Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals - PubMed (original) (raw)

. 2005 Aug;17(8):1131-40.

doi: 10.1093/intimm/dxh275. Epub 2005 Jul 19.

Cheng-Rong Yu, Rashid M Mahdi, Daniel C Douek, Gregory B Dirusso, Frank M Midgley, Rajpreet Dogra, Gloria Allende, Eliot Rosenkranz, Alberto Pugliese, Charles E Egwuagu, Igal Gery

Affiliations

Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals

Hiroshi Takase et al. Int Immunol. 2005 Aug.

Abstract

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)-PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RT-PCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules.

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Figures

Fig. 1

Fig. 1

Detection of mRNA transcripts of the four retinal antigens in human thymi. (A) RT–PCR reactions were performed as detailed in Methods, using total RNA from the 18 human thymus samples. The thymic samples were tested for the β-actin transcript by 30 cycles of PCR or by 30, 33, 37 and 40 cycles for the four retinal antigens and the controls. The controls included ‘R’, an RNA preparation of normal human retina, and ‘N’, negative control with no cDNA. (B) The calculated intensity levels of the RT–PCR bands of individual thymic samples when tested for transcripts of the four retinal antigens by 37 cycles of PCR. The intensity levels were measured using NIH Image, followed by compensating the relative intensity with both β-actin and positive control (‘R’) bands.

Fig. 2

Fig. 2

Detection of mRNA transcripts of the four melanocyte antigens in human thymi. (A) RT–PCR reactions were performed as detailed in Methods, using total RNA from 17 human thymus samples (thymic sample no. 5 was not tested for these transcripts). The samples were tested for the β-actin transcript by 30 cycles and for the melanocyte transcripts and controls by 35 or 40 cycles of PCR. The controls included ‘R’, an RNA preparation of normal human retina, ‘N’, negative control without cDNA, and ‘M’, RNA preparation from the melanoma cell line ‘1088 mel’. Each antigen was tested on a double comb gel. (B) The calculated intensity levels of the RT–PCR bands of the thymic samples when tested by 40 PCR cycles. The intensity levels were measured using NIH Image, followed by compensating the relative intensity with both β-actin and the positive melanocyte line control.

Fig. 3

Fig. 3

Reproducibility of the RT–PCR assay is confirmed. Three tissue pieces (‘a’, ‘b’ and ‘c’) were collected from different areas of each of thymic samples ’1’, ‘2’ and ‘3’ and total RNA extracted from each of these pieces was tested by RT–PCR for transcripts of β-actin, IRBP and RPE65, using 30 PCR cycles for the β-actin and 40 PCR cycles for the antigens, as detailed in Methods. Please note the close similarity between the pattern of response among the tissue pieces of the three thymic samples, as well as between these responses and the corresponding responses shown in Fig. 1(A).

Fig. 4

Fig. 4

A comparison by quantitative PCR between the levels of S-Ag transcript in human retina and thymus. RNA preparations of human thymus sample no. 18 and a normal human retina were tested by real-time PCR assay, as described in Methods. Relative gene expression levels of S-Ag in the thymus was calculated to be 1.4 × 105-fold lower than that of the human retina.

Fig. 5

Fig. 5

Thymic expression of retinal antigens shown by immunostaining. (A) Frozen thymus sections were stained with rabbit antibodies against S-Ag, IRBP or RPE65, as described in the Methods. The original magnification values, ×20 or ×60, are indicated. The predominant location of stained cells is in the medulla (‘M’) or corticomedullary junction, but not in the cortex (‘C’). No staining was detected in the negative controls, sections stained with no primary antibody. (B) Immunostaining of positive cells is blocked by the specific antigen. Serial sections of thymic tissue were stained with mAb against S-Ag (left panel) or with the antibody following its incubation for 1 h with S-Ag (right panel). A positive staining of a cell in the medulla (white arrow) is eliminated by blocking with the antigen. (C) Identification of positively stained cells to be thymic epithelial cells. A thymic section was double stained with antibodies against RPE65 and against cytokeratin (‘AE3’), as detailed in the Methods. A cell positive for RPE65 (orange) also stained for cytokeratin (green) and produced the yellow color when the two colors were merged.

References

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