TUMOR LYMPHOKINES, TCR PEPTIDES&PERFORIN TUMOR REJECTION

  • Podack, Eckhard R, (PI)

Project: Research project

Description

The interrelationship, in tumor rejection, between (1) lymphokine
production, (2) T-cell receptor usage, and (3) cytotoxicity, as measured
in perforin transgenic mice, will be investigated in this proposal. An
immunological response to a normally nonimmunogenic tumor is enforced by
lymphokinic transfection of the tumor cell prior to transplantation into
mice. IL2 transfection of the non-immunogenic Lewis lung cell carcinoma
(LLC) renders the tumor immunogenic in such a way that it is rejected in
normal mice but not in immunodeficient mice lacking T-cells (Nude,SCID) or
NK-cells (Beige). Hence, both a T-cell and an NK-cell response are
required for tumor rejection of this particular tumor producing IL2 (LLC-
IL2). Even though IL2 producing LLC can retard the growth of normal LLC,
a complete immunity resulting in rejection of the non-lymphokine producing
tumor has not been achieved. By transfecting LLC with various lymphokines
singly, or in a new double expression vector, with two lymphokine cDNAs
simultaneously, the induction of an immune response giving complete tumor
immunity is sought. Combination of lymphokines will focus on IL2 with
IL4, IL6, I18 or gamma interferon. Studies with singly transfected LLC
gave rise only to partial immunity. In rejected or retarded tumors the T-
cell response will be determined by measuring the presence of T-cell
receptor alpha and beta v-region gene families present in tumor
infiltrating T-cells. Using a set of already tested (in NOD mice) v-
region gene primers for both TCR-chains and the corresponding c-region
primer, the cDNAs of the TCRs, generated by reverse transcription, will be
amplified by PCR and cloned. The sequence of the most predominant TCR
families, present at the tumor site under the influence of various tumor
produced lymphokines, will be established for the v-, D-, and J-segments.
To manipulate the T-cell response and uncover rejecting or suppressing T-
cell clones, mice will be immunized with synthetic peptides corresponding
to the TCR sequence. The ensuing anti idiotype antibody and cellular
response will be evaluated with regard to its effect on tumor rejection.
These studies will be complemented by the isolation and cloning of T-cells
specific for lymphokine producing and non-producing LLC. The role of
cytotoxicity, specifically the role of perforin, in tumor rejection in
this model will be studied by comparing the tumor response of perforin
deficient transgenic mice to that of perforin sufficient mice. Perforin
deficiency has been achieved in embryonal stem cells by homologous
recombination and perforin deficient chimeras are currently under study.
These studies will be complemented by reconstituting perforin deficient
mice, as well as normal mice, with human perforin transgenes. Human
perforin, detectable by antibody and Northern analysis and distinguishable
from mouse perforin, is expressed under the murine perforin promoter and
under various promoter deletions, designed to enhance perforin expression
in T-cell subsets. Improved perforin expression in transgenic mice will be
analyzed with regard to its effect on tumor rejection.
StatusFinished
Effective start/end date9/1/928/31/01

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $243,319.00
  • National Institutes of Health: $261,804.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $246,753.00

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Lymphokines
Perforin
T-Lymphocytes
Lewis Lung Carcinoma
Neoplasms
Peptides
Apoptosis
Interleukin-2
Genes
Immunity
Transgenic Mice
Product Packaging
Transfection
Cell Survival
Inbred NOD Mouse
Pharmaceutical Preparations
Pharmacology
Tacrolimus Binding Proteins
Natural Killer Cells
T-Lymphocyte Subsets

ASJC

  • Medicine(all)