Signal regulatory protein alpha (SIRPalpha) cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)alpha (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+) (SIRPalpha-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1). Sorted CD172a(+) cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+), CD172a(+), and CD3(+) cells, including CD172a-expressing multi-nucleated giant cells. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+) cells, bind to CD172a(+) cells, and induce multi-nucleated giant cells expressing CD172a.

An ESAT-6:CFP10 DNA vaccine administered in conjunction with Mycobacterium bovis BCG confers protection to cattle challenged with virulent M. bovis.

The potency of genetic immunization observed in the mouse has demonstrated the utility of DNA vaccines to induce cell-mediated and humoral immune responses. However, it has been relatively difficult to generate comparable responses in non-rodent species. The use of molecular adjuvants may increase the magnitude of these suboptimal responses. In this study, we demonstrate that the co-administration of plasmid-encoded GM-CSF and CD80/CD86 with a novel ESAT-6:CFP10 DNA vaccine against bovine tuberculosis enhances antigen-specific cell-mediated immune responses. ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinated animals exhibited significant (p<0.01) antigen-specific proliferative responses compared to other DNA vaccinates. Increased expression (p< or =0.05) of CD25 on PBMC from ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinates was associated with increased proliferation, as compared to control DNA vaccinates. Significant (p<0.05) numbers of ESAT-6:CFP10-specific IFN-gamma producing cells were evident from all ESAT-6:CFP10 DNA vaccinated animals compared to control DNA vaccinates. However, the greatest increase in IFN-gamma producing cells was from animals vaccinated with ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA. In a low-dose aerosol challenge trial, calves vaccinated as neonates with Mycobacterium bovis BCG and ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA exhibited decreased lesion severity in the lung and lung-associated lymph nodes following viruluent M. bovis challenge compared to other vaccinated animals or non-vaccinated controls. These data suggest that a combined vaccine regimen of M. bovis BCG and a candidate ESAT-6:CFP10 DNA vaccine may offer greater protection against tuberculosis in cattle than vaccination with BCG alone.

In vitro effects of 1,25-dihydroxyvitamin D3 on interferon-gamma and tumor necrosis factor-alpha secretion by blood leukocytes from young and adult cattle vaccinated with Mycobacterium bovis BCG.

Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are critical in the development of an effective immune response. Vitamin D, essential in short-term calcium homeostasis and recently shown to modulate proliferation and function of blood mononuclear cells from adult dairy cattle, may be an effective modulator of the calf's immune system. Effects of antigen sensitization and 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] on cytokine secretion by cells from calves vaccinated with Bacille Calmette-Guérin (BCG) were examined. One-week-old dairy calves (n = 6) and yearling heifers (n = 4) were vaccinated concurrently with BCG and boosted six weeks later. Ten weeks after primary vaccination, cells from vaccinated calves and adults, and nonvaccinated, age-matched calves (n = 4) were evaluated in vitro for their capacity to produce IFN-gamma and TNF-alpha. Cells were stimulated with pokeweed mitogen (PWM) or recall antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] in the presence of 0, 0.1, 1.0, and 10 nM of 1,25-(OH)2D3 for 20, 44, and 68 hours, respectively. IFN-gamma and TNF-alpha concentrations in culture supernatants harvested at these times were quantified by enzyme-linked immunosorbent assay (ELISA). PPD-induced IFN-gamma and TNF-alpha responses of cells from vaccinated calves and adults were greater than responses of autologous unstimulated cells. In contrast, PPD-specific responses of calf and adult cells collected immediately before primary vaccination were substantially lower and comparable to responses in resting (i.e., unstimulated) cultures. At ten weeks, the PPD-specific response of vaccinates exceeded the response of nonvaccinated calves; however, responses of vaccinated calves were more vigorous than corresponding responses of vaccinated adults. Incubation period also influenced the magnitude of both IFN-gamma and TNF-alpha, responses in PPD- and PWM-stimulated cultures. Effects of 1,25-(OH)2D3 on antigen-induced secretion of IFN-gamma and TNF-alpha were marginal. Only IFN-gamma responses of vaccinated adults were affected by 1,25-(OH)2D3. Vitamin D caused a concentration-dependent decrease in IFN-gamma response and an increase in TNF-alpha response in PWM-stimulated cultures. These results indicate that animal maturity (i.e., age) and antigenic experience affect IFN-gamma and TNF-alpha secretion by bovine leukocytes and suggest that 1,25-(OH)2D3 can alter secretion of both cytokines under specific conditions of culture.

Effects of mastectomy on composition of peripheral blood mononuclear cell populations in periparturient dairy cows.

There is an increased incidence of infectious disease in periparturient dairy cows. During the periparturient period there is a decline in T-lymphocyte cell subsets, which parallels a reduction in functional capacities of blood lymphocytes and neutrophils. Mechanisms responsible for these changes in immune function during the periparturient period are poorly characterized. Ten mastectomized and eight intact multiparous Jersey cows were used to determine whether the periparturient changes in peripheral blood mononuclear cell populations are the result of the physiological demands associated with the onset of lactation or whether they are a result of the act of parturition. Blood mononuclear cells were phenotyped with monoclonal antibodies against T-cell subsets, B-cells, and monocytes. Blood samples were taken frequently from before 4 to 4 wk after parturition. In intact cows, all T-cell subset populations (i.e., CD3-, CD4-, CD8-, and gamma-delta positive cells) decreased at the time of parturition, while the percentage of monocytes increased. Mastectomy eliminated the changes in leukocyte subset populations (CD3-, CD4-, and gamma-delta positive cells, and monocytes) observed in intact cows around parturition. These results indicate that the mammary gland and metabolic stresses associated with lactation influence the composition of peripheral blood mononuclear cell populations in dairy cows during the periparturient period.