Neo-epitopes are required for immunogenicity of the La/SS-B nuclear antigen in the context of late apoptotic cells

Abstract

Mechanisms responsible for the induction of anti-nuclear autoantibodies (ANA) following exposure of the immune system to an excess of apoptotic cells are incompletely understood. In this study, the immunogenicity of late apoptotic cells expressing heterologous or syngeneic forms of La/SS-B was investigated following subcutaneous administration to A/J mice, a non-autoimmune strain in which the La antigenic system is well understood. Immunization of A/J mice with late apoptotic thymocytes taken from mice transgenic (Tg) for the human La (hLa) nuclear antigen resulted in the production of IgG ANA specific for human and mouse forms of La in the absence of foreign adjuvants. Preparations of phenotypically healthy cells expressing heterologous hLa were also immunogenic. However, hLa Tg late apoptotic cells accelerated and enhanced the apparent heterologous healthy cell-induced anti-La humoral response, while non-Tg late apoptotic cells did not. Subcutaneous administration of late apoptotic cells was insufficient to break existing tolerance to the hLa antigen in hLa Tg mice or to the endogenous mouse La (mLa) antigen in A/J mice immunized with syngeneic thymocytes, indicating a requirement for the presence of heterologous epitopes for anti-La ANA production. Lymph node dendritic cells (DC) but not B cells isolated from non-Tg mice injected with hLa Tg late apoptotic cells presented immunodominant T helper cell epitopes of hLa. These studies support a model in which the generation of neo-T cell epitopes is required for loss of tolerance to nuclear proteins after exposure of the healthy immune system to an excess of cells in late stages of apoptosis.

Fig. 1

Redistribution of human La (hLa) antigen in hLa transgenic (Tg) thymocytes. (a) Confocal images showing morphological changes of hLa Tg thymocytes that were stained at various time-points following γ-irradiation (600 rad) with the SW5 anti-hLa-specific monoclonal antibody (mAb). No staining of control non-Tg thymocytes by SW5 mAb was observed. (b) Fluorescence activated cell sorter (FACS) dot-plots showing annexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining of portions of the irradiated cells from (a).

Fig. 2

Anti-La/SS-B responses elicited by human La (hLa) transgenic (Tg) or non-Tg late apoptotic thymocytes. (a) Detection of IgG antibodies binding to six-histidine fusion protein (6xhis-hLa), -mouse La (-mLa) and control -dihydrofolate reductase (DHFR) proteins by enzyme-linked immunosorbent assay (ELISA) in immune (days 14, 28, 42, 55) sera (1 : 50 dilutions) from non-Tg A/J mice immunized with hLa Tg or non-Tg late apoptotic thymocytes. (b) Detection of IgG antibodies binding to recombinant hLa-GST and control GST proteins by ELISA in preimmune (day 0) and immune (day 42) sera (1 : 100 dilutions) of hLa Tg and non-Tg mice immunized with hLa Tg late apoptotic thymocytes. (c) Detection of IgG antibodies binding to 6xhis-hLa and control -DHFR proteins by ELISA in preimmune (day 0) and immune (days 14, 28, 42) sera (1 : 100 dilutions) of (BALB/c × A/J)F₁ mice immunized with BALB/c-derived 3T3 cells transfected with a plasmid containing the hLa gene (hLa-3T3) or a control plasmid (c-3T3). (d) Lack of detection of anti-La antibodies in the sera of control mice serially immunized with phosphate-buffered saline (PBS) buffer alone.

Fig. 3

Anti-Ro antibodies occur only in the sera of mice that make antibodies to La. Non-transgenic (Tg) mice were immunized with human La (hLa) Tg thymocytes (hLa Tg), hLa transfected 3T3 cells (hLa-3T3), completely syngeneic apoptotic thymocytes (non-Tg) or 3T3 cells transfected with control plasmid alone (c-3T3). Individual serial serum samples were screened for IgG antibodies to purified bovine La (top panel) or 60 kDa Ro (bottom panel) by enzyme-linked immunosorbent assay. Data from individual mice are presented in the same order for each panel. Data from two independent experiments are shown.

Fig. 4

Late apoptotic cells expressing heterologous La/SS-B are immunogenic. A/J mice (five mice per group) were immunized on days 0, 10 and 24, with cell preparations containing numbers of the cell phenotypes listed below each panel and enclosed by the boxes, and serial serum samples were tested for IgG antibody reactivity to the hLa-GST antigen (top panel) or the control GST protein (bottom panel). N/A = none added; *injections in this group also contained 1 × 10⁷ non-transgenic (Tg) annexinV^–propidium iodide (PI)^– cells.

Fig. 5

Human La-specific T cell hybridomas KD9B9 and KD3C5. T cell hybridomas generated by immortalization of six-histidine fusion protein (6xhis-hLa)-primed lymph node (LN) T cells responded to the 6xhis-hLa antigen but not the 6xhis-mLa antigen or an irrelevant 6xhis-linked control protein. Proliferation of the interleukin (IL-4) and/or IL-2-dependent HT-2 cell line to T cell hybridoma supernatants is reported. Inset: Coomassie-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing the purity and expected mobility of the antigens used in this assay (2 µg protein per lane).

Fig. 6

Presentation of immunodominant T cell epitopes of human La (hLa) by lymph node (LN) dendritic cells (DC) from mice injected with hLa transgenic (Tg) late apoptotic thymocytes. (a) Positively selected CD11c + LN DC taken from mice immunized once with hLa Tg late apoptotic thymocytes (filled bars, left panel) or non-Tg late apoptotic thymocytes (open bars, left panel) were assayed for the ability to present immunodominant H-2^a-restricted T cell epitopes of the hLa antigen to hLa-specific T cell hybridomas. Peptide specificity of KD3C5 (3C5) for hLa 61–84 and KD9B9 (9B9) for hLa 288–302 was verified by culture of the hybridomas with the indicated synthetic peptides and irradiated syngeneic splenocytes from unimmunized A/J mice (right panel). Responses were assessed by measuring tritiated thymidine uptake of the HT-2 cell line when incubated with cell culture supernatants from T cell hybridomas cultured alone or with the indicated LN DC. *Indicates significant differences in responses compared to T cells cultured without DC or with DC exposed to late apoptotic cells in vivo (P < 0·05, Student's t-test). (b) CD11c⁺ DC and CD19⁺ B cells were purified from the draining LN of mice injected once with hLa Tg or non-Tg late apoptotic thymocytes, then assayed for the ability to present the hLa 61–84 immunodominant T cell epitope to the KD3C5 T cell hybridoma. Fluorescence activated cell sorter (FACS) histograms show anti-CD11c or -CD19 staining of LN cells before purification (open plots) or after purification (filled plots). Presentation of the immunodominant I-E^k-restricted T cell epitope recognized by the KD3C5 hybridoma by DC but not by B cells was detected (centre panel). Presentation of this epitope was inhibited in the presence of a blocking anti-I-E^k monoclonal antibody (mAb) but not by an isotype control mAb (right panel).