Berylliosis Candidates for Intervention in Chronic and Anti-LFA-1

The Journal of Immunology

Activation Pathways Implicate Anti-HLA-DP and Anti-LFA-1 Antibodies as Lead Candidates for Intervention in Chronic Berylliosis1

Yuan K. Chou,2*§ David M. Edwards,2* Andrew D. Weinberg,¶ Arthur A. Vandenbark,*‡§ Brian L. Kotzin,ʈ Andrew P. Fontenot,ʈ and Gregory G. Burrows3*†

CD4؉ T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4؉ T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the -presence of APCs, beryllium induced strong proliferation responses of BAL CD4؉ T cells, production of superoptimal concen trations of secreted proinflammatory cytokines, IFN-␥, TNF-␣,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4؉ T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4؉ T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4؉ T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease. The Journal of Immunology, 2005, 174: 4316–4324.

etailed characterization of resident T cells in tissues un- extremely vague, and in the majority of cases a consensus is far dergoing chronic inflammation presents a number of dif- from being established. D ficulties, not the least being the inaccessibility of the Chronic beryllium disease (CBD)4 is an occupational disease target organ. T cells involved in autoimmune disease of the CNS caused by exposure to the metal beryllium (molecular weight ϭ 9), (multiple sclerosis) and pancreas (type 1 diabetes) present extreme characterized by the formation of noncaseating granuloma and examples of this inaccessibility (1, 2). In addition, studies of mononuclear cell infiltration into the lung (8, 9). Immunological chronic inflammation and the characterization of the molecular studies have suggested that CD4ϩ T cells are important in the players involved have primarily focused on protein-derived pep- initiation and perpetuation of the beryllium-induced chronic in- tide Ags. However, T cell activation and pathogenesis can also be flammation (10, 11). Recognition of beryllium by CD4ϩ T cells in triggered by a wide variety of low molecular weight Ags, including CBD patients appears to involve certain HLA class II molecules, metals, frequently resulting in allergic hyperreactivity (3, 4) and and specific HLA-DP molecules have been associated with beryl- the ability of metals to induce T cell activation in a MHC-restricted lium sensitization and CBD severity (12–14). Beryllium-specific manner has been clearly established (5). Although several inter- CD4ϩ T cells have been found to express a Th1-type phenotype in esting models explaining this metal-induced activation of T cells response to beryllium sulfate (BeSO4) (15, 16), and nearly all have been proposed, a detailed understanding of the precise nature bronchoalveolar lavage (BAL) T cells from patients with CBD of the epitope recognized by metal-specific T cells remains in al- exhibit an effector memory cell phenotype (17–19). The develop- most all cases unknown (6, 7). Furthermore, the detailed immu- ment of granulomatous inflammation is associated with the accu- nological characteristics defining the T cell response to metals is mulation of activated CD4ϩ T cells in the BAL (20). The majority of beryllium-specific CD4ϩ T cells are compartmentalized in the lung, with up to 20–30% of the BAL CD4ϩ T cells being beryl- Departments of *Neurology, †Biochemistry and Molecular Biology, and ‡Molecular lium specific and expressing Th1-type cytokines upon beryllium Microbiology and Immunology, Oregon Health & Science University, Portland, OR 97239; §Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR exposure in culture (17). In a number of CBD patients studied, the 97207; ¶Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, T cells showed oligoclonal expansion with particular TCRs spe- ʈ Providence Portland Medical Center, Portland, OR 97213; and Department of Med- cific for CBD (21), and analysis of TCR expression on BAL CD4ϩ icine and Immunology, University of Colorado Health Sciences Center, Denver, CO 80206 T cells has revealed the presence of TCR motifs specific for be- Received for publication October 8, 2004. Accepted for publication January 24, 2005. ryllium-reactive T cells (22). We have chosen to study CBD and ϩ The costs of publication of this article were defrayed in part by the payment of page beryllium-induced molecular changes in CD4 T cells both as a charges. This article must therefore be hereby marked advertisement in accordance model of metal-induced T cell activation in the lung and as an with 18 U.S.C. Section 1734 solely to indicate this fact. opportunity to characterize resident CD4ϩ T cells from tissues 1 This work was supported by the National Institutes of Health Grants ES10554 (to undergoing chronic inflammation. Our long-term goal is to map G.G.B), HL062410, and P01ES011810 (to A.P.F.) and by the Department of Neu- rology, OHSU, and the Veterans Affairs Medical Center (Portland, OR). the cascade of events that control the resident T cell populations’ 2 Y.K.C. and D.M.E. contributed equally to this project. 3 Address correspondence and reprint requests to Dr. Gregory G. Burrows, Depart- 4 Abbreviations used in this paper: CBD, chronic beryllium disease; AMLR, autolo- ment of Neurology, UHS-46, Oregon Health & Science University, 3181 SW Sam gous MLR; BAL, bronchoalveolar lavage; CBA, cytokine bead array; MOG, myelin Jackson Park Road, Portland, OR 97239. E-mail address: address:[email protected] oligodendrocyte glycoprotein; OX40L, OX40 ligand; SI, stimulation index.

behavior as a means of obtaining new insights focused on how to the same concentration range as that used for autologous APCs. As irrel- prevent this chronic inflammatory and eventually debilitating evant Ag controls, NiSO4 (Sigma-Aldrich) and the myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (New England Peptide) were used. disease. ϩ Morphological features of BAL CD4 T cell activation in the culture, with With regard to Ag-specific T cell activation, naive T cells appear either EBV-transformed autologous APCs or DAP3(DP2)-transfected ␮ to require two signaling events, TCR engagement with Ag/MHC APCs upon stimulation with 10 M BeSO4, were studied by microscopy and a second costimulatory signal, which is most frequently me- (Leica Microsystems). diated by the engagement of CD28 on the T cell with its ligands Cell surface marker phenotyping B7-1 or B7-2, on APCs (23, 24). The BAL beryllium-specific ϩ CD4ϩ T cells are effector memory cells that proliferate and secrete BAL CD4 T cell surface marker phenotyping in an unstimulated state was followed by stimulation with 10 ␮M BeSO at 24, 48, 72, 96, 120, and cytokines after TCR engagement without an absolute requirement 4 144 h in the presence of EBV-transformed autologous APCs and IL-2. All for CD28-mediated costimulation (25). This result suggested either Abs were purchased from BD Pharmingen and BD Biosciences, with the no dependence on costimulation or the involvement of a different exception of anti-CD28 (CALTAG Laboratories) and anti-OX40L (R&D costimulation pathway such as OX40/OX40 ligand (OX40L). Systems). All markers were costained with either CD4-FITC or CD4-PE OX40 (CD134), a membrane-bound member of the TNFR super- (clone RPA-T4; catalogues nos. 555346 and 555347, respectively). The ϩ surface markers were stained with the following mAbs: CD3-PE (clone family, is expressed primarily on activated CD4 T cells (26). UCHT1; catalogue no. 555333), CD8-PE (clone RPA-T8; catalogue no. Following engagement of OX40 with OX40L expressed on the 555367), CD45RO-FITC (clone UCHL1; catalogue no.555492), APC, OX40 delivers a costimulatory signal that leads to potent, ␣␤TCR-PE (clone T10B9.1A-31; catalogue no. 555548), CD25-PE (clone proinflammatory effects (27, 28). However, whether or not human M-A251; catalogue no.555432), CD69 (clone FN50; catalogue no. beryllium-specific CD4ϩ T cells expressed OX40 and, if so, 555529), CD134-PE (OX40) (clone L106; catalogue no. 340420), OX40L (TNFSF4) (clone 159403; catalogue no. MAB10541), CD28 (clone 15E8; whether the OX40/OX40L costimulatory pathway could play a catalogue no. MHCD2800–4), CD152-PE (CTLA-4) (clone BNI3; cata- ϩ role in beryllium-induced CD4 T cell activation has not been logue no.555853), CD80 (B7-1) (clone L307.4; catalogue no. 557227), explored previously. CD86 (B7-2) (clone 2331; catalogue no. 555658), CD50 (ICAM-3) (clone In the current study, we evaluated a beryllium-reactive T cell TU41; catalogue no. 555957), CD11a (LFA-1) (clone HI111; catalogue no. 555382), HLA-DR (clone L243, catalogue no. 347360), HLA-DP (clone line from the BAL of a CBD patient for the level and the duration B7/21; catalogue no. 347730), and HLA-DQ (clone SK10; catalogue no. of T cell proliferation and cytokine secretion, MHC restriction, and 347450). EBV-transformed APCs and DP2- or DP4-transfected APCs were changes in the expression of T cell adhesion molecules, activation stained appropriately with CD-19-PE (clone HIB19; catalogue no. markers, and costimulatory molecules upon stimulation with be- 555413), CD11b-PE (Mac-1) (clone ICRF44; catalogue no. 555388), ryllium. These studies substantially enhance our understanding of CD209 (DC-SIGN) (clone DCN46; catalogue no. 551186), CD11a (LFA- 1), and anti-HLA-DR, -DP, and -DQ mAbs. Purified mAbs were labeled the immunological features of beryllium-induced activation of with secondary pan-IgG PE polyclonal Ab (catalogue no. 550589). Each ϩ BAL CD4 T cells and provide new data suggesting potential staining sample, with 100,000 cells, was stained appropriately with Abs for therapeutic avenues for intervention in CBD. 30 min at 4°C. After washing three times with sterile PBS supplemented with 2% FBS (pH 7.2), the cells were analyzed using CellQuest software on a FACScan (BD Biosciences). Cell viability was monitored using An- Materials and Methods nexin VPE and 7-aminoactinomycin D from Apoptosis Detection Kit I (cat- Generation of beryllium-specific BAL CD4ϩ T cells and APCs alogue no. 559763; BD Pharmingen) on FACScan. The BAL CD4ϩ T cell line from a representative HLA-DP2 homozygous Cytometric bead array (CBA) assay used to detect and quantify CBD patient was developed as previously described (29). Briefly, the cytokine production diagnosis of CBD was established using previously defined criteria, including a history of beryllium exposure, the presence of granulomatous The CBA assay was performed using the human Th1/Th2 cytokine II CBA inflammation upon lung biopsy, and a positive beryllium lymphocyte-pro- kit (catalogue no. 551809; BD Biosciences), which quantifies the following liferation test (30, 31). The BAL cells from an HLA-DP2 homozygous cytokines: IL-2, IL-4, IL-6, IL-10, IFN-␥, and TNF-␣. CBA cytokine quan- CBD patient were collected and briefly stimulated with 10 ␮M(1ϫ 10Ϫ5 tification assay sensitivity for each cytokine is listed as follows: IL-2 Ϯ Ϯ Ϯ M) BeSO4 (Sigma-Aldrich) in the presence of EBV-transformed autolo- (2.6 0.2 pg/ml), IL-4 (2.6 0.2 pg/ml), IL-6 (3.0 0.2 pg/ml), IL-10 gous APCs at a ratio of 1:1 (T:APC) for 3 days, followed by the addition (2.8 Ϯ 0.2 pg/ml), IFN-␥ (7.1 Ϯ 0.3 pg/ml), and TNF-␣ (2.8 Ϯ 0.2 pg/ml). of 10 ng/ml rIL-2 (R&D Systems) twice weekly. The induction of EBV- To obtain an accurate value for absolute IFN-␥ production within the limits transformed APCs from patient’s PBMC was achieved by using the meth- of the CBA system, a 1/25 dilution of culture supernatants was necessary. ods as previously reported (32, 33). HLA typing was performed by stan- Briefly, 50 ␮l of supernatant, 50 ␮l of a mixture of capture beads, and 50 dard molecular techniques as previously described (29). ␮l of Th1/Th2 PE detection reagent were combined and incubated for 3 h at room temperature in the dark. The samples were thoroughly washed and BAL CD4ϩ T cell line proliferation assay subsequently resuspended in 200 ␮l of wash buffer and analyzed by flow ϩ cytometry (FACScan). Standard curves were generated for each cytokine The BAL CD4 T cell line was selected by stimulating BAL cells with 10 using a mixed bead standard to interpolate the concentration of cytokine in ␮ M BeSO4 in the presence of irradiated (9000 rad) EBV-transformed au- the supernatant in picograms per milliliter. tologous APCs at a ratio of 1:1 in culture medium of RPMI 1640 with 10% FBS supplemented with 2 mM L-glutamine,1 mM sodium pyruvate, 0.2 Blocking T cell response to beryllium by using mAbs and mM nonessential amino acids (Invitrogen Life Technologies), 100 ␮g/ml recombinant CTLA-4Ig penicillin G, and 100 ␮g/ml streptomycin (Invitrogen Life Technologies) followed by the addition of 10 ng/ml rIL-2 twice weekly. The proliferation The purified forms of anti-OX40 (CD134), anti-OX40L (TNFS4 Ab), anti- assay was performed using 4 ϫ 104 T cells/well in triplicate in 96-well CD28, anti-CTLA-4 (CD152), anti-HLA-DR, -DP, and -DQ were dialyzed U-bottom culture plates plus irradiated (9000 rad) EBV-transformed au- against RPMI 1640 with three buffer changes to remove any potential

tologous APCs at a ratio of 1:1, alone or in the presence of BeSO4,ata cytotoxic sodium azide. All blocking Abs were subsequently tested in a concentration ranging from 0.2 to 80 ␮M, for 3 days with 0.5 ␮Ci [3H]thy- proliferation assay with PBMCs stimulated with Con A as a control to midine (Amersham) incorporation for the last6hoftheculture, before verify that no residual sodium azide remained. In our mAb blocking pro- ␮ ϫ 4 harvesting. The radioactivity was determined by a liquid scintillation liferation assay, T cells were stimulated with 10 M BeSO4 at 4 10 counter 1205 Betaplate (Wallac) and proliferation was expressed by either cells/well plus irradiated EBV-transformed autologous APCs at a ratio of stimulation index (experimental cpm Ϭ control cpm) or net cpm (experi- 1:1 in triplicate in the presence or absence of mAbs at concentrations mental cpm Ϫ control cpm). Student’s t test was used for determining ranging from 2 to 25 ␮g/ml for 3 days, followed by [3H]thymidine incor- statistical signiﬁcance between different cpm (mean Ϯ SD) values. The poration for the last6hoftheculture. Percent inhibition was calculated by transfected mouse L cell line DAP3(DP2) encoding HLA-DP2 genes was subtracting the cpm of mAb treated samples from cpm of 10 ␮M berylli- also used as APCs, irradiated (4500 rad) (33), and cocultured with CD4ϩ um-stimulated cells with no mAbs, followed by dividing with the cpm of ϫ T cells at a ratio of 1:1 for 3 days, alone or in the presence of BeSO4,at no mAb 100%. Recombinant human CTLA-4Ig (catalogue no. 325-CT; 4318 ACTIVATION PATHWAYS IN CHRONIC BERYLLIOSIS

Ͼ ϫ ␮ R&D Systems) was prepared in aliquots of RPMI 1640/1% serum and a maximal stimulation index (SI) 11 at 20 M BeSO4. The ensured of noncytotoxicity in a Con A stimulation assay as described above choice of APCs used was of particular importance. Using autolo- for purified mAbs. gous EBV-transformed cells as APCs (primarily B cells; see Fig. 2C), the CD4ϩ T cell line responded significantly to 0.2 ␮M Results ϫ ϩ BeSO4 withaSIof4 that increased dramatically with increasing Specificity and proliferation level of BAL CD4 T cells ϫ concentrations of BeSO4. A maximal SI of 11 occurred at 20 ϩ ␮ ϫ The TCR repertoire of blood and BAL CD4 T cells from patients M, then gradually dropped down to a SI of 7–8 , even at BeSO4 with CBD are different. From this particular CBD patient, there is concentrations as high as 80 ␮M (Fig. 1A). Use of a transfected ϩ an increased percentage of V␤8.1 T cells in ex vivo BAL (16.8% mouse fibroblast cell line DAP3(DP2) encoding human HLA-DP2

compared with 5.1%). After five cycles of BeSO4 stimulation, the (HLA-DPA1*0103/DPB1*0201) genes as APCs resulted in a max- ϩ increased percentage of V␤8.1-expressing CD4 T cells persisted. imal SI of 4ϫ, 70% lower than that induced by autologous APCs. ϩ In addition, a V␤5.1 population markedly increased in proportion In contrast, use of a transfected mouse fibroblast cell line ϩ to the other CD4 T cells in culture following continued cycles of DAP3(DP4) encoding the human HLA-DP4 gene (HLA- ϳ ϩ BeSO4 stimulation. This subset accounted for 30% of the CD4 DPA1*0103/DPB1*0401; identical to HLA-DP2 except at resi- T cells after the fifth cycle of stimulation, and junctional region dues 36, 55/56, and 69 within the ␤-1 domain) as APCs resulted in nucleotide sequencing of V␤5.1-expressing BAL T cell clones a maximal SI of only 2ϫ, 85% lower than that induced by autol- from this line revealed two related clonal T cell populations with ogous APCs, and no greater than that of T cells alone in the pres-

an identical CDR3 length (A. P. Fontenot, unpublished observa- ence of BeSO4. The cells were highly speciﬁc for beryllium and tion). These data indicate that as expected, continued selection of showed virtually no proliferation to NiSO4 or to an irrelevant Ag

BAL cells in the presence of BeSO4 resulted in an oligoclonal peptide, MOG35–55 peptide, which can bind with high affinity to ϩ expansion of the most highly reactive T cells. BAL CD4 T cells HLA-DP2 (R. Meza-Romero, personal communication). from this representative HLA-DP2 homozygous CBD patient pro- Potent secretion of IFN-␥ and TNF-␣ by beryllium-stimulated liferated strongly in response to BeSO4. As shown in Fig. 1A, CD4ϩ T cells BeSO4-specific proliferation was observed over a dose range of almost three orders of magnitude (0.2–80 ␮M) of beryllium with Activation requirements for optimal production of inflammatory cytokines are shown in Table I. Although BAL CD4ϩ T line cells or APCs alone secreted very low levels of cytokines, the combination of T cells plus APCs produced a synergistic 3- to 10-fold increase in TNF-␣ and IFN-␥, but no change in IL-2. This beryllium-independent activation conceivably could represent an autologous MLR (AMLR), perhaps driven by recognition of self-MHC class II molecules. A similar degree of cytokine secretion occurred when BAL CD4ϩ T line cells but not APCs alone were activated

by BeSO4. This beryllium-dependent response could result from self-presentation of BeSO4 by T-T interactions or through limiting numbers of BAL-derived APCs that persisted in the T cell line. Of importance, beryllium-dependent activation of BAL CD4ϩ T cells with added APCs resulted in the superoptimal production of IFN-␥, TNF-␣, and IL-2 (Table I). More specifically, BAL CD4ϩ T cells were stimulated with 10 ␮ M BeSO4 in the presence of EBV-transformed autologous APCs at a ratio of 1:1 for 3 days and the supernatants were collected and analyzed for Th1/Th2 cytokines using a quantitative CBA assay. BAL CD4ϩ T cells (40,000/well) cultured in 0.2 ml of medium generated Ͼ30 ng/ml IFN-␥ and 1 ng/ml TNF-␣ by 72 h (Fig. 1B). The normalized production of IFN-␥ and TNF-␣ was calculated to be Ͼ150 ng IFN-␥ and 5 ng TNF-␣ produced per 1 million BAL beryllium-stimulated CD4ϩ T cells within 72 h. Along with pro- ϩ ϩ duction of IFN-␥ and TNF-␣, BAL CD4 T cells produced IL-2 at FIGURE 1. Beryllium-specific proliferation of BAL CD4 T cell line Ͼ cells selected from a HLA-DP2 homozygous patient with CBD. A, BAL 3 ng per million T cells. IL-4, IL-6, and IL-10 were essentially ϩ undetectable following stimulation. Thus, beryllium-specific CD4 T cell line cells (40,000/well in triplicate) were stimulated with ϩ ␮ CD4 T cells showed a predominant Th1 cytokine profile with BeSO4 ranging from 0.2 to 80 M in the presence of EBV-transformed autologous PBMC irradiated (9000 rad) (as APCs) or HLA-DP2- and unexpectedly high levels of secreted IFN-␥ and TNF-␣. Surpris- HLA-DP4-transfected fibroblast cells irradiated (4500 rad) (as APCs) at a ingly, IFN-␥ and TNF-␣ production by beryllium-stimulated T ␮ ␮ ratio of 1:1 for 72 h. In brief, 10 M NiSO4 and 10 g/ml MOG35–55 cells persisted for an extended period (12 wk in culture), when the peptide were added into 40,000 T cells/well plus EBV-transformed autol- cells were split with fresh medium containing exogenous IL-2 ogous APCs at a ratio of 1:1 in triplicate for 72 h, respectively. Forty without washing out the residual beryllium (data not shown). thousand T cells alone with 10 ␮M BeSO ,without adding APCs, were 4 These data suggested that chronic exposure to even low levels of used as a control. B, Cytokine production (picograms per milliliter) of BAL ϩ beryllium caused persistent production of IFN-␥ and TNF-␣. CD4 T cell line cells was measured (in triplicate) by the CBA method using a human Th1/Th2 cytokine CBA kit. Supernatants were collected ϩ ϩ Phenotypic analysis of BAL CD4 T cells and EBV-transformed from BAL CD4 T cells cultured at 40,000/well with EBV-transformed autologous APC ␮ autologous APCs at a ratio of 1:1, with and without 10 M BeSO4 for 72 h. Serial dilution of supernatants was used to determine the actual amount of Cell surface molecules were analyzed by FACS with a focus on IFN-␥ present. characterizing the beryllium-induced activation profile of surface The Journal of Immunology 4319

FIGURE 2. Phenotypic analysis of BAL CD4ϩ T cell line cells and EBV- transformed autologous APCs. A, Sur- face expression of CD3, CD4, TCR␣␤ chain, CD45RO, LFA-1 (CD11a), ICAM-3 (CD50), and HLA class II, including HLA-DR, -DP, and -DQ molecules by BAL CD4ϩ T cells resting in IL-2-containing medium before restimulation with beryllium. B, Changing surface expression of OX40, IL-2R (CD25), B7-1, B7-2, and CD28 on BAL CD4ϩ T cells upon stimula- ␮ tion with 10 M BeSO4 at 24, 48, 72, and 144 h in the presence of EBV- transformed APCs at a ratio of 1:1 in comparison to unstimulated cells. Dis- tinct differential expression of each cellular marker at particular time points presented as histograms with both unstimulated and isotype mAb controls is shown on the left. C, EBV- transformed autologous APC cell surface expression of CD19, CD11b, OX40L, LFA-1 (CD11a), B7-1, B7-2, DC-SIGN (CD209), and HLA-DR, -DP, and -DQ molecules.

markers on BAL T cells cultured ex vivo. In addition to having within 24 h after beryllium exposure. B7-2, however, was already relatively high levels of ␣␤-TCR (87%), the resting CD3ϩCD4ϩ highly expressed on resting BAL T cells before stimulation (Fig. BAL T cells were virtually 100% positive for CD45RO. Unstimu- 2B). Similar to the B7-1 pattern, resting BAL T cells had a mod- lated BAL T cells also expressed high levels of MHC class II erate expression of the costimulation molecule CD28 on resting molecules, HLA-DR, -DP, and -DQ, as well as LFA-1 (CD11a) and ICAM-3 (CD50) (Fig. 2A). Upon exposure to beryllium, BAL T cells showed distinct cell Table I. Activation Requirements surface molecular changes that provided clear insight into their behavior and, potentially, avenues for therapeutic intervention. Cytokine (pg/ml)a OX40 (CD134) and CD25 (IL-2R␣) were both at low levels in Condition IFN-␥ TNF-␣ IL-2 IL-2-supported resting culture, but dramatically increased to levels Ͼ90% positive within 24 h (CD25) and 48 h (OX40) in the pres- T only 16 2 0 APC only 102 5 0 ence of BeSO4 and APCs. The activated BAL T cells maintained T ϩ APCs 2,305 24 0 these high expression levels until at least 144 h, the last time point ϩ T ϩ Be2 1,728 16 0 evaluated (Fig. 2B). OX40L expression was not visible on T cells APCs ϩ Be2ϩ 113 4 0 either before or after stimulation (data not shown). Costimulatory T ϩ APCs ϩ Be2ϩ 38,000b 1,050 710 molecules B7-1 (CD80) and B7-2 (CD86), both of which were a Representative of three individual experiments. Each experimental point reﬂects expressed on APCs at very high levels (Fig. 2C), were differen- the mean of two independent measurements. Forty thousand T cells per well with 0.2 tially expressed and up-regulated on BAL T cells upon beryllium ml of medium in triplicate were cultured with APCs at a ratio of 1:1 with or without 10 ␮M BeSO4 for 72 h. stimulation (Fig. 2B). B7-1 was expressed at moderate levels be- b Five thousand picograms per milliliter is the detection limit. Serial dilution of fore beryllium stimulation and rapidly increased to high levels supernatants was used to determine the actual amount of IFN-␥ present. 4320 ACTIVATION PATHWAYS IN CHRONIC BERYLLIOSIS

Table II. Blocking Experiments

Effect (% change)a

Proliferation IFN-␥ TNF-␣ IL-2 Blocking mAb Used (%) (%) (%) (%)

Antiadhesion molecules Anti-LFA-1 Ϫ53 Ϫ36 Ϫ19 Ϫ40 Anti-MHC restriction molecules Anti-HLA-DP Ϫ99 Ϫ53 Ϫ67 Ϫ71 Anti-HLA-DR Ϫ72 nc nc nc Anticostimulation molecules Anti-OX40 nc nc nc nc Anti-OX40L Ϫ99 nc nc Ϫ24 Anti-CD28 nc nc nc nc CTLA4-lg Ϫ15 Ϫ19 nc Ϫ23

a For clarity, only results using the highest dose of blocking mAb are presented. nc, No change from control. Each experimental point reﬂects the data collected from individual experiments.

FIGURE 3. Blocking BAL CD4ϩ T cell response to beryllium by a mAb (clone HI111) specific for human LFA-1. A, Blocking BAL CD4ϩ T cells that increased strongly after 24 h of stimulation (Fig. 2B). cell proliferation to beryllium by anti-LFA-1. BAL CD4ϩ T cells (40,000/ CTLA-4 (CD152) was not significantly expressed on BAL T cells well in triplicate) were cultured with EBV-transformed autologous APCs at ␮ (data not shown). Staining of T cells with annexin V and 7-ami- ratio of 1:1 with and without 10 M BeSO4 for 72 h. Anti-human LFA-1 noactinomycin D before and after beryllium stimulation docu- Ab at concentrations of 10 and 2 ␮g/ml were added into cultures of T cells mented survival rates typically observed for effector/memory T stimulated with beryllium in the presence of APCs for 72 h. Mouse IgG1 ␮ cells (data not shown) (34, 35). at 10 g/ml was used as isotype Ab control. T cell proliferation was determined by [3H]thymidine incorporation for the last6hoftheculture. The The EBV-transformed autologous APCs used for these studies Ͼ ϩ percent block of proliferation after addition of anti-LFA-1 Ab was calcu- were 95% CD19 , suggesting that they were predominantly B lated relative to that of the isotype control Ab. Significance was determined cells, with high levels of expression of LFA-1 (CD11a), MHC by Student’s t test. B, Blocking Th1 cytokine production of beryllium- class II molecules, B7-1 and B7-2, and unlike the BAL T cells an stimulated BAL CD4ϩ T cells by anti-LFA-1. TNF-␣ and IL-2 production alternative costimulation molecule, OX40L (Fig. 2C). (picograms per milliliter) were measured using the CBA method on supernatant samples collected from BAL CD4ϩ T cell cultures in triplicate as Blocking LFA-1 prevented proliferation and dramatically described above. The percent block of cytokine production by anti-LFA-1 reduced effector cytokine production Ab was calculated relative to that of the isotype control Ab and significance We used a series of mAb blocking experiments, summarized in was determined by Student’s t test. Table II, to characterize the requirement and relative importance of particular cell surface molecules for proliferation and effector cytokine production of beryllium-specific BAL T cells. In the presence of anti-LFA-1 Ab, both proliferation and, to a lesser degree, secretion of cytokines (IFN-␥, TNF-␣, and IL-2) by beryllium- activated BAL T cells were inhibited in a dose-dependent manner (Fig. 3). These data indicate that interactions between the adhesion/trafficking molecule LFA-1 and its ligands contributes to the activation of beryllium-specific T cells.

Blocking HLA-DP prevented proliferation and dramatically reduced effector cytokine production The absolute requirement of MHC class II molecules for activation of beryllium-specific T cells was clearly documented using Ab blocking experiments. As shown in Fig. 4A, addition of anti- HLA-DP Ab blocked T cell proliferation in a dose-dependent manner, with complete inhibition at 20 ␮g/ml, strongly reinforcing previous data showing that MHC class II molecules are critical for beryllium-specific proliferation of BAL T cells. Moreover, anti-DP FIGURE 4. Anti-HLA-DP and anti-HLA-DR mAbs significantly Ab blocked IFN-␥ 53% and TNF-␣ and IL-2 by ϳ70% (Fig. 4B). blocked the BAL CD4ϩ T cell response to beryllium. A, BAL CD4ϩ T Interestingly, anti-HLA-DR Abs also had a blocking effect on pro- cells (40,000/well in triplicate) were cultured with EBV-transformed APCs ␮ liferation of BAL T line cells (Fig. 4A), but anti-DR Ab had vir- at a ratio of 1:1 with and without 10 M BeSO4 for 72 h. Predialyzed tually no effect on inhibition of cytokine secretion (Fig. 4B). The nontoxic anti-HLA-DP (clone B7/21) and anti-HLA-DR (clone L243) were added into cultures of T cells stimulated with beryllium at Ab concentra- anti-HLA-DR Ab used in these experiments reacts with a nonpoly- tions of 20 and 5 ␮g/ml for 72 h. Mouse IgG1 at 20 ␮g/ml served as isotype morphic HLA-DR epitope (36–38) and does not cross-react with Ab control. B, Cytokine production (picograms per milliliter) was deter- HLA-DQ or HLA-DP molecules (38). Moreover, we have inde- mined in triplicate using the CBA method. The supernatant samples were pendently confirmed that the anti-DR Ab does not cross-react with collected from T cell cultures as described above. The percent block after the Ag-binding/TCR recognition domains of either HLA-DP2 or addition of Abs was calculated relative to that of the isotype control Ab. HLA-DP4 by ELISA (data not shown). Significance was determined by Student’s t test. The Journal of Immunology 4321

beryllium stimulation before declining back to the resting distribution (Fig. 2B). Similar to a previous report (25), blockade of signaling through CD28 using soluble CTLA4-Ig to ligate B7-1 and B7-2 molecules on APCs had a relatively modest inhibition of proliferation (15%), IFN-␥ (19%) and IL-2 (23%) secretion, but little effect on TNF-␣ (Fig. 5B). Moreover, addition of anti-CD28 Ab had no signiﬁcant effect on activation (Fig. 5A). These data suggest a relatively minor contribution of the CD28/B7 costimulation pathway on beryllium-induced T cell activation. In contrast to the transient up-regulation of CD28, beryllium- activated T cells had long-lasting high levels of OX40 expression (Fig. 2B), possibly implicating this costimulation pathway. Indeed, blockade of the OX40-OX40L interaction using anti-OX40L Ab resulted in complete (99%) inhibition of proliferation (Fig. 5, A and B). However, there was only a modest inhibitory effect of anti-OX40L Ab on IL-2 secretion (24%) and no detectable effect on IFN-␥ or TNF-␣ levels (Fig. 5C). Similar to anti-CD28 Ab, anti-OX40 Ab also did not inhibit beryllium-induced T cell proliferation (Fig. 5A). These ﬁndings suggest a dominant role of the OX40/OX40L costimulation pathway on beryllium-induced proliferation, but further indicate that cytokine release is relatively independent of costimulation by either of these pathways.

Discussion Characterization of pathogenic T cells from patients with CBD has revealed that beryllium-specific CD4ϩ T cells play a major role in FIGURE 5. Involvement of costimulation molecules in beryllium-spe- initiating and perpetuating granulomatous inflammation (10, 11). cific activation of BAL CD4ϩ T cell line cells in the presence of EBV- The results presented above demonstrate that activation of BAL ϩ transformed autologous APCs. A, Blocking beryllium-induced BAL CD4ϩ CD4 T cells is a complex process, involving beryllium-depen- T cell proliferation in the presence of EBV-transformed autologous APCs. dent HLA-DP and HLA-DR-restricted stimulation through the ϩ Forty thousand BAL CD4 T cells were cultured in the presence of EBV- TCR, partial dependence on the OX40/OX40L costimulation path- transformed APCs at a ratio of 1:1 in triplicate with and without 10 ␮M way, and involvement of cell-cell adhesion mediated through li- ␮ BeSO4 for 72 h. Anti-OX40 (25 g/ml), anti-OX40L, anti-CD28, CTLA4- gation of LFA-1. Moreover, activation may also be induced by Ig, and an isotype Ab control (mouse IgG1) were added into cultures of T beryllium-independent interactions between T cells and APCs, cells stimulated with beryllium for 72 h. Proliferation was expressed as net reminiscent of an AMLR involving MHC class II recognition. An cpm above background. The percent block after addition of Abs was calculated relative to that of the isotype control Ab. Significance was deter- inherent weakness of our system is the reliance on observations mined by Student’s t test. B, Blocking assay on BAL CD4ϩ T cell prolif- made using mass T cell culture in vitro to faithfully duplicate what ␮ occurs in vivo. That being said, in preliminary experiments, ex eration to 10 M BeSO4 in the presence of EBV-transformed autologous APCs at a ratio of 1:1 was repeated, with and without adding 25 and 10 vivo BAL cells from CBD subjects express high levels of LFA-1 ␮g/ml anti-OX40L and CTLA4-Ig, respectively, for 72 h. Proliferation was and up to 30% of direct ex vivo BAL CD4ϩ T cells up-regulate expressed as net cpm above background. C, Blocking IL-2 production of OX40 expression by 72 h after beryllium exposure in culture (A. P. ϩ beryllium-stimulated BAL CD4 T cells by both CTLA-4Ig and anti- Fontenot, unpublished observation). These results indicate that our OX40L. Cytokine production (picograms per milliliter) was determined in cultured BAL cell line retained at least two important character- triplicate by the CBA method. The supernatant samples were collected istics of freshly isolated BAL T cells from additional CBD pa- from T cell cultures as described above. The percent block after addition of tients, and thus this line appears to be generally representative of Abs was calculated relative to that of the isotype control Ab. Significance was determined by Student’s t test. beryllium-driven T cells. Consistent with the severe granulomatous inflammation known to occur in lung tissue in CBD, the in vitro combination of beryllium, APCs, and BAL CD4ϩ T cells induced strong proliferation

ϩ responses; superoptimal concentrations of the secreted proinflam- Costimulation involved in beryllium-induced CD4 T cell matory cytokines IFN-␥, TNF-␣, and IL-2; and up-regulation of activation: OX40/OX40L pathway plays a dominant role in numerous T cell surface markers that would promote T-T Ag pre- proliferation sentation. Ab blocking experiments revealed that anti-HLA-DP or Memory T cells can proliferate and secrete cytokines after TCR anti-LFA-1 Ab strongly reduced proliferation responses and, to a engagement without CD28-mediated costimulation (39), and the lesser extent, cytokine secretion by BAL CD4ϩ T cells. In con- CD28 costimulation independence of BAL beryllium-specific T trast, anti-HLA-DR or anti-OX-40L Ab mainly affected beryllium- cells has previously been reported (25). We demonstrated above induced proliferation responses with little impact on cytokines that 98% of BAL beryllium-specific CD4ϩ T line cells were of the other than IL-2, thus implying that nonproliferating BAL CD4ϩ T CD45ROϩ memory phenotype, responding to beryllium in the cells may still contribute to inflammation. Blockade with presence of autologous APCs by proliferation and secretion of CTLA4-Ig had a minimal effect on proliferation and cytokine re- high levels of IFN-␥ and TNF-␣. Although CTLA-4 was not sponses, confirming that activation was independent of B7/CD28 present at an appreciable level on resting or beryllium-activated costimulation. Because T cell proliferation and secretion of proin- BAL T cells (data not shown), the level of CD28 on the BAL T cell flammatory cytokines appear to play a dominant role in the chronic population changed significantly (from 54 to 92%) for 24 h after inflammatory process that defines CBD, it would appear that Abs 4322 ACTIVATION PATHWAYS IN CHRONIC BERYLLIOSIS specific for HLA-DP and LFA-1 would be well suited for further tions with CD28 and CTLA-4 on T cells. These molecules are investigation as a therapeutic strategy for intervention in CBD as expressed on APCs as well as on activated CD4ϩ cells at late well as other hypersensitivity pneumonitides. stages after activation (47). We found very high-level expressions T cell proliferation to beryllium salt in vitro was beryllium spe- of B7-2 even on resting BAL CD4ϩ T cells, and B7-1 was dra- cific and dose dependent (Fig. 1A), and our analysis clearly ex- matically up-regulated within 24 h upon beryllium stimulation cluded nonspecific T cell stimulation by other metal salts, although (Fig. 2B). Previous studies suggested that beryllium-specific mem- the molecular details of metal-protein interactions underlying the ory CD4ϩ T cells in blood continued to require CD28 costimula- transport and delivery of metal ions to APCs during their early tion for proliferation and cytokine responses, whereas BAL effec- sensitization phase and the interaction of beryllium with HLA and tor memory cells were functionally independent of CD28

TCR is still unclear (40). The BeSO4 dose-dependent T cell pro- costimulation (25). One possibility that warrants further investiga- liferation curve consistently showed a decreased maximal stimu- tion is that constitutively high levels of B7-2 allow direct T:T ␮ high high lation above the peak responsive concentration of 20 M BeSO4, costimulation between resident activated B7-2 CD28 BAL T but concentrations up to 80 ␮M still showed almost 70% of the cells and effector/memory B7-2highCD28low CD4ϩ T cells re- maximal response. These data are consistent with previous obser- cruited to the lung from the blood, with T-T interactions providing vations regarding beryllium toxicity to APCs (41) and suggest that both the primary signal as well as costimulation. Ab blocking stud- beryllium-specific T cells are truly tolerant of high doses of ies have clearly helped to identify the most important pathways for ϩ ϩ BeSO4. Our later studies documented that BAL CD4 T cells activation of BAL CD4 T cells. The ability of anti-HLA-DP to retained the ability to secrete large amounts of IFN-␥ and contin- abolish proliferation as well as eliminate 70% of both IFN-␥ and uously live for months under conditions in which exogenous IL-2 TNF-␣ effector cytokine and IL-2 autocrine secretion point to the was added (Y. K. Chou, personal communication). central role played by HLA-DP in T cell activation. Future studies Our studies documented that the BAL CD4ϩ T cell line was will be directed at evaluating how beryllium and HLA-DP interact strongly polarized toward Th1, secreting detectable amounts of and further the molecular recognition of beryllium as an HLA- IFN-␥ even in the absence of beryllium stimulation (Table I). In DP-restricted Ag. Blockade of LFA-1 interaction using mAb the absence of APCs, addition of beryllium increased secretion of HI111 (48) consistently inhibited proliferation and IL-2 secre- IFN-␥ by Ͼ100-fold, directly suggesting T-T interactions play a tion by Ͼ50% and also significantly reduced secretion of IFN-␥ role in maintaining the chronic milieu characteristic of CBD. In and TNF-␣. The known ligands of LFA-1 include ICAM-1 contrast, addition of beryllium to APCs alone resulted in no change (CD54), ICAM-2 (CD102), and ICAM-3 (CD50) (49–52). in IFN-␥ and TNF-␣ production. However, IFN-␥ secretion in- ICAM-1 and ICAM-2 are expressed on endothelium and have creased 100-fold merely by culturing the T cells with autologous been implicated in leukocyte adhesion and transendothelial mi- APCs at a 1:1 ratio, indicating a beryllium-independent activation gration. ICAM-3 is exclusively present on T and B leukocytes reminiscent of an AMLR, perhaps involving recognition of self- where it appears to be important for the initial scanning of the MHC class II molecules. Unexpectedly, upon beryllium addition APC surface by T cells, as well as playing a role in augmenting to a 1:1 culture ratio of T:APC, the activated T line cells produced LFA-1- and ICAM-1-mediated adhesion and signaling via low- an astounding 38,000 pg/ml secreted IFN-␥, as well as Ͼ1000 affinity interactions (53–55). It is noteworthy that ICAM-1, pg/ml TNF-␣ and 700 pg/ml IL-2. Importantly, in the absence of which has an ϳ10,000-fold higher affinity for LFA-1 than APCs no IL-2 secretion was observed, clearly documenting the ICAM-3 (56), is also up-regulated on BAL T cells upon APC requirement of APCs for the full activation profile of these BAL T plus beryllium exposure (data not shown). cells. A novel observation from this study is that OX40 expression by The memory phenotype of BAL beryllium-specific T cells has beryllium-stimulated CD4ϩ T cells was strongly up-regulated been reported previously (16–18), and is confirmed by our staining within 24–48 h after stimulation with beryllium and was main- data, with the vast majority of the CD4ϩ T cells staining positive tained at high levels throughout the 144-h observation period (Fig. for CD45RO. The features of high-level expression of CD134 2B). This activation profile of BAL T cells upon BeSO4 stimula- (OX40; upon stimulation) and HLA-DR, -DP, and -DQ on the cell tion is similar to changes observed with peptide-Ag-activated surface and the ability to secrete extremely high levels of IFN-␥ all CD4ϩ T cells (57) and suggests that the engagement of OX40 with confirm that this beryllium-specific BAL CD4ϩ T cell line was OX40L expressed on APCs may play a role in activation and in- highly activated withaTeffector/memory phenotype (42–44). The creased survival of beryllium-specific memory T cells (58). Inter- survival of CD4ϩ T effector/memory cells depends on their ability estingly, blockade with anti-OX40L Ab inhibited proliferation re- to produce IL-2, whereas cells producing only effector cytokines sponses but not cytokine secretion by BAL CD4ϩ T cells, (e.g., IFN-␥) may be short-lived (45). The level of CD25 (IL-2R␣) suggesting an uncoupling of the cell cycle from cytokine produc- expression is considered to be a reflection of recent Ag stimulation tion. This finding implies that interruption of the OX40/OX40L and plays a crucial role in the regulation of T cell proliferation costimulation pathway might limit the number of beryllium-reac- (46). In the current study, we found that CD25 was very rapidly tive T cells without affecting their secretion of inflammatory cy- up-regulated during the course of stimulation with beryllium, with tokines. A similar pattern showing inhibition of proliferation but 5–10% of the resting cells expressing CD25, increasing to Ͼ90% not cytokine responses was observed using anti-HLA-DR Ab, and within 24 h and remaining at an elevated level for at least 144 h. the possibility exists that BAL CD4ϩ T cells may exhibit a unique CD25 expression on the cell surface finally declined 2–3 wk after activation pathway involving TCR ligation through HLA-DR with beryllium stimulation in direct correlation to the concentration of OX40/OX40L costimulation.

BeSO4 depleted from the culture medium by serial dilution. T cell In summary, our data reinforce previous work concerning the proliferation continued as long as IL-2 was added to the culture central importance of HLA-DP in beryllium disease, with blockade medium. Of possible diagnostic importance, these data suggest of HLA-DP being the best approach to eliminate proliferation abil- that CD25 can be used as a functional marker for BAL CD4ϩ T ity and cytokine production. The ability of anti-LFA-1 to partially cell activation and exposure to BeSO4. inhibit these activities is also of potential therapeutic importance. CD80 (B7-1) and CD86 (B7-2) are members of the Ig supergene Since T cell expansion and secretion of proinflammatory cytokines family and are coregulators of T cell activation through interac- appear to play a dominant role in the chronic inflammatory process The Journal of Immunology 4323 that defines CBD, it would appear that Abs specific for HLA-DP 23. Bluestone, J. A. 1995. New perspectives of CD28–B7-mediated T cell costimu- and LFA-1 would be well suited for further investigation as a lation. Immunity 2:555. 24. Michel, F., G. Attal-Bonnefoy, G. Mangino, S. Mise-Omata, and O. Acuto. 2001. therapeutic strategy for CBD. CD28 as a molecular amplifier extending TCR ligation and signaling capabilities. Immunity 15:935. 25. Fontenot, A. P., L. Gharavi, S. R. Bennett, S. J. Canavera, L. S. Newman, and Acknowledgments B. L. Kotzin. 2003. CD28 costimulation independence of target organ versus We thank Dr. Dennis Bourdette for meaningful discussion regarding CD4ϩ circulating memory antigen-specific CD4ϩ T cells. J. Clin. Invest. 112:776. T cell-mediated chronic inflammation in the CNS and Dorian LaTocha for 26. Sugamura, K., N. Ishii, and A. D. Weinberg. 2004. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nat. Rev. 4:420. his assistance with the T cell phenotyping work. 27. Watts, T. H., and M. A. DeBenedette. 1999. T cell co-stimulatory molecules other than CD28. Curr. Opin. Immunol. 11:286. Disclosures 28. Gramaglia, I., A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft. 2000. The OX40 costimulatory receptor determines the development of CD4 Senior author G. G. Burrows has filed provisional patents with the Oregon memory by regulating primary clonal expansion. J. Immunol. 165:3043. Health & Science University Technology and Research Collaboration Of- 29. Fontenot, A. P., M. T. Falta, B. M. Freed, L. S. Newman, and B. L. Kotzin. 1999. fice, and coauthors Y. K. Chou, A. A. Vandenbark, and D. Edwards are Identification of pathogenic T cells in patients with beryllium-induced lung dis- listed as co-inventors. ease. J. Immunol. 163:1019. 30. Newman, L. S. 1996. Significance of the blood beryllium lymphocyte proliferation test. Environ. Health Perspect. 104:953. References 31. Newman, L. S., K. Kreiss, T. E. King, Jr., S. Seay, and P. A. Campbell. 1989. 1. Martin, R., and H. F. McFarland. 1995. Immunological aspects of experimental Pathologic and immunologic alterations in early stages of beryllium disease: re- allergic encephalomyelitis and multiple sclerosis. Crit. Rev. Clin. Lab. Sci. examination of disease definition and natural history. Am. Rev. Respir. Dis. 32:121. 139:1479. 2. Atkinson, M. A., and G. S. Eisenbarth. 2001. Type 1 diabetes: new perspectives 32. Sharif-Askari, E., L. M. Fawaz, P. Ahmad, and J. Menezes. 2001. Interleukin on disease pathogenesis and treatment. Lancet 358:221. 15-mediated induction of cytokine effector cells capable of eliminating Epstein- 3. Devouassoux, G., and C. Brambilla. 2002. Effect of diesel particles on allergic Barr virus-transformed/immortalized lymphocytes in culture. J. Natl. Cancer inflammatory response: cellular targets and molecular mechanisms. Rev. Mal. Inst. 93:1724. Respir. 19:467. 33. Chou, Y. K., A. D. Weinberg, A. Buenafe, D. N. Bourdette, R. Whitham, J. A. 4. Thierse, H. J., C. Moulon, Y. Allespach, B. Zimmermann, A. Doetze, S. Kuppig, R. Kaleeba, I. F. Robey, D. G. Kavanagh, H. Offner, and A. A. Vandenbark. D. Wild, F. Herberg, and H. U. Weltzien. 2004. Metal-protein complex-mediated 1996. MHC-restriction, cytokine profile, and immunoregulatory effects of human transport and delivery of Ni2ϩ to TCR/MHC contact in nickel-specific human T T cells specific for TCR VB CDR2 peptides: comparison with myelin basic cell activation. J. Immunol. 172:1926. protein-specific T cells. J. Neurosci. Res. 45:838. 5. Sinigaglia, F. 1994. The molecular basis of metal recognition by T cells. J. Invest. 34. Strauss, G., I. Knape, I. Melzner, and K. M. Debatin. 2003. Constitutive caspase Dermatol. 102:398. activation and impaired death-inducing signaling complex formation in CD95- 6. Mandervelt, C., F. L. Clottens, M. Demedts, and B. Nemery. 1997. Assessment resistant, long-term activated, antigen-specific T cells. J. Immunol. 171:1172. of the sensitization potential of five metal salts in the murine local lymph node 35. Gorak-Stolinska, P., D. M. Kemeny, and A. Noble. 2002. Activation-induced cell assay. Toxicology 21:161. death in human T cells is a suicidal process regulated by cell density but supe- 7. Budinger, L., and M. Hertl. 2000. Immunologic mechanisms in hypersensitivity rantigen induces T cell fratricide. Cell Immunol. 219:98. reactions to metal ions: an overview. Allergy 55:108. 36. Lampson, L. A., and R. Levy. 1980. Two populations of Ia-like molecules on a 8. Newman, L. S., L. A. Maier, and B. Nemery. 1998. Interstitial lung disorders due human B cell line. J. Immunol. 125:293. to beryllium and cobalt. In Interstitial Lung Disease, 3rd ed. 37. Brodsky, F. M. 1984. A matrix approach to human class II histocompatibility M. I. Schwarz and T. E. king, Jr., eds. Dekker, Hamilton, Ontario, Canada. p. 367. antigens: reactions of four monoclonal antibodies with the products of nine hap- 9. Infante, P. F., and L. S. Newman. 2004. Beryllium exposure and chronic beryl- lotypes. Immunogenetics 19:179. lium disease. Lancet 363:415. 38. Robbins, P. A., E. L. Evans, A. H. Ding, N. L. Warner, and F. M. Brodsky. 1987. 10. Saltini, C., K. Winestock, M. kirby, P. Pinkston, and R. G. Crystal. 1989. Main- Monoclonal antibodies that distinguish between class II antigens (HLA-DP, DQ, tenance in patients with chronic beryllium disease by beryllium-specific helper T and DR) in 14 haplotypes. Hum. Immunol. 18:301. cells. N. Engl. J. Med. 320:1103. 39. Croft, M., L. M. Bradley, and S. L. Swain. 1994. Naive versus memory CD4 T 11. Fontenot, A. P., M. W. Torres, H. Marshall, L. S. Newman, and B. L. Kotzin. cell response to antigen: memory cells are less dependent on accessory cell co- 2000. Beryllium presentation to CD4ϩ T cells underlies disease susceptibility stimulation and can respond to many antigen-presenting cell types including rest- HLA-DP alleles in chronic beryllium disease. Proc. Natl. Acad. Sci. USA 97: ing B cells. J. Immunol. 152:2675. 12717. 40. Weinberg, A. D., D. E. Evans, C. Thalhofer, T. Shi, and R. A. Prell. 2004. The 12. Richeldi, L., R. Sorrentino, and C. Saltini. 1993. HLA-DPB1 glutamate 69: a generation of T cell memory: a review describing the molecular and cellular genetic marker of beryllium disease. Science 262:242. events following OX40(CD134) engagement. J. Leukocyte Biol. 75:962. 13. Maier, L. A., D. S. McGrath, H. Sato, P. Lympany, K. Welsh, R. du Bois, 41. Chaudhary, A., N. N. Sauer, and G. Gupta. 2004. Beryllium-specific immune L. Silveira, A. P. Fontenot, R. T. Sawyer, E. Wilcox, and L. S. Newman. 2003. response in primary cells from healthy individuals. Toxicology 201:9. Influence of MHC class II in susceptibility to beryllium sensitization and chronic 42. Sawyer, R. T., D. E. Doherty, B. A. Schumacher, and L. S. Newman. 1999. beryllium disease. J. Immunol. 171:6910. Beryllium-stimulated in vitro migration of peripheral blood lymphocytes. Toxi- 14. Tinkle, S. S., and L. S. Newman. 1997. Beryllium-stimulated release of tumor cology 138:155. necrosis factor-␣, interleukin-6, and their soluble receptors in chronic beryllium 43. Newman, L. S., C. Bobka, B. Schumacher, E. Daniloff, B. Zhen, M. M. Mroz, and disease. Am. J. Respir. Crit. Care Med. 156:1884. T. E. King Jr. 1994. Compartmentalized immune response reflects clinical se- 15. Tinkle, S. S., L. A. Kittle, B. A. Schumacher, and L. S. Newman. 1997. Beryllium verity of beryllium disease. Am. J. Respir. Crit. Care Med. 150:135. induces IL-2 and IFN-␥ in berylliosis. J. Immunol. 158:518. 44. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two 16. Rossman, M. D., J. A. Kern, J. A. Elais, M. R. Cullen, P. E. Epstein, O. P. Preuss, subsets of memory T lymphocytes with distinct homing potential and effector T. N. Markham, and R. P. Daniele. 1988. Proliferative response of bronchoal- functions. Nature 401:708. veolar lymphocytes to beryllium. Ann. Intern. Med. 108:687. 45. Younes, S.-A., B. Yassine-Diab, A. R. Dumont, M.-R. Boulassel, Z. Grossman, 17. Fontenot, A. P., S. J. Canavera, L. Gharavi, L. S. Newman, and B. L. Kotzin. J.-P. Routy, and R.-P. Sekaly. 2003. HIV-1 viremia prevents the establishment of ϩ 2002. Target organ localization of memory CD4ϩ T cells in patients with chronic interleukin 2-producing HIV-specific memory CD4 T cells endowed with pro- beryllium disease. J. Clin. Invest. 110:1473. liferative capacity. J. Exp. Med. 198:1909. 18. Fontenot, A. P., L. A. Maier, S. J. Canavera, T. B. Hendry-Hofer, 46. Ladanyi, A., B. Somlai, K. Gilde, Z. Fejos, I. Gaudi, and J. Timar. 2004. T-cell M. Boguniewicz, E. A. Barker, L. S. Newman, and B. L. Kotzin. 2002. Beryllium activation marker expression on tumor-infiltrating lymphocytes as prognostic fac- skin patch testing to analyze T cell stimulation and granulomatous inflammation tor in cutaneous malignant melanoma. Clin. Cancer Res. 10:521. in the lung. J. Immunol. 168:3627. 47. Linsley, P. S., W. Brady, L. Grosmaire, A. Aruffo, N. K. Damle, and 19. Saltini, C., M. Kirby, B. C. Trapnell, N. Tamura, and R. G. Crystal. 1990. Biased J. A. Ledbetter. 1991. Binding of the B cell activation antigen B7 to CD28 accumulation of T lymphocytes with “memory”-type CD45 leukocyte common costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. antigen gene expression on the epithelial surface of the human lung. J. Exp. Med. Med. 173:721. 171:1123. 48. Ma, Q., M. Shimaoka, C. Lu, H. Jing, C. V. Carman, and T. A. Springer. 2002. 20. Huang, H., K. C. Meyer, L. Kubai, and R. Auerbach. 1992. An immune model of Activation-induced conformational changes in the I domain region of lymphocyte beryllium-induced pulmonary granulomata in mice: histopathology, immune re- function-associated antigen 1. J. Biol. Chem. 277:10638. activity, and flow-cytometric analysis of bronchoalveolar lavage-derived cells. 49. Marlin, S. D., and T. A. Springer. 1987. Purified intercellular adhesion mole- Lab. Invest. 67:138. cule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA- 21. Fontenot, A. P., B. L. Kotzin, C. Comment, and L. S. Newman. 1998. Expansion 1). Cell 51:813. of T-cell subsets expressing particular T cell receptor variable regions in chronic 50. Nortamo, P., R. Salcedo, T. Timonen, M. Patarroyo, and C. G. Gahmberg. 1991. beryllium disease. Am. J. Respir. Cell Mol. Biol. 18:581. A monoclonal antibody to the human leukocyte adhesion molecule intercellular 22. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, and J. P. Allison. 1992. adhesion molecule-2: cellular distribution and molecular characterization of the CD28-mediated signaling co-stimulates murine T cells and prevents induction of antigen. J. Immunol. 146:2530. anergy in T-cell clones. Nature 356:607. 51. Rothlein, R., M. L. Dustin, S. D. Marlin, and T. A. Springer. 1986. A human 4324 ACTIVATION PATHWAYS IN CHRONIC BERYLLIOSIS

intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J. Immunol. and D. N. J. Hart. 1995. Intercellular adhesion molecule-3 is the predominant 137:1270. co-stimulatory ligand for leukocyte function antigen-1 on human blood dendritic 52. De Fougerolles, A. R., and T. A. Springer. 1992. Intercellular adhesion molecule cells. Eur. J. Immunol. 25:2528. 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 56. Shimaoka, M., C. Lu, R. T. Palframan, U. H. Von Andrian, A. McCormack, 1 on resting lymphocytes. J. Exp. Med. 175:185. J. Takagi, and T. A. Springer. 2001. Reversibly locking a protein fold in an active ␣ conformation with a disulfide bond: integrin L I domains with high affinity and 53. Montoya, M. C., D. Sancho, G. Bonello, Y. Collette, C. Langlet, H. He, P. Aparicio, antagonist activity in vivo. Proc. Natl. Acad. Sci. USA 98:6009. A. Alcover, D. Olive, and F. Sańchez-Madrid. 2002. Role of ICAM-3 in the initial 57. Gramaglia, I., A. D. Weinberg, M. Lemon, and M. Croft. 1998. OX40 ligand: a interaction of T lymphocytes and APCs. Nat. Immunol. 3:159. potent costimulatory molecule for sustaining primary CD4 T cell responses. 54. Hauss, P., F. Selz, C. M. Cavazzana, and A. Fischer. 1995. Characteristics of J. Immunol. 161:6510. antigen-independent and antigen-dependent interaction of dendritic cells with ϩ 58. Maxwell, J. R., A. Weinberg, R. A. Prell, and A. T. Vella. 2000. Danger and CD4 T cells. Eur. J. Immunol. 25:2285. OX40 receptor synergize to enhance T cell survival by inhibiting peripheral de- 55. Starling, G. C., A. D. Mclellan, W. Egner, R. V. Sorg, J. Fawcett, D. L. Simmons, letion. J. Immunol. 164:107.