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To identify activated T cell subset in the asthmatic bronchia, we developed a triple-colour immunohistofluorescence labelling technique on cryo-section to discriminate activated CD4+ CD25+ T cells, (effector T cells) from Foxp3+ regulatory T cells ( Treg). Additional coexpression of activation and proliferation markers was also examined in situ. Bronchial biopsies were taken from 20 aluminium potroom workers (12 smokers) with asthma (>12% reversibility), 15 non-asthmatic potroom workers (7 smokers) and 10 non-smoking, non-exposed controls. Non-smoking asthmatics had significantly higher subepithelial density of both Tregs, effector T cells, activated ( HLA- DR+) CD8+ and activated CD4+ T cells. Moreover, both Tregs, effector T cells and CD8+ T cells proliferated in the non-smoking asthmatics, only. Although smoking asthmatics had no asthma-associated increase in bronchial T cell, both had a significantly increase in effector T cell to Treg ratios. The significantly increased bronchial density of Tregs, effector T cells, proliferative T cells and activated CD8+ T cells in non-smoking asthmatics clearly showed that both the effector T cells and the inhibitory Treg system were activated in asthma. [ABSTRACT FROM AUTHOR]

Salivary gland specimens from 10 patients with primary Sjögren's syndrome (pSS) were examined by two-colour immunofluorescence with various combinations of monoclonal and polyclonal antibody reagents of the following specificities; human leucocyte antigen (HLA) class I and II (DR, DP and DQ), CD3. CD45 (leucocyte common antigen), various cytokeratins, and factor VIII-related antigen. Tissue specimens from 10 normal glands and 10 glands with obstructive sialadenitis (no known autoimmunity) served as controls. Only some intercalated ducts and scattered acini of the normal major glands expressed HLA class II determinants( < 5% of total epithelial area); the relative proportion of positive elements indicated differential expression (DR > DP > DQ). SS glands contained substantial T cell infiltrates and increased numbers of activated (DR+ ) T cells; adjacent epithelium showed extensive differential expression of HLA class II determinants (DR >> DP > DQ). Glands with obstructive sialadenitis showed similarly increased epithelial expression of H LA-DR but with surprisingly small amounts of concomitant HLA-DP and -DQ expression. Epithelial HLA class 11 expression probably depends on cytokines as an inductive event, which is not unique for SS but particularly prominent in this disorder. Our results suggest that epithelial expression of HLA-DP or -DQ. rather than -DR. might be a prerequisite for the autoimmune process of SS to develop in genetically susceptible individuals. [ABSTRACT FROM AUTHOR]

Expression of the γ/δ T cell receptor (TCR) on CD3+ intraepithelial lymphocytes (IELs) was studied by two-colour immunofluorescence in duodenal tissue sections from healthy (n = 6) or infection-prone (n=7) subjects with selective IgA deficiency (IgAD), and subjects (n = 4) with combined IgAD and IgG subclass deficiency. TCRγ/δ+ IEL proportions in selective IgAD subjects (median 6.3%, range 1.0-41%.) and in those with combined deficiency (median 4.5%, range 1.2-33%) were well within the range (0.3-38%) for histologically normal controls (n=11), but the healthy IgAD subgroup tended to show raised TCRγ/δ+ IEL proportions (median 13.6%) compared with the other two subgroups. Also the number of TCRγ/δ+ IELs per intestinal length unit was relatively high (median 13.9/mm) in the healthy IgAD subjects, and significantly raised (P<0.03) compared with controls (median .12 'mm). Paired staining revealed that most TCRγ/δ+ IELs in both selective IgAD (98%) and combined deficiency (99%) were CD8-, and a large fraction (median 84% and 63%, respectively) expressed the Vδ1/Jδl-encoded epitope. The total number of CD3+ IELs (mostly CD8+) was similar to controls. IgAD subjects, and especially the healthy subgroup, had significantly increased serum concentrations of soluble CD8 (P<0.0002). neopterin (P<0.005), and β2-microglobulin (P<0.007), which was similar to our previous observations in common variable immunodeficiency, and probably reflected stimulation of cell-mediated immunity. In addition, the increased TCRγ/δ+ IELs might reflect a component of compensatory surface protection in the healthy IgAD subgroup. [ABSTRACT FROM AUTHOR]

A revived interest in intraepithelial lymphocytes (IEL) has been elicited by several recent reports suggesting that murine and avian intestinal epithelium contains mainly CD3+ CD8+ cells expressing the γ/δ T-cell receptor (TcR) for antigen; this contrasts with systemically distributed T cells which preferentially employ the TcRα/β. An anatomical dichotomy in the distribution of these two T-cell lineages has hence been proposed Here we report that this concept does not hold true in man. In situ studies with monoclonal TcR-framework antibodies showed that most (70-90%) human intestinal IEL (which are mainly CD3+CD8) expressed TcRα/β, Moreover, almost half of the intraepithelial CD3+ cells were positive for the smallest (180 kDa) CD45 molecule (UCHLl); this probably reflected that they are antigen-primed and thus represent traditional CD3+ CD8+ α/β+ memory T cells. [ABSTRACT FROM AUTHOR]

Interactions between homing receptors on circulating leucocytes and endothelial addressins regulate tissue-specific cellular extravasation. Although integrin α4β7 appears to be the main receptor for gut-homing T lymphocytes, less is known about molecules mediating mucosal B cell homing. Expression of integrin α4β7 on B lymphocytes, B cell blasts, and plasma cells in human gut-associated lymphoid tissue (GALT; the Peyer's patches and appendix) and lamina propria was studied by multi-colour immunofluorescence applied on cryosections. Isolated mononuclear cells from the same tissue compartments were examined by flow cytometry and compared with peripheral blood B cells. Integrin α4β7 was expressed by IgA+ B cell blasts and plasma ceils (CD38high) in the lamina propria, B cell blasts in GALT, and sIgD+ B lymphocytes in peripheral blood. In contrast, GALT sIgD+ B lymphocytes were negative or only weakly positive for β4β7. These results suggested that B lymphocytes down-regulate α4β7 upon extravasation in GALT but up-regulate this integrin after antigen-priming. Thus, α4β7 may be a homing receptor also for B cell blasts extravasating in the gut lamina propria, where this integrin is maintained on plasma cells, perhaps as a local retention factor. [ABSTRACT FROM AUTHOR]

Jejunal biopsy specimens from 10 patients with treated coeliac disease and seven non-coeliac controls were challenged in vitro with peptic-tryptic gluten digest. Mucosal T cells were examined in situ by three-colour immunofluorescence staining for expression of the activation marker CD25 (the p55 α-chain of interleukin-2 receptor) and the nuclear proliferation marker revealed by monoclonal antibody Ki-67. Intraepithelial T cells expressed CD25 rarely whereas the proportion of activated lamina propria T cells increased (P < 0.002) from median 2.8% (cultured with 20% fetal calf serum alone for 24-48 h) to 10.0% after 24 h with gluten (n = 10; range 1.1-17.4%) and to 10.4% after 48 h (n = 7; range 1.4-17.5%). Such gluten-induced increase of CD25+ T cells was not observed in specimens from non-coeliac control subjects. Crypt-cell hyperplasia and T-cell proliferation (Ki-67+) were observed neither in the coeliac nor in the control mucosae after gluten stimulation. Three-colour staining combining a polyclonal antibody reagent to CD3 and a monoclonal antibody to CD25 with a monoclonal antibody to CD45R0, CD4, CD8, the p75 β-chain of interleukin-2 receptor, integrin αEβ7, or HLA-DR showed that most of the CD25+ T cells (> 90%) were CD4+CD8-, co-expressed CD45R0 and the p75 β-chain, and often also the integrin αEβ7 but not HLA-DR. In addition to these activated T cells, a dominating population of CD25+ CD3- CD4+ subepithelial pan-HLA-class II+ macrophages (CD68+) with variable expression of the p75 β-chain was often induced by gluten challenge. [ABSTRACT FROM AUTHOR]

T-cell receptor γ/&delta+ (TcRγ/&delta+) lymphocytes in human Peyer's patches (PP) adjoining ileal mucosa were studied by monoclonal antibodies with paired immunofluorescence staining in situ and by flow-cytometric phenotyping of isolated cells. The proportion of γ/&delta+ T cells in the follicle-associated epithelium outside the M-cell areas (median 4.1%, range 2.2-30.1%) was similar to that in mucosal villous epithelium (median 4.4%, range 0.5-30.5%). Most intraepithelial γ/&delta) cells (∼90%) expressed CD45R0 but only a few expressed CD8 (< 10%) and none L-selectin; a dominating subset (median 46%) employed the V&delta1/J&delta1 gene product (range 22-100%). The M-cell areas lacked γ/δ cells but contained clustered CD20+ and CD3+ lymphocytes. The subepithelial PP dome area and interfollicular (T-cell) zones, as well as the mucosal lamina propria, contained very few γ/&delta cells (median 1.7%, range 0.4-8.9%.) which were dominated (88-100%) by the Vδ2-encoded subset. Those in the dome area and lamina propria were often (∼75%) CD45R0+ (range 44-90%) while very few (∼2%) expressed L-selectin (range 0-15%). By contrast, CD45R0 expression on γ/&delta cells in the PP T-cell zones was relatively low (∼46%) and that of L-selectin relatively high (∼43%). In conclusion, TcR γ/&delta+ cells are quite rare in human PP and belong mainly to the V&delta2-encoded subset, thus being different from most intraepithelial γ/&delta cells that probably have another origin. The L-selectin+ fraction of PP γ/&delta cells presumably represent newly recruited 'naive' T lymphocytes while CD45R0+ γ/&delta cells both in PP and lamina propria are probably antigen-primed. [ABSTRACT FROM AUTHOR]

Fetal (n = 20) and postnatial (n = 40) parotid glands were examined by two-colour immunohistochemistry combining monoclonal and polyclonal antibody reagents to study the expression of HLA class I and II (DR, DP, and DQ), CD45 and CD3. The epithelium kicked class II during fetal life, whereas class I determinants appeared in some acini and most major ducts. Fetal vessels were positive for both class I and class II (mainly DR), suggesting constitutive expression. Some class II-positive (DR > DP > DQ) histiocytic cells, scattered CD45+ leucocytes, and very few CD3+ T cells were present in the fetal stroma. The epithelium remained DR-negative the first few weeks after birth, but brisk expression was seen subsequently, DP and DQ remained virtually negative in the epithelium throughout the first year. A slight postnatal increase of class II expression (DR > DP > DQ), along with an apparent decrease in class I. was observed in the endothelium. The number of class II-positive histiocytic cells. CD45+ leucocytes and CD3+ T cells, as well as the proportion of presumably activated (DR+) T cells, increased a few weeks after birth, The local immune system hence seemed to be stimulated by extrinsic factors, but the overall number of T cells nevertheless remained small. Stimuli other than T cell-derived lymphokines. therefore, probably explained the brisk postnatal epithelial DR induction. [ABSTRACT FROM AUTHOR]