Beryllium Skin Patch Testing to Analyze T Cell Stimulation and Granulomatous Inflammation in the Lung

Abstract

Chronic beryllium disease (CBD) is characterized by granulomatous inflammation and the accumulation of CD4+ T cells in the lung. Patch testing of CBD patients with beryllium sulfate results in granulomatous inflammation in the skin. We investigated whether the T cell clonal populations present in the lung of CBD patients would also be present in the involved skin of a positive beryllium patch test and thus mirror the granulomatous process in the lung. CBD patients with clonal TCR expansions in bronchoalveolar lavage (BAL) were selected for study. All three CBD patients studied had a positive response to beryllium sulfate application and a negative patch test to normal saline. Immunohistochemistry showed extensive infiltration with CD4+ T cells and few, if any, CD8+ T cells both at 3 days and at later times when granulomas were apparent. T cell infiltration early after skin testing appeared to be nonspecific with the TCR repertoire of infiltrating T cells being distinct from that present in BAL. At later times when granulomas were present, T cell clones in skin overlapped with those in BAL in all patients tested. Total TCR matches in skin and BAL were as high as 40% in selected Vβ T cell subsets. Studies of peripheral blood T cells before and after patch testing provided evidence for mobilization of large numbers of pathogenic beryllium-reactive T cells into the circulating pool. These studies using skin patch testing provide new insight into the dynamics of T cell influx and mobilization during granulomatous inflammation.

Oligoclonal CD4+T Cells in the Lungs of Patients with Severe Emphysema

“…The tissue was agitated at 37ЊC for 90 minutes, passed through mesh, and washed with balanced salt solution. Mononuclear cells were isolated from the pulmonary cell suspension and from heparinized blood using Ficoll-Hypaque density gradient separation as previously described (14)(15)(16).…”

“…These same cells were stained with mAbs directed against 16 different TCR-V␤ regions as previously described (see online supplement) (16,17). The labeled cells were analyzed using a FACScalibur cytometer (Becton Dickinson, San Jose, CA).…”

“…RNA was isolated using a commercially available kit (RNAid Plus; BIO 101, La Jolla, CA). cDNA was prepared and polymerase chain reaction amplification was performed as previously described (14,16). Each polymerase chain reaction product was ligated, transformed, and sequenced as previously described (see online supplement) (14,16).…”

Influence of MHC CLASS II in Susceptibility to Beryllium Sensitization and Chronic Beryllium Disease

“…These CD4 ϩ T cells express a biased repertoire of TCRs, confirming the specificity of the Be-stimulated response in CBD (7,8). Evaluating these TCRs in the development of granulomas after Be skin patch testing of CBD subjects suggests that clonal T cells, similar to those found in the lung, are mobilized from the blood and infiltrate the affected tissue during granuloma formation (9). In early studies, Abs to MHC class II but not class I molecules blocked the ability of Be to induce CD4 ϩ T cell proliferation (6).…”

Regulatory Role of γδ T Cells in the Recruitment of CD4+ and CD8+ T Cells to Lung and Subsequent Pulmonary Fibrosis

“…For example, in berylliosis, large populations of beryllium-specific CD4 ϩ T cells are recruited to the lung in response to the persistent presence of the beryllium Ag (11,30,31,37). These T cells recognize beryllium in an MHC II-restricted manner (38 -40), and their recruitment to lung precedes the development of granulomatous inflammation (41). Although berylliosis is considered a progressive disease with one-third of affected subjects historically progressing to respiratory insufficiency (42), the specific role of beryllium-specific CD4 ϩ T cells in the development of fibrosis is unclear.…”