Identification and characterization of protective T cells in hsp65 DNA-vaccinated and Mycobacterium 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-gamma). 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-gamma-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.

FIG. 1

Expression of CD44 on CD4⁺/CD8⁻ and CD8⁺/CD4⁻ splenocytes freshly purified from hsp65 DNA-immunized, M. tuberculosis-infected, or normal control mice. The cells were stained with MAb against CD4 or CD8 (to confirm purity) and against CD44 and then analyzed by FACScan. The percentage of cells showing CD44^hi fluorescence is shown in the upper right corner of each panel.

FIG. 2

Expression of CD44 on CD4⁺/CD8⁻ and CD8⁺/CD4⁻ splenocytes when the cells had been amplified by 7-day culture with J774-hsp65 after purification from hsp65 DNA-immunized or M. tuberculosis-infected mice; the cells were stained with MAb against CD44 and analyzed by FACScan. The percentage of cells showing CD44^hi fluorescence is shown in the upper right corner of each panel.

FIG. 3

ELISPOT estimates of the frequencies of cells that produced IL-4 or IFN-γ in response to hsp65 among splenocytes from hsp65 DNA-immunized or M. tuberculosis-infected mice. Purified CD4⁺/CD8⁻ and CD8⁺/CD4⁻ cells were amplified by culture for 14 days on J774-hsp65 cells before assay in the presence of J774-hsp65 cells. The results shown are mean estimates (± SD) from triplicate wells at 3 different dilutions after subtraction of estimates obtained by assay in the presence of J774 vector cells (about 10 per 1,000 for CD4⁺/CD8⁻ cells and 12 per 1,000 for CD8⁺/CD4⁻ cells).

FIG. 4

Association of IFN-γ production with CD44^hi cells. Purified CD4⁺/CD8⁻ and CD8⁺/CD4⁻ cells were amplified by culture for 14 days on J774-hsp65 cells and then separated into CD44^hi and CD44^lo by FACSort and amplified for a further 12 days before assay in the presence of J774-hsp65 cells as described in the legend to Fig. 3.

FIG. 5

Adoptive transfer of protective immunity against tuberculosis by bulk transfer of CD4⁺/CD8⁻ or CD8⁺/CD4⁻ cells to naive mice. T-cell subsets were obtained by negative and positive selection from spleens of M. tuberculosis-infected or hsp65 DNA-immunized mice. Recipient mice were gamma irradiated and then injected intravenously with 5 × 10⁶ T cells and 1 × 10⁵ M. tuberculosis cells. The numbers of live bacteria in the spleens were determined 4 weeks after infection. Control mice were either untreated or were irradiated and reconstituted with nonspecific splenic T cells enriched from normal mice. Results are shown as mean CFU ± SD from groups of five animals.

FIG. 6

Association of protection with CD8⁺/CD44^hi hsp65-reactive T cells. Purified CD4⁺/CD8⁻ and CD8⁺/CD4⁻ cells were amplified by culture for 14 days on J774-hsp65 cells and then separated into CD44^hi and CD44^lo types by FACSort and amplified for a further 12 days. The cells were tested for the ability to protect naive recipient mice against challenge with M. tuberculosis as described in the legend to Fig. 5.

FIG. 7

Protection by T-cell clones. Four strongly growing hsp65-responsive clones of CD4⁺/CD44^lo and four of CD4⁺/CD44^hi, four of CD8⁺/CD44^lo, and four of CD8⁺/CD44^hi phenotypes were selected from spleens of M. tuberculosis-infected and from hsp65 DNA-immunized mice. After characterization for hsp65 antigen-dependent cytotoxicity and IL-4 and IFN-γ production, they were tested for the ability to protect naive mice from challenge with M. tuberculosis as described in the legend to Fig. 5. The numbers of live bacteria in spleens 4 weeks after challenge are shown as mean log₁₀ ± SD for groups of five animals. Dark-shaded and unshaded bars represent data from clones that produced IL-4 and IFN-γ, respectively. Controls were as follows: 1, untreated; 2 and 3, reconstituted, respectively, with CD4⁺/CD8⁻ and CD8⁺/CD4⁻ clones having irrelevant antigenic specificity.