PMC

Derivation of Chlamydia-specific CD8 T-cell clones. CD4 and CD8 staining of polyclonal T-cell populations expanded from immune mice using irradiated immune splenocytes with (a) UV-inactivated Chlamydia muridarum (uvMoPn), (b) Elementary bodies-depleted Chlamydia antigen. (c) CD8β staining of CD8 T-cell clones 8uvmo-2, 8uvmo-3, 8sAg1 and 8sAg3 compared with alloreactive CD8 T-cell clone CD8bm1.

Chlamydia-specific CD8 T-cell clones express perforin but not Plac8. Total mRNA was isolated from each of the T-cell clones and tested for presence of β-actin, perforin and Plac8. In the bottom panel, previously published Chlamydia-specific CD4 T-cell clones uvmo-2 and uvmo-4 were included as positive and negative controls, respectively, for the Plac8 RT-PCR. No RT, no reverse transcriptase control. 2% agarose gel stained with ethidium bromide; image inverted for presentation.

Cytokine profiles of the CD8 T-cell clones. (a) CD8 T-cell clones were activated with immobilized anti-CD3 antibody. Supernatants were collected at 24 hr and indicated cytokine levels were determined by ELISA. Aggregate data from two independent experiments. (b) Interleukin-12 (IL-13) facilitates Chlamydia muridarum replication in epithelial cells. C57epi.1 epithelial cells untreated, and treated with tumour necrosis factor-α (TNF-α (5 µg/ml), IL-13 (30 µg/ml) and TNF-α/IL-13 (5/30 ηg/ml) for 10 hr then infected with C. muridarum at 3 inclusion forming units (IFU) per cell. Monolayers were harvested 25 hr later; recovered IFU were quantified on McCoy monolayers. Aggregate data from six independent experiments; **P < 0·005.

Specificity of CD8 T-cell clones. Each T-cell clone was activated with irradiated (2000 rads) immune splenocytes mock-pulsed, UV-inactivated Chlamydia muridarum (uvMoPn)-pulsed, and elementary bodies-depleted antigen (sAg)-pulsed. At 36 hr culture supernatants were harvested and [³H]thymidine was added; wells were harvested at 48 hr to score proliferation. (a) Proliferation in counts per minute (CPM). (b) Interferon-γ (IFN-γ) production as determined by ELISA. Data are means and SD for one experiment performed as quadruplicates. For each T-cell clone the experimental wells were compared with their mock-pulsed control and with the antigen-presenting-cells-only control (No T) for the relevant antigen. The higher P-value of those two comparisons (the least significant) was assigned and graphed. **P < 0·005; ***P < 0·0005.

Mapping the restriction element for the CD8 T-cell clones. (a) Irradiated (1000 rads) immune and naive splenocytes from C57BL/6 mice were pulsed with UV-inactivated Chlamydia muridarum (uvMoPn) and co-cultured without T-cell clone (No CD8 clone) and with non-specific bystander CD8bm1 T cells in quadruplicate. Supernatants were collected at 72 hr and levels of interferon-γ (IFN-γ) determined by ELISA. CD8bm1 experimental wells were compared with the relevant ‘No CD8 clone’ control wells; mean IFN-γ levels shown in parentheses. (b) CD8 T-cell clones were mock-activated and uvMoPn-activated with irradiated (1000 rads) naive C57BL/6 and K^bD^b knockout (KO) splenocytes antigen-presenting cells. Supernatants were collected at 72 hr and levels of IFN-γ were determined by ELISA. Data presented are aggregate data from two independent experiments. IFN-γ released by 8sAg1, 8sAg2 and 8sAg3 in response to uvMoPn-pulsed irradiated splenocytes was at least fivefold higher than the bystander effect shown in (a). **P < 0·005; ***P < 0·0005

CD8 T clone recognition and termination of Chlamydia muridarum replication in epithelial cells. (a) C57ep.1 epithelial cells, untreated (a) or treated with IFN-γ (10 ng/ml for 10 hr) (b), were infected with three inclusion forming units (IFU) C. muridarum per cell. After 4 hr the inocula were removed, monolayers were washed then T cells were added. Wells were harvested 32 hr after infection; recovered IFU were quantified on McCoy monolayers. Top panels of (a) and (b) = experiment #1; bottom panels = experiment #2. For IFU values below 100 000, actual recovered IFU are shown immediately above the data bars. (c) Supernatants in experiments #1 and #2 were collected immediately before harvesting monolayers; interferon-γ (IFN-γ) levels were determined by ELISA. Aggregate data from experiments 1 and 2. **P < 0·005; ***P < 0·0005.