Splenic but not thymic autoreactive T cells from New Zealand Black mice respond to a dominant erythrocyte Band 3 peptide

Abstract

Previous work from our laboratory suggested that erythrocyte Band 3 peptide 861-874 is the dominant epitope recognized by splenic T cells from adult New Zealand Black (NZB) mice that are developing autoimmune haemolytic anaemia (AIHA). Here, it is shown that splenic T cells from 6-week-old NZB mice mount a vigorous in vitro proliferative response to peptide 861-874 and some other selected Band 3 peptides. As the donors grow older, splenic T cells respond to an increasing number of Band 3 peptides and the magnitude of their response also becomes greater. Splenic T cells from 3-week-old NZB mice still responded vigorously to peptide 861-874 and Band 3. By contrast, neither thymocytes nor single-positive CD4-enriched thymus cells from NZB mice responded to peptide 861-874 or Band 3, although they responded to concanavalin A (Con A). However, thymocytes from mice expressing a transgenic T-cell receptor (TCR)-specific for myelin basic protein (MBP) peptide Ac 1-9 responded vigorously to Ac 1-9. It is considered that the T-cell response of NZB mice to Band 3 is initially focused on peptide 861-874 and later spreads to other Band 3 peptides as the disease progresses and that peptide 861-874-reactive T cells are primed in the periphery rather than the thymus.

Figure 1

Proliferative responses of splenic T cells derived from NZB mice of different ages to Band 3 and selected Band 3-derived peptides. Splenic T cells were obtained from 1·5-, 3-, 4·5- and 6-month-old NZB mice, and were cultured with syngeneic APC. Each bar represents the mean stimulation index (SI) of the peak responses. Each peptide was tested against two or three individual splenic T-cell pools. The dotted line indicates a SI of 3.

Figure 2

Kinetics of proliferation driven by peptide 861–874 and Band 3 in splenic T-cell cultures derived from two 3-week-old female NZB mice. Each point represents the mean of duplicates.

Figure 3

Proliferative responses of NZB thymocytes cultured with Band 3 or peptide 861–874. Thymocytes were pooled from two female 3-week-old NZB mice, and cultured with syngeneic APC (thymocytes, irradiated thymocytes, or irradiated spleen cells) in the presence of Band 3, peptide 861–874 or mitogen Con A. No antigen was added to the negative control wells. Data represent mean values of the responses on day 2 of culture, at which time the response to Con A peaked.

Figure 4

Proliferative responses to Ac 1–9 of thymocytes and splenocytes were derived from a male F₄ B10.PL transgenic mouse aged 10 weeks. These thymocytes were cultured with syngeneic APC (either irradiated thymocytes or spleen cells) with or without Ac 1–9 of MBP. Cell proliferation was determined daily from day 2 to day 4 of culture wherever possible by incorporation of [³H]thymidine over 6 hr. Peptide Ac 1–9 was used at a final concentration of 5 μg/ml. Data represent mean values of the peak responses on day 2 of culture.