Flow cytometric identification of virally infected cells and altered production of cytokines in viremic B10.F mice

Author

Ming-Yu Yang

Date of Award

12-1994

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Raymond A. Popp

Committee Members

Steve J. Kennel, Margaret Terzaghi-Howe, Michael L. Mucenski

Abstract

The balance of cellular and humoral immune responses to various pathogenic infections is regulated mainly by CD4⁺ T cells, which are divided into two distinct subsets of cloned helper T cells, T_H1 and T_H2, defined by the pattern of cytokines they secrete in response to antigen or mitogen activation. Studies of acquired immunodeficiency syndrome (AIDS) patients have suggested that the resistance to HIV infection and progression to AIDS are dependent on a T_H1> T_H2 dominance. It has also been suggested that persistent activation of CD4⁺ T cells with the T_H2 cytokine secretion pattern is responsible for chronic B cell stimulation, impaired cytotoxic function of CD8⁺ T cells, and down-regulation of T_H1 cytokines in mouse AIDS (MAIDS). B10FV, a unique ecotropic murine retrovirus, infects newborn B10.F congenic mice and integrates into the DNA of lymphoid cells, but the B10 inbred partner is resistant to B10FV. B10.F mice pass this virus from mother to offspring and exhibit a phenotype of splenic hyperplasia, lymphocyte subset perturbation, immunodeficiency, and life shortening. A rat anti-MuLV monoclonal antibody, which is specific for a MuLV envelope glycoprotein, was obtained from Dr. Leonard Evans, Rocky Mountain Laboratories. A flow cytometric method using this anti-MuLV antibody to detect MuLV antigen on the surface of B10FV-infected cells was established and infectivity was confirmed by the XC plaque assay. The incidence of lymphoid cells that expressed MuLV antigen increased markedly between one and four weeks of age. At four weeks of age, MuLV antigen was detected on approximately 30% of the cells, and this level of expression of viral antigen was shown to represent the persistent steady-state of infection established by B10FV. Almost no MuLV antigen was detected on lymphoid cells from B10.F babies that were foster-nursed on B10 mother. In contrast, significant levels of MuLV antigen were detected on spleen and mesenteric lymph node cells from B10 babies that were foster-nursed on B10.F mothers. Data from both neonatal and foster nursing studies were consistent with the hypothesis that the primary route of the B10FV mother-to-offspring transmission is via milk. Flow cytometric analysis on CD4⁺ and CD8⁺ T cell subsets revealed an early CD8⁺ T cell depletion and a high CD4⁺/CD8⁺ ratio in spleen cells from B10.F mice. The levels of T_H2-type cytokines, IL-4 and IL-10, in supernatants of stimulated spleen cell cultures and in the T2 clone of CD4⁺ T cells in spleen cell suspensions were both substantially higher in B10.F than in B10 mice through ten months of age. The distinction became even more significant at and beyond six months of age, although a classical T_H 1-type cytokine profile was not found in either B10 or B10.F mice. Most B10.F mice become immunodeficient and develop diseases after six months of age; the elevation of a dominant T_H2-type immune response in six-month and older B10.F mice is correlated with the lymphocyte subset perturbations and immunodeficiency observed in viremic B10.F mice.

Recommended Citation

Yang, Ming-Yu, "Flow cytometric identification of virally infected cells and altered production of cytokines in viremic B10.F mice. " Master's Thesis, University of Tennessee, 1994.
https://trace.tennessee.edu/utk_gradthes/11737