A method for testing the specificity of influenza A virus-reactive memory cytotoxic T lymphocyte (CTL) clones in limiting dilution cultures

Identification and targeting of novel immunobiological factors that regulate the induction of Th1 cells are crucial for designing effective vaccines against certain intracellular pathogens, including Chlamydia. IL-10-deficient dendritic cells (DC) are potent APCs and effective cellular vaccines that activate a high frequency of specific Th1 cells. To elucidate the molecular basis for the potency of the IL-10-deficient APC system, we tested the hypothesis that Chlamydia Ag-primed IL-10 knockout (IL-10KO) DC are quantitatively and qualitatively distinct in their metabolic characteristics relating to T cell activation. Using a combination of RT-PCR, two-dimensional gel electrophoresis, and MALDI-TOF-based proteomics analyses, the transcriptional and translational activities of Chlamydia-pulsed DC from wild-type and IL-10KO mice were assessed. IL-10 deficiency caused early maturation and activation of pulsed DC (i.e., high CD11c, CD40, CD80, CD83, CD86, IL-1, IL-12, and the T cell-attracting chemokine CCL27/CTACK) and consequently an enhanced ability to process and present Ags for a rapid and robust T cell activation. Supporting comparative proteomics revealed further that IL-10 deficient DC possess specific immunobiological properties, e.g., the T cell-attracting chemokine CCL27/CTACK, calcium-dependent protein kinase, and the IL-1/IL-12 inducer, NKR-P1A (CD161), which differentiated them immunologically from wild-type DC that express molecules relating to anti-inflammatory, differentiative, and metabolic processes, e.g., the anti-IL-12 molecule peroxisome proliferator-activated receptor-α and thymidine kinase. Collectively, these results provide a molecular basis for the high Th1-activating capacity of IL-10KO APC and may provide unique immunomodulation targets when designing vaccines against pathogens controlled by T cell immunity.

Section: Measurement Of Frequency Of Chlamydia-specific Th1 Cells (Thmentioning

confidence: 99%

Molecular Basis for the Potency of IL-10-Deficient Dendritic Cells as a Highly Efficient APC System for Activating Th1 Response

Moore

Eko

et al. 2005

“…Purified lymphoid cells contained at least 95% CD3 ϩ cells, as determined by FACS. A modified procedure for the limiting dilution technique reported previously (45,46) was used to measure the frequency of Chlamydia-specific Th1 cells (assayed as Ag-specific IFN-␥ production) by each cell population. In brief, purified T cells were seeded in a serial doubling dilution into 96-well round-bottom tissue culture plates at 24 wells/dilution.…”

Section: Measurement Of the Frequency Of Chlamydia-specific Th1 Cellsmentioning

confidence: 99%

Section: Measurement Of the Frequency Of Chlamydia-specific Th1 Cellsmentioning

confidence: 99%

A Novel Recombinant Multisubunit Vaccine against Chlamydia

Eko

Brown

et al. 2004

The administration of an efficacious vaccine is the most effective long-term measure to control the oculogenital infections caused by Chlamydia trachomatis in humans. Chlamydia genome sequencing has identified a number of potential vaccine candidates, and the current challenge is to develop an effective delivery vehicle for induction of a high level of mucosal T and complementary B cell responses. Vibrio cholerae ghosts (VCG) are nontoxic, effective delivery vehicles with potent adjuvant properties, and are capable of inducing both T cell and Ab responses in mucosal tissues. We investigated the hypothesis that rVCG could serve as effective delivery vehicles for single or multiple subunit chlamydial vaccines to induce a high level of protective immunity. rVCG-expressing chlamydial outer membrane proteins were produced by a two-step genetic process, involving cloning of Omp genes in V. cholerae, followed by gene E-mediated lysis of the cells. The immunogenicity and vaccine efficacy of rVCG-expressing single and multiple subunits were compared. Immunologic analysis indicated that i.m. immunization of mice with either vaccine construct induced a strong mucosal and systemic specific Th1 response against the whole chlamydial organism. However, there was an immunogenic advantage associated with the multiple subunit vaccine that induced a higher frequency of Th1 cells and a relatively greater ability to confer protective immunity, compared with the single subunit construct. These results support the operational theory that the ability of a vaccine to confer protective immunity against Chlamydia is a function of the level of Th1 response elicited.

“…Therefore, we investigated the hypothesis that a specific anti-chlamydial Th1 response is induced more rapidly, and greater numbers of specific Th1 cells are recruited into the genital mucosa of infected IL-10 Ϫ/Ϫ mice compared with those in wild-type IL-10 ϩ/ϩ mice. Accordingly, limiting dilution analysis (26,27) was used to compare the kinetics of induction and recruitment of chlamydial-specific Th1 cells into the genital tract of infected IL-10 Ϫ/Ϫ and IL-10 ϩ/ϩ mice. By calculating the frequency of Ag-specific IFN-␥-secreting T cells (i.e., Th1 cells) in the genital tract T cell preparations, it was found that IL-10 Ϫ/Ϫ mice exhibited a greater and more rapid Th1 response than control mice (Table I).…”

Section: High Resistance Of Il-10ko Mice To Genital Chlamydial Infectmentioning

confidence: 99%

“…Three times the value of the SD was added to the mean value, and the sum was the baseline for positive experimental wells. Following determination of the number of positive and negative wells per dilution of each T cell preparation, the data were analyzed by a limiting dilution computer program (LIDIA) (26,27) that provided both the Th1 frequency and the conformity of the input data with a single-hit Poisson model. Genital tract T cells from naive IL-10 ϩ/ϩ mice have a Th1 frequency of 15 (range, 9 -21).…”

Section: ) Micementioning

confidence: 99%

Suppression of Endogenous IL-10 Gene Expression in Dendritic Cells Enhances Antigen Presentation for Specific Th1 Induction: Potential for Cellular Vaccine Development

Igietseme

Ananaba

Bolier

et al. 2000

133

116

A new paradigm for designing vaccines against certain microbial pathogens, including Chlamydia trachomatis, is based on the induction of local mucosal Th1 response. IL-10 is an anti-inflammatory cytokine that exerts negative immunoregulatory influence on Th1 response. This study investigated whether biochemical modulation of endogenous IL-10 expression at the level of APCs is a practical strategy for enhancing the specific Th1 response against pathogens controlled by Th1 immunity. The results revealed that the high resistance of genetically engineered IL-10−/− (IL-10KO) mice to genital chlamydial infection is a function of the predilection of their APCs to rapidly and preferentially activate a high Th1 response. Thus, in microbiological analysis, IL-10KO mice suffered a shorter duration of infection, less microbial burden, and limited ascending infection than immunocompetent wild-type mice. Also, IL-10KO were resistant to reinfection after 8 wk of the primary infection. Cellular and molecular immunologic evaluation indicated that IL-10KO mice induced greater frequency of chlamydial-specific Th1 response following C. trachomatis infection. Moreover, IL-10KO APCs or antisense IL-10 oligonucleotide-treated wild-type APCs were potent activators of Th1 response from naive or immune T cells. Furthermore, both Ag-pulsed dendritic cells from IL-10KO mice and IL-10 antisense-treated dendritic cells from wild-type mice were efficient cellular vaccines in adoptive immunotherapeutic vaccination against genital chlamydial infection. These findings may furnish a novel immunotherapeutic strategy for boosting the Th1 response against T cell-controlled pathogens and tumors, using IL-10-deficient APCs as vaccine delivery agents.