The head-associated lymphoid tissues of the chicken, composed of the harderian gland and the conjunctiva-associated lymphoid tissue (CALT), were studied to determine whether changes occurred in lymphocyte subpopulations as chickens age from 1 week to 8 weeks. The B cells and subpopulations of T-lymphocytes of the head-associated lymphoid tissues were identified using in situ immunohistochemical staining. Monoclonal antibodies specific for various lymphocyte surface antigens were used. The concentration of T-lymphocytes, particularly CD3+ and CD4+ cells, within the harderian gland increased with age, whereas the concentration of B cells remained the same. B-lymphocytes were observed within the germinal centers of the CALT of 4-week-old birds. The T-lymphocytes within the CALT surrounded the B-cell-rich germinal centers. CD3+ T-lymphocytes were the predominant cell population in all age groups examined. Increasing concentrations of CD3+, CD4+, and CD8+ T-lymphocytes were observed within the CALT as chicks developed from 1 week to 4 weeks of age. Finally, no changes were observed in lymphocyte populations within the CALT as chicks developed from 4 weeks to 8 weeks of age.

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A blastogenesis microassay employing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was adapted to measure blastogenic responses of lymphocytes from the chicken's head-associated lymphoid tissues (i.e., the harderian gland and conjunctiva-associated lymphoid tissue) to T- and B-cell mitogens. Lymphocytes isolated from peripheral blood, spleen, and the harderian gland had highly significant (P < 0.01) responses to T- and B-cell mitogens compared with control lymphocytes cultured without mitogens. Cultured lymphocytes obtained from the harderian gland had highly significant mitogenic responses to the T-cell mitogen concanavalin A (25 to 100 micrograms/ml) and to the B-cell mitogen Salmonella typhimurium lipopolysaccharide (1.25 to 5.0 micrograms/ml) compared with the control lymphocytes. Mitogenic responses of cultured lymphocytes obtained from the conjunctiva-associated lymphoid tissue could not be measured within the given parameters of the blastogenesis microassay. This was primarily due to the low yield of lymphocytes, which proved to be a limiting factor. The ability of the MTT blastogenesis microassay to detect blastogenic responses of the harderian gland to mitogens may be indicative of its usefulness for measuring cell-mediated immunity responses to other antigens.

Birds Experimental design Statistical analysis RESULTS In vitro lymphoproliferation responses of systemic lymphocytes .... Ill In vitro lymphoproliferation responses of Harderian gland lymphocytes In situ immunocytochemistry of the HALT DISCUSSION 122

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