Clustering of distinct autoimmune diseases associated with functional abnormalities of T cell survival in children

Abstract

To ascertain whether alterations of lymphocyte switching off may be associated with clustering of autoimmune diseases in children, Fas- and C2-ceramide-induced cell death was evaluated on T cell lines derived from three patients affected by clustering of autoimmune disorders. Three patterns were found: patient 3 was resistant to Fas- and C2-ceramide, patient 1 was resistant to Fas, but sensitive to C2-ceramide, patient 2 was resistant to C2-ceramide, but sensitive to Fas. By contrast, Fas- and C2-ceramide-induced cell death was normal in five children with systemic juvenile rheumatoid arthritis, five children with insulin-dependent diabetes and 10 age-matched healthy controls. Surface expression of Fas was low in patient 1, but normal in patients 2 and 3. Together with normal Fas transcripts, patients 2 and 3 displayed a transcript 152 bp longer than the normal one retaining intron 5. Our data indicate that polyreactive autoimmune syndromes may be associated with heterogeneous alteration of the immune response switching-off system.

Fig. 1

Cell survival on treatment with anti-Fas MoAb, C2-ceramide, or methylprednisolone (PDN) of T cell lines derived from the three patients with polyreactive autoimmunity, five controls with systemic juvenile rheumatoid arthritis (JRA; ▴), five controls with insulin-dependent diabetes mellitus (IDDM; •) and 10 age-matched normal controls (Δ). Long-term T cell lines were treated with the indicated reagent and survival was assessed after 18 h. Results are expressed as specific cell survival percentage. In the patients' group, each point represents the mean percentage value of three distinct experiments. Vertical bars indicate 2 s.d. The horizontal lines indicate the upper limit of the normal range calculated as the mean + 2 s.d. from data obtained from 65 non-age-matched normal donors. ○, Patient 1; ▪, patient 2; □, patient 3.

Fig. 2

Fas expression in the long-term T cell lines derived from the three patients with polyreactive autoimmunity and the sister of patient 2. Fas expression was evaluated by direct immunofluorescence and cytofluorometric analysis. Quadrants show the cytofluorometric plots (negative control and Fas staining) from the indicated subject. In each quadrant, the number indicates the median fluorescence intensity ratio (MFI-R), calculated as described in PATIENTS and METHODS. The mean ± s.d. of MFI-R obtained from T cell lines derived from 10 age-matched controls was 18 ± 8 (range 5·6–33·4).

Fig. 3

Fas transcripts identified in patients 2 and 3. (a) Schematic depiction of transcripts identified in patients 2 and 3 and the encoded protein. The insertion of intron 5 would, if translated, originate a truncated protein lacking exons 6–9, but including 32 new amino acids after glycine 169. A stop codon is located at base 99 of the intronic sequence. Exonic sequence is indicated in capital letters, intronic sequence in lower case. (b) Polymerase chain reaction (PCR) products obtained using primers E and F [21] from a normal donor (lane 1), patient 2 (lane 2), patient 3 (lane 3), normal brother of patient 3 (lane 4), and patient 1 (lane 5). Patient 2, patient 3 and his healthy brother show fragments of 211 bp (normal) and 363 bp (abnormal). Sequencing showed that the 363-bp fragment includes read-through of intron 5. The PCR product from patient 1 was obtained after two rounds of PCR: nevertheless, the 211-bp band intensity is lower than the others (the multiple spurious bands in this lane are due to the two rounds of PCR). M, Marker ladder.