependence on isotype, Fc portion, and macrophage FcgammaR. Scand J Immunol 47:136-45
Szczepanik, M; Nowak, B; Askenase, P W et al. (1998) Cross-talk between gammadelta T lymphocytes and immune cells in humoral response. Immunology 95:612-7
Murray, A G; Schechner, J S; Epperson, D E et al. (1998) Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin. Am J Pathol 153:627-38
Szczepanik, M; Anderson, L R; Ushio, H et al. (1997) Gamma/delta T cells from tolerized alpha/beta-TCR-deficient mice antigen specifically inhibit contact sensitivity in vivo and IFN-gamma production in vitro. Int Arch Allergy Immunol 113:373-5
Matsuda, H; Ushio, H; Geba, G P et al. (1997) Human platelets can initiate T cell-dependent contact sensitivity through local serotonin release mediated by IgE antibodies. J Immunol 158:2891-7
Tsuji, R F; Geba, G P; Wang, Y et al. (1997) Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells. J Exp Med 186:1015-26
Paliwal, V; Ptak, W; Sperl, J et al. (1997) Recombinant soluble alphabeta T cell receptors protect T cells from immune suppression: requirement for aggregated multimeric, disulfide-linked alphabeta heterodimers. J Immunol 159:1718-27
Ptak, W; Szczepanik, M; Ramabhadran, R et al. (1996) Immune or normal gamma delta T cells that assist alpha beta T cells in elicitation of contact sensitivity preferentially use V gamma 5 and V delta 4 variable region gene segments. J Immunol 156:976-86

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