Identification and Characterization of Protective T Cells in hsp65 DNA-Vaccinated andMycobacterium tuberculosis-Infected Mice

Abstract

Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4 ؉ /CD8 ؊ and CD8 ؉ /CD4 ؊ hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44 lo ) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44 hi ) and produced gamma interferon (IFN-␥). In adoptive transfer of protection to naive mice, the total CD8 ؉ /CD4 ؊ cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4 ؉ /CD8 ؊ and CD8 ؉ /CD4 ؊ types and then into CD44 hi and CD44 lo types, CD44 lo cells were essentially unable to transfer protection, the most protective CD44 hi cells were CD8 ؉ /CD4 ؊ , and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44 lo IL-4-producing phenotype prevailed during infection, protection was associated with the CD8 ؉ /CD44 hi IFN-␥-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.

Cytotoxic T cells and mycobacteria

“…Protection partly re£ected the ability of the cells to produce IFN-Q; IL-4 producing cells were not protective and protection with IFN-Q producing cells was decreased by administering antibody against IFN-Q. However, the most protective CD4 and CD8 T cell clones also displayed antigen-speci¢c cytotoxicity in vitro and selectively lysed macrophages that were infected with M. tuberculosis [46,47]. This is consistent with the view that cytotoxicity also has a positive role in protection [11].…”

“…We have also found precursors of the protective hsp65-speci¢c CD8 /IFN-Q producing/cytotoxic/CD44 hi phenotype to persist at elevated frequency in spleens in parallel with persistence of protection for at least 8 months after DNA vaccination [44]. This also occurs after BCG vaccination, or during M. tuberculosis infection, but then the persistent response is dominated by T cells with the non-protective Th2 phenotype (IL-4 producing, CD44 lo , non-cytotoxic) [47]. To better understand the role of T cells in protection against tuberculosis we further characterized 28 CD4 and 28 CD8 hsp65-speci¢c T cell clones in vitro and in vivo and test whether lysis of M. tuberculosis-infected target macrophages by these clones can cause death of the bacteria by either the perforin-or Fas^FasL-dependent pathway [57].…”

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon‐γ recovery and reduction of lung injury

“…This strategy of evaluation was considered essential by our group because our previous reports focused on the characterization of spleen cell activation in vitro. 13,14 According to our model of infection, after the first 30 days of infection, mice developed a response more related to the Th1 pattern, characterized by increased levels of IFN-c and IL-12 in comparison with uninfected mice, which are crucial to the development of protective immunity. 19,23,24 Levels of IL-4 and IL-10 were similar to those of uninfected mice.…”

“…[9][10][11][12] A DNA vaccine based on the M. leprae heat-shock protein 65 gene (pHSP65) protected mice against subsequent M. tuberculosis challenge by establishing a cellular immune response characterized by antigen-specific CD4 + and CD8 + T lymphocytes, which both produce interferon-c (IFN-c) and are cytotoxic to infected cells. 13 Recently, a therapeutic effect of this vaccine in mice during the course of experimental TB was described. 14 The development of a TB vaccine depends on knowledge of the protective immune response that follows infection.…”

High‐frequency vaccine‐induced CD8+ T cells specific for an epitope naturally processed during infection with Mycobacterium tuberculosis do not confer protection

“…(A, B) Mice (CB6F1) were aerosol challenged with Mycobacterium tuberculosis Erdman and euthanized 6 weeks later to evaluate responses in perfused lungs. (A) Cells isolated from perfused lung tissue were subjected to intracellular flow cytometry after stimulation (1 + 5 (with brefeldin A) h) with either medium, rTB10.4, TB10.4 P1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] or the 9-meric peptide TB10.4 [3][4][5][6][7][8][9][10][11] . Cells were gated as follows: singlets > lymphocytes > CD4 + /CD8 + .…”