S Regulates Class II MHC Processing in Human CD4 + HLA-DR+ T Cells

This information is current as Cristina Maria Costantino, Hidde L. Ploegh and David A. of September 26, 2021. Hafler J Immunol 2009; 183:945-952; Prepublished online 24 June 2009; doi: 10.4049/jimmunol.0900921

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cathepsin S Regulates Class II MHC Processing in Human CD4؉ HLA-DR؉ T Cells1

Cristina Maria Costantino,* Hidde L. Ploegh,† and David A. Hafler2*

Although it has long been known that human CD4؉ T cells can express functional class II MHC molecules, the role of lysosomal in the T cell class II MHC processing and presentation pathway is unknown. Using CD4؉ T cell clones that constitutively express class II MHC, we determined that cathepsin S is necessary for invariant chain proteolysis in T cells. CD4؉HLA-DR؉ T cells down-regulated cathepsin S expression and activity 18 h after activation, thereby ceasing nascent class II MHC product formation. This blockade resulted in the loss of the invariant chain fragment CLIP from the cell surface, suggesting that—like professional APC—CD4؉ HLA-DR؉ cells modulate self-Ag presentation as a consequence of activation. Furthermore, cathepsin S expression and activity, and concordantly cell surface CLIP expression, was reduced in HLA-DR؉ CD4؉ T cells as compared

with B cells both in vitro and ex vivo. The Journal of Immunology, 2009, 183: 945–952. Downloaded from

D4ϩ T cells are activated by TCR engagement of pep- surrogate substrate and trafficking chaperone (11). As the MHC:Ii tide/class II MHC complexes on APCs to initiate an complex migrates through the endo/lysosomal compartment, res- C adaptive immune response, but can themselves also ex- ident proteases systematically degrade the this chaperone, leaving press class II MHC (1, 2). The expression of class II MHC on only the Ii fragment CLIP to occlude the class II MHC binding CD4ϩ T cells occurs in most mammalian species (3), the exception pocket. These same proteases hydrolyze self and foreign http://www.jimmunol.org/ being mice, which do not transcribe the CIITA promoter III in to generate peptide epitopes, which ultimately displace CLIP and CD4ϩ T cells (4, 5). are loaded into the class II MHC binding pocket with the aid of the In the human system, expression of HLA-DR, the most preva- loading molecule HLA-DM (12). lent class II MHC molecule, was first described as a marker of Key proteolytic regulators of class II MHC processing have activated T cells (2). Patients with chronic autoimmune disease, been identified in professional APC with the use of knock-out mice inflammation, and the recent recipients of immunizations exhibited and specific inhibitors (13, 14). Blockade of Ii degrada- a higher frequency of HLA-DRϩ T cells in the peripheral blood as tion results in the accumulation of Ii intermediates and can lead to compared with healthy donors (6). Yet, for human CD4ϩ T cells, a corresponding decrease in surface expression of class II MHC

HLA-DR is more than a biomarker of activation. Class II MHC on products (15, 16). In human lines, treatment with the pan- by guest on September 26, 2021 these cells is functional and can be used to present peptide Ag to inhibitor leupeptin or the cathepsin S inhibitor activate responder CD4ϩ T cells in vitro (7–9). Furthermore, re- leucine-homophenylalanine-vinyl sulfone (LHVS) blocks success- cent studies have identified HLA-DR expression on CD4ϩ T cells ful degradation of Ii (17). Characterization of cathepsin S (CatS) in the blood of healthy donors, specifically a subset of CD4ϩ knock-out mice has further implicated CatS in the terminal cleav- CD25high FoxP3ϩ natural regulatory T cells, and suggest that age of Ii to yield CLIP in professional APC (13). Further studies, HLA-DR may have a functional role in these cells (10). however, have demonstrated a cell type-specific role for cysteine Although the class II MHC processing and presentation pathway proteases in these later stages of Ii processing. CatL in thymic has been studied extensively in professional APC, this pathway in epithelial cells and CatF in can also perform this human CD4ϩ T cells has not been characterized. This is not a cleavage (18) (19). trivial issue, as many of the involved in the generation of Although B and T cells are derived from a common precursor, antigenic peptides are not ubiquitously expressed. HLA-DR mat- these cells ultimately differentiate into functionally unique lineages uration is regulated by the invariant chain (Ii),3 which acts as a with distinct trafficking pathways, organization, and composition within their intracellular processing compartments. This prompted us to explore in detail the biosynthesis of human class II MHC products *Division of Molecular Immunology, Center for Neurologic Diseases, Brigham and in MHC-identical B and T cells. In this study, we demonstrate, using Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; ϩ † CD4 T cell clones, that CatS is a key required for proteol- Whitehead Institute, Department of Biology, Massachusetts Institute of Technology, ϩ ϩ Cambridge, Massachusetts 02142 ysis of Ii in CD4 HLA-DR T cells. We find that activation-induced Received for publication March 23, 2009. Accepted for publication May 19, 2009. regulation of CatS expression and activity leads to the down-regula- ϩ ϩ The costs of publication of this article were defrayed in part by the payment of page tion of CLIP expression in CD4 HLA-DR T cells both in vitro and ϩ ϩ charges. This article must therefore be hereby marked advertisement in accordance ex vivo. Our data indicates that CD4 HLA-DR T cells modulate with 18 U.S.C. Section 1734 solely to indicate this fact. peptide epitope presentation postactivation, and furthermore suggests 1 This work was supported by National Institutes of Health grants. that presentation of non-CLIP self-peptide may be integral to the func- 2 Address correspondence and reprint requests to Dr. David Hafler, Harvard Medical tion of class II MHC on these cells. School, 77 Avenue Louis Pasteur, Room 641, Boston, MA 02115. E-mail address: dhafl[email protected] 3 Abbreviations used in this paper: Ii, invariant chain; LHVS, leucine-homopheny- Materials and Methods lalanine-vinyl sulfone; Cat, cathepsin; AEP, ; EBV, Epstein- Cell culture reagents and Abs Barr Virus. Cells were cultured in RPMI 1640 medium supplemented with 2 mM Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 L-glutamine, 5 mM HEPES, 100 U/ml penicillin/streptomycin (all from www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900921 946 INVARIANT CHAIN PROTEOLYSIS IN HUMAN CD4ϩ T CELLS

BioWhittaker), 0.5 mM sodium pyruvate, 0.5 mM nonessential amino ac- Results 2 ids (from Life Technologies) in 96-well U-bottom plates or 25 cm vented Establishment of CD4ϩ HLA-DRϩ T cell clones flasks (CoStar). T cell clone medium additionally received 5% human AB serum (Meditech) and 25 U/ml recombinant human IL-2 (Tecin, National Class II MHC is a traditional biomarker of activated human CD4ϩ Cancer Institute). The medium for the Epstein-Barr Virus (EBV)-trans- T cells (2), but relatively little is known about endogenous class II formed B cell lines was supplemented with 8% FBS. The ␣CD3 (UCHT1 ␣ ␣ ␣ expression, processing, and Ag presentation in these adaptive, non- and Hit3a), CD4 (RPA-T4), CD28 (CD28.2 and 3D10), CLIP/ ϩ HLA-DR (CerCLIP), ␣HLA-DR (L243 and Tu¨36), ␣ class II MHC (Tu¨39), professional APC. To assess class II MHC expression in CD4 T ϩ ␣HLA-DM (MaP.DM1), and ␣CD19 (1D3) Abs were purchased from BD cells at the single cell level, we generated CD4 T cell clones from Pharmingen. the peripheral blood of healthy donors. We propagated these Cell isolation clones in APC-free cultures to ensure that our analysis was re- stricted to endogenous class II MHC expression and would ex- Whole mononuclear cells were isolated from healthy individuals after in- clude acquisition of class II from traditional APC. We compared formed consent in green-capped, heparinized tubes by Ficoll-Hypaque (GE Healthcare) gradient centrifugation. CD19ϩ B cells were isolated using class II MHC synthesis in these clones to genetically identical CD19 microbeads (Miltenyi Biotec). Total CD4ϩ T cells were isolated via EBV-transformed B cell lines. This comparison allowed us to con- the CD4ϩ T cell negative isolation kit II (Miltenyi Biotec) and incubated trol for donor-to-donor variability in expression, enzyme with an excess volume of fluorochrome-labeled Abs against HLA-DR activity, and MHC haplotype. (L243 PerCP), CD62L (Dreg 56 APC), CD25 (M-A251 PE), CD32 (3D3 Consistent with previous reports (24, 25), chronically activated FITC), CD14 (M5E2 FITC), CD116 (M5D12 FITC), and CD20 (2H7 ϩ FITC) all from BD Pharmingen. The FITC-labeled Abs were used as a CD4 T cell clones acquired constitutive cell surface expression of ϩ ϩ the class II MHC determinants HLA-DR, HLA-DP, and HLA-DQ

combined mixture to ensure that no APCs were isolated. HLA-DR CD4 Downloaded from T cell populations were sorted on a FACS ARIA (BD Biosciences) to (Fig. 1, A and B, data not shown). This basal level of class II Ͼ typically 98% purity in post sort analysis. expression was up-regulated by polyclonal activation of T cell CD4ϩ T cell clones and generation of EBV transformed B cell clones with ␣CD3 and ␣CD28 (Fig. 1). After 5 days of activation, lines these clones expressed cell surface class II MHC equivalent to that of EBV-transformed B cells. Additionally, activated CD4ϩ T cell T cell clones were generated from the peripheral blood of healthy individ- clones up-regulated CIITA and HLA-DR␣ mRNA after treatment uals (DRB1*01,03, DRB3*; DRB1*15,13, DRB3*, DRB5*; DRB1*15,07, http://www.jimmunol.org/ DRB4*, DRB5*). CD62LϩCD25ϪHLA-DRϪCD4ϩ cells were sorted at with ␣CD3 and ␣CD28 (Fig. 1, D and E), in tandem with cell one cell per well into 96-well U-bottom plates containing 2 ϫ 105 irradi- surface protein expression. We confirmed that expression of class ated (5000 rad) PBMC, 50 U/ml recombinant human IL-2, 1 ␮g/ml ␣CD3 II MHC was endogenous by metabolic labeling with [35S]-methi- ␮ ␣ (Hit3a), and 1 g/ml CD28, a modification to previously published pro- onine, followed by immunoprecipitation of HLA-DR in resting cedures (10). Clones were expanded for 30 days before restimulation in ␣ 96-well U-bottom plates coated with 50 ␮lof1␮g/ml each ␣CD3 and activated T cell clones (Fig. 1F). Synthesis of HLA-DR and (UCHT1) and ␣CD28 (3D10) diluted in PBS, incubated for2hat37°C, ␤-chains, as well as Ii isoforms p41 and p31, was up-regulated at and then washed once in PBS. Clones were expanded for 4 wk in 3 and 5 days postactivation in these cells, although SDS-stable 2 XVIVO-15 medium and then subsequently restimulated in 25 cm flasks dimer formation was not observed for all individuals assayed. coated with ␣CD3 and ␣CD28. Clones underwent at least two more rounds ϩ of expansion and restimulation with ␣CD3 and ␣CD28 before assay. EBV- These results indicate that human CD4 T cell clones synthesize by guest on September 26, 2021 transformed B cell lines were generated from PBMC as previously de- and express class II MHC, corroborating previous reports of ϩ scribed (20). HLA-DR expression by CD4 T cell clones (7, 9). expression analysis Class II MHC processing in CD4ϩ T cell clones requires RNA was isolated by the RNeasy Mini Kit, RNase-free DNase procedure cysteine proteases (Qiagen), converted to cDNA via reverse transcription by random hexam- ϩ ers and Multiscribe RT using the TaqMan RT-PCR kit from Applied Bio- We detected the Ii fragment CLIP on the surface of CD4 T cell systems and diluted 1/10 before use. TaqMan PCR were performed in clones (Fig. 1C) and Ii isoforms bound to HLA-DR complexes triplicate using TaqMan Fast Universal PCR Master Mix to amplify human immunoprecipitated from [35S]-methionine-labeled cells (Fig. 1F). CIITA (Hs00172106_m1), HLA-DR␣ (Hs00219575_m1), CatB ϩ (Hs00157194_m1), CatL (Hs00377632_m1), CatS (Hs00356423_m1), and Given these findings, we hypothesized that CD4 T cell clones, ␤ like professional APC, use the endosomal class II MHC processing CatV (Hs00426731_m1), GADPH (Hs99999905_m1), or 2M (4326319E) on an ABI 7500 instrument (all from Applied Biosystems). Difference in Ct pathway. In this pathway, HLA-DR ␣- and ␤-chains are assembled ␤ values normalized to 2M for each sample as per the formula: Normalized on the Ii chaperone, which must be processively cleaved by resi- expression ϭ 0.5ˆ((-C value of ␤ M–C value of target)*1000). t 2 t dent proteases in the endo-lysosomal compartment to allow pep- Pulse-chase and immunoprecipitation tide loading. To identify a proteolytic requirement for class II MHC ␣␤ dimer Radiolabeling experiments were conducted as previously described (21). In ϩ brief, cells were washed and incubated in methionine-free medium (Life formation in CD4 T cell clones, we treated these cells with the Technologies) for1hat37°C. During the last 10 min of starvation, cells pan-cysteine protease inhibitor leupeptin or the pan-aspartyl pro- were treated with control amounts of DMSO, 1 mM leupeptin (all from tease inhibitor pepstatin, pulsed the treated cells for 45 min with Sigma-Aldrich), or LHVS as indicated. These concentrations of inhibitor [35S]-methionine to label nascent proteins, and then chased the were maintained throughout the remainder of the pulse and chase. Follow- radiolabeled proteins for up to 3 h before immunoprecipitation of ing starvation, cells were pulsed with 0.5 mCi of [35S]methionine (PerkinElmer) for 45 min and then chased with regular culture medium for HLA-DR complexes and associated Ii fragments. In EBV-trans- the time indicated. After the chase, cells were washed once with PBS and formed B cells, treatment with leupeptin but not pepstatin inhibited lysed. Precleared lysates were incubated with Tu¨36 and protein A-agarose Ii cleavage; blockade of cysteine proteases in these cells generated beads (Roche) to immunoprecipitate class II MHC molecules. Samples a 22 kDa peptide (the p22 leupeptin induced peptide) and pre- were boiled in sample buffer before analysis on a 12.5% SDS-PAGE gel. vented successful SDS-stable dimer formation (␣␤:peptide) (16) Substrate-specific protease activity assays and active-site (Fig. 2A). Likewise, leupeptin treatment, but not pepstatin treat- labeling ment, inhibited HLA-DR maturation in a donor-matched CD4ϩ T The enzyme activity of , cathepsin B plus L, cathepsin S, and cell clone (Fig. 2B). After2hofchase, the Ii fragments p22 and asparagine endopeptidase (AEP) was measured in vitro as described (21, p24 could be resolved in T cell clones treated with leupeptin alone 22). DCG-04 labeling was performed as described (21, 23). but not in those treated with pepstatin. Leupeptin treatment also The Journal of Immunology 947 Downloaded from http://www.jimmunol.org/

FIGURE 2. Although Ii processing in both CD4ϩ T cells and B cells is leupeptin-dependent, CD4ϩ T cell clones exhibit a faster rate of ␣␤:peptide formation and increased Iip24 fragment formation. A donor-matched EBV- transformed B cell line (A) and CD4ϩ T cell clone (B) were treated with leupeptin (1 mM), pepstatin A (10 ␮M), or control amounts of DMSO before pulse with [35S]-methionine and chase with unlabeled medium for the times indicated. HLA-DR complexes and associated Ii fragments were by guest on September 26, 2021 immunoprecipitated and analyzed by SDS-PAGE under denaturing condi- tions. The class II MHC ␣- and ␤-chains are indicated, as well as the Ii isoforms p41 and p31, and the Ii degradation intermediates p24, p22, and p10. C, Quantification of Ii fragment formation (mean Ϯ SD). FIGURE 1. The constitutive expression of class II MHC in CD4ϩ T cell clones is up-regulated following activation. Donor-matched BCL and a representative CD4ϩ T cell clone were activated with cross-linking ␣CD3/ ␣CD28 and assayed for cell surface expression of total class II MHC (A), processing in both B and T cells, but our data argues for differ- HLA-DR (B), and HLA-DR:CLIP (C) complexes. Isotype control shown in ences in the proteolytic repertoire between these two cellular sub- gray. Also shown, quantification of relative mRNA expression in BCL and sets that could subtly alter class II processing. CD4ϩ T cell clones by Taqman RT-PCR of the class II transcriptional ␣ activator (D) and HLA-DR -chain (E) in BCL and T cell clones. F, En- ϩ dogenous synthesis of HLA-DR␣, HLA-DR␤, and Ii was confirmed by AEP does not contribute to Ii processing in CD4 T cell clones metabolic [35S]-methionine labeling, followed by immunoprecipitation of We have previously reported that asparagine endopeptidase inhi- HLA-DR complexes with the conformationally specific Ab Tu¨36, and bition results in development of p24 in leupeptin-treated BCL and SDS-PAGE under the conditions indicated (B, denatured by boiling; NB, loss of p22 in BCL treated with both leupeptin and pepstatin (21). nonboiled). Histogram and mean fluorescence intensity (MFI) of represen- ϩ As we observed similar Ii cleavage fragment patterns in CD4 T tative samples shown for A–D; for E and F, graphs represent mean Ϯ SEM, n ϭ 4 donors. cell clones treated with leupeptin and leupeptin/pepstatin (Fig. 3A), we hypothesized that these cells lack AEP activity. Indeed, AEP mRNA was barely detectable in CD4ϩ T cell clones, as compared with donor matched BCL, or in CD19ϩ and CD4ϩ HLA-DRϩ T disrupted SDS-stable dimer formation in T cell clones. These find- cells ex vivo (Fig. 3A). Furthermore, we could not detect AEP ings indicate that cysteine protease activity is required for success- ϩ activity in CD4 T cell clones by direct enzymatic assay (Fig. 3B). ful class II MHC processing in both B and CD4ϩ T cells. These findings show that AEP is not significantly expressed in Although we found cysteine protease activity necessary for class ϩ CD4 T cells and therefore does not play a role in class II MHC II MHC maturation in both B and T cells, other proteases can processing and Ag presentation in these cells. cleave Ii and alter the kinetics of class II processing. We observed in some clones the formation of p24 upon leupeptin treatment and ϩ reduced formation of p22 with upon treatment with both leupeptin Cathepsin S inhibition blocks Ii cleavage in CD4 T cell clones and pepstatin (Fig. 2, B and C), phenomena that were not observed CatS plays a critical role in class II MHC processing in murine and in donor-matched BCL. Cysteine proteases may dominate class II human B cells, dendritic cells, and macrophages (17, 26, 27). 948 INVARIANT CHAIN PROTEOLYSIS IN HUMAN CD4ϩ T CELLS

tin resulted in blockade of invariant chain degradation and the formation of the fragments Iip22 and Iip24 (Fig. 4, A and B). Leupeptin furthermore significantly reduced the total percentage of SDS-stable dimer formation (to 26.3 Ϯ 8.02%; mean Ϯ SD; n ϭ 4) (Fig. 4C), confirming a requisite role for cysteine proteases in class II MHC maturation in T cell clones. Similar to treatment with leupeptin, selective inhibition of CatS successfully impaired HLA-DR maturation in both CD4ϩ T cells and BCL (Fig. 4, data not shown). This treatment resulted in both FIGURE 3. CD4ϩ T cells do not express AEP. A, Quantification of invariant chain cleavage fragment generation and the reduction of ϩ ϩ Ϯ AEP mRNA in B cell lines, CD4 T cell clones, and ex vivo CD19 B SDS-stable dimers (to 27.5 12.4% SDS-stable dimer formation; cells and CD3ϩ CD4ϩ HLA-DRϩ T cells isolated from two donors mean Ϯ SD; n ϭ 4) (Fig. 4C). Given the impact of 5 nM LHVS (mean Ϯ SD). B, AEP activity in postnuclear lysate of donor-matched BCL treatment on nascent ␣␤:peptide formation, we conclude that CatS ϩ (B cell) and a CD4ϩ T cell clone (T cell), as measured by hydrolysis of the is required for successful Ii cleavage in CD4 T cells. synthetic peptide substrate Z-Ala-Ala-Asn-AMC (mean Ϯ SD).

Cathepsin S is down-regulated in activated CD4ϩ T cell clones Given the blockade in invariant chain proteolysis imposed by cys- Dendritic, B, and ␥␦ϩ T cells modulate class II MHC processing ϩ teine protease inhibition in CD4 T cell clones (Fig. 2B), we hy- and presentation early postactivation (30, 31). To determine the Downloaded from pothesized that CatS activity is required for processing of class II effect of short-term activation on class II MHC expression in MHC in these nontraditional APC. CD4ϩ T cells, we stimulated T cell clones with PMA and iono- To test this hypothesis, we measured Ii processing and SDS- mycin or ␣CD3 and ␣CD28 for 18 h and then stained for cell ϩ stable dimer formation in CD4 T cell clones treated either control surface HLA-DR (Fig. 5A). At this early timepoint, activated amounts of DMSO, leupeptin, or 5 nM LHVS (Fig. 4). We used CD4ϩ T cell clones expressed less HLA-DR than resting clones

this low concentration of LHVS to selectively inhibit CatS (17, 28, (down to 34.7 Ϯ 1.0% from 54.7 Ϯ 1.6%; p Ͻ 0.0001; mean Ϯ http://www.jimmunol.org/ 29). We pulsed inhibitor-treated T cell clones with [35S]-methio- SEM; n ϭ 35 clones). These findings are consistent with studies nine and then chased the radiolabeled proteins for 6 h with unla- showing that PMA treatment of previously activated, and therefore beled medium. After chase, we immunoprecipitated properly class II MHCϩ, T cells reduces class II MHC expression on these folded HLA-DR ␣␤ and Ii complexes from these lysates and re- cells (32). solved both Ii cleavage fragments and SDS-stable dimer formation The loss of HLA-DR from the cell surface coincides with the with SDS-PAGE. As observed previously, treatment with leupep- reduction of both CatS expression and activity, which were sig- nificantly decreased in T cell clones after 18 h of activation (Fig. 5, C and D). Down-regulation of cysteine protease expression and activity was restricted to CatS, as CatB and, to a lesser extent, CatL by guest on September 26, 2021 were up-regulated post activation in both clones and HLA-DRϩ CD4ϩ T cells ex vivo (data not shown). As CatS is required for the optimal formation of nascent HLA-DR complexes (Fig. 4), down- regulation of this protease could account for the reduction of cell surface class II MHC postactivation. Such a direct relationship would imply that CatS actively maintains class II MHC on the cell surface or indicate that T cells rapidly internalize and degrade class II molecules. To determine the consequence of CatS ablation on HLA-DR expression, we treated CD4ϩ T cell clones for 18 h with leupeptin or LHVS and stained for cell surface HLA-DR. Treatment of CD4ϩ T cell clones with either leupeptin or LHVS was insufficient to reduce the percentage of HLA-DRϩ cells in the clones tested (Fig. 5, E–G). The mean density of HLA-DR molecules was re- duced in some clones after treatment. Significant down-regulation of intracellular HLA-DR was observed only in clones treated with leupeptin (from 88.06 Ϯ 15.92 to 60.48 Ϯ 11.3; mean Ϯ SEM; n ϭ 12; p ϭ 0.0365), but not LHVS (65.78 Ϯ 15.6 and 74.48 Ϯ 18.59), and down-regulation of extracellular HLA-DR expression was not statistically significant (from 20.42 Ϯ 5.97 to 13.8 Ϯ 3.22; mean Ϯ SEM; n ϭ 12; p ϭ 0.0576) (Fig. 5H). Therefore, short- FIGURE 4. Inhibition of cathepsin S blocks nascent ␣␤:peptide dimer term cysteine protease inhibition did not directly reduce cell sur- ϩ ϩ formation in CD4 T cell clones. A and B, CD4 T cell clones were face HLA-DR expression. Of course, continued inhibition of CatS 35 pulsed with [ S]-methionine and chased for 6 h after treatment with contributes to loss of class II MHC over time because nascent leupeptin, 5 nM LHVS, or control amounts of DMSO. HLA-DR ␣, ␤, complex formation is blocked (data not shown), but our data in- and associated Ii fragments were then immunoprecipitated, denatured by boiling (where indicated, B), and resolved via SDS-PAGE (NB, dicates that this mechanism cannot by itself account for the ob- nonboiled). A and B are representative autoradiographs of single T cell served loss of HLA-DR early postactivation. These results are con- clones derived from two different healthy donors. C, Quantification of sistent with the extended half-life of class II MHC molecules (33) p ϭ 0.0014; mean Ϯ SEM; and do not provide evidence for rapid turnover of class II in T ,ءء .relative SDS-stable dimer formation n ϭ 4 clones from two donors). cells. The Journal of Immunology 949

FIGURE 5. Down-regulation of cathepsin S ex- pression and activity decreases cell surface CLIP ex- pression in recently activated CD4ϩ T cell clones. A and B, Cell surface CLIP and HLA-DR expression in a panel of CD4ϩ T cell clones from a single donor (A) and in a single representative clone (B), at rest and after 18 h of mitogenic activation with PMA and iono- p Ͻ 0.0001). C, CatS mRNA expression in ,ءءء) mycin a panel of CD4ϩ T cell clones from a single donor, at p ϭ 0.0026; relative ,ءء) rest and after 18 h of activation to BCL expression). D, Down-regulation of Cat S in two representative clones after 18 h of activation, as indicated by DCG-04 labeling and mRNA expression ϩ ␤ Downloaded from (relative to 2 microglobulin). E and F, CD4 T cell clones treated for 18 h with the pan-cysteine protease inhibitor leupeptin (1 mM), 200 nM LHVS (CatB/L/S inhibition), 5 nM LHVS (CatS alone), or control amounts of DMSO before staining and flow cytometry. G, Percentage of CLIP positive cells and HLA-DR pos- ;p ϭ 0.0007 ,ءءء) itive cells after 18 h of inhibition mean Ϯ SD). H, MFI of CLIP and HLA-DR expression http://www.jimmunol.org/ ;p ϭ 0.0365 ,ء ;p ϭ 0.0063 ,ءء) after 18 h of inhibition mean Ϯ SD). by guest on September 26, 2021

Although short-term cysteine protease blockade did not signif- regulation of cell surface CLIP but not reduction of total class icantly reduce cell surface HLA-DR expression, LHVS treatment II MHC. did result in the loss of the invariant chain fragment CLIP from the cell surface (Fig. 5, E, G, and H). Indeed, inhibition of CatS alone ϩ with 5 nM LHVS reduced expression of CLIP in the HLA-DR B, L, and S are differentially expressed in CD4 binding pocket (from 21.6 Ϯ 0.6% to 10.2 Ϯ 0.4%; p ϭ 0.0007; T cell clones and BCL Ϯ ϭ mean SEM; n 32 clones) (Fig. 5G). Continued treatment with Cysteine proteases other than CatS have been implicated in both LHVS for 48 h also resulted in significant down-regulation of invariant chain proteolysis and peptide epitope generation (18, 36, Ϯ Ϯ ϭ CLIP (34.7 1.9% control to 7.2 0.5% 5 nM LHVS; p 37). We wished to identify differences in lysosomal protease ex- 0.0005; mean Ϯ SEM; n ϭ 12 clones, data not shown), without pression and activity that could contribute to class II MHC pro- concomitant loss of total cell surface HLA-DR. As cell surface ϩ cessing in CD4 T cells, as compared with B cells. We focused CLIP was also down-regulated early postactivation, even in indi- our work on cathepsins B, L, and S, as these lysosomal cysteine vidual clones with limited total HLA-DR down-regulation (Fig. proteases have been implicated in class II MHC processing and 5B), CatS likely plays a role in the maintenance of CLIP on the cell presentation (38) and are also expressed in CD4ϩ T cells (Fig. 6A surface. A ϩ ϩ CLIP fragments bound in the peptide-binding groove of class II and Fig. 7 ). We found that resting CD4 HLA-DR T cell clones MHC heterodimers are exchanged for antigenic peptide through express less CatS mRNA (Fig. 6A) and contain less CatS activity the action of the loading molecule HLA-DM (12), although CLIP (Fig. 6B) than donor-matched BCL. We verified these patterns of exchange can also occur in the absence of HLA-DM (34, 35). To expression in cells ex vivo and confirmed that peripheral blood HLA-DRϩ CD4ϩ T cells contained fewer CatS transcripts and less verify that loss of CLIP from the cell surface was not due to dif- ϩ ferences in HLA-DM expression, we stained protease inhibitor- active CatS than CD19 B cells (Fig. 6, C and D). ϩ treated CD4ϩ T cell clones for intracellular HLA-DM. HLA-DM Conversely, CD4 T cell clones expressed more CatB and CatL expression remained constant, while CLIP expression decreased in message and activity than BCL, but this significant difference in these cells (Fig. 5F). Although the possibility remains that changes expression could not be extended to ex vivo cell populations (Fig. in HLA-DM localization or kinetic activity could impact peptide 6, C and D; Fig. 7; data not shown). Furthermore, CatL transcripts ϩ editing, there is no evidence to date that protease inhibitors affect were only found in a subset of CD4 T cell clones; lack of CatL such action. We therefore conclude that short-term inhibition of mRNA did not correlate with the absence of cell surface HLA-DR CatS activity in activated CD4ϩ T cell clones results in down- on a given clone. 950 INVARIANT CHAIN PROTEOLYSIS IN HUMAN CD4ϩ T CELLS

FIGURE 6. Cathepsin S expression and activity are reduced in CD4ϩ T cells, as compared with CD19ϩ B cells. A, CatS mRNA expression in CD4ϩ T cell clones (T cell) as compared with a donor-matched EBV- transformed B cell line (B cell) (mean Ϯ SD). B, Measurement of CatS enzyme activity in postnuclear lysates of a CD4ϩ T cell clone (F) and donor matched B cell line (E) as indicated by cleavage of the substrate Downloaded from Bos-Val-Leu-Lys-AMC. C, CatS and CatB mRNA expression in periph- eral blood CD19ϩ B cells and CD3ϩCD4ϩHLA-DRϩ T cells (mean Ϯ ϭ ␤ SEM; n 5; relative to 2 microglobulin). D, Cysteine protease activity in a BCL, CD4ϩ T cell clone, ex vivo CD19ϩ B cells and CD3ϩCD4ϩ T cells from a representative donor, assayed with the activity-based probe

DCG-04. ϩ ϩ

FIGURE 8. Peripheral blood CD4 HLA-DR T cells express less http://www.jimmunol.org/ CLIP than CD19ϩ B cells. A, Cell surface expression of CLIP and intra- HLA-DRϩCD4ϩ T cells ex vivo express less CLIP than CD19ϩ cellular expression of HLA-DM in PBMC purified from a representative donor, gated on CD3ϩCD4ϩHLA-DRϩ T cells or CD19ϩHLA-DRϩ B T cells ϩ cells (CD3 gate shown for CD4 cells; MFI indicated; isotype control ;p ϭ 0.001 ,ءء ;We observed that HLA-DRϩCD4ϩ T cells express less active shown in gray). B, CLIP mean fluorescence intensity (MFI ϩ CatS than CD19ϩ B cells (Fig. 4, C and D). To determine the mean Ϯ SEM; n ϭ 3); C, relative frequency of CLIP cells (expressed as ϩ ϩ p ϭ 0.002; mean Ϯ SEM; n ϭ 5) in ,ءء ; consequence of this reduced CatS expression on CLIP presentation %CLIP /%HLA-DR CD3ϩCD4ϩ T cells or CD19ϩ B cells ex vivo; and D, the ratio of CLIP to in these cells, we stained for cell surface CLIP in peripheral blood. ϩ ϩ ϩ ϩ ϩ ϩ HLA-DR in CD3 CD4 HLA-DR T cells vs CD19 B cells (expressed

HLA-DR CD4 T cells ex vivo express less cell surface CLIP by guest on September 26, 2021 .(p ϭ 0.0047; mean Ϯ SD ,ءء ;ϩ ϩ as (CLIP (MFI)/HLA-DR (MFI))*100 than HLA-DR CD19 B cells (12.4 Ϯ 0.3 vs 60.4 Ϯ 0.1 CLIP MFI, mean Ϯ SEM, n ϭ 3) (Fig. 8). The low level of CLIP ex- ϩ ϩ pression on HLA-DR CD4 T cells was not due to overexpres- surface, while CD19ϩ B cells, which express higher levels of sion of HLA-DM in this subset (Fig. 8A). Indeed, intracellular CatS, constitutively express cell surface CLIP. HLA-DM expression was lower in CD4ϩ T cells than in CD19ϩ B cells. These data suggest that the modest CatS activity in HLA- ϩ ϩ Discussion DR CD4 T cells is not sufficient to maintain CLIP on the cell ϩ HLA-DR expression on CD4 T cells was observed several de- cades ago, but the mechanisms of class II MHC proteolytic regu- lation in these cells remain undefined. With the use of protease inhibitors, we examined the requirements for successful Ii process- ing and class II MHC complex presentation in constitutively HLA- DRϩCD4ϩ T cell clones. Our results demonstrate that CatS is crucial for Ii proteolysis in these cells. Specific inhibition of CatS with low concentrations of LHVS resulted in the formation of Ii cleavage intermediates and blocked the successful generation of ␣␤:peptide complexes (Fig. 4). Furthermore, we find that down-regulation of CatS in early ac- tivated HLA-DRϩ CD4ϩ T cells results in the loss of CLIP from the cell surface but does not significantly reduce total class II MHC presentation (Fig. 5). Therefore the prevailing consequence of variable CatS expression is alteration of the peptide repertoire: HLA-DRϩ CD4ϩ T cells both in vitro and ex vivo express less CatS and therefore maintain less CLIP on the cell surface than donor-matched B cells (Figs. 6 and 8). A lower level of CatS ϩ ϩ expression in CD4 T cells does not necessarily precipitate equiv- FIGURE 7. CD4 T cells express more active cathepsin B and cathep- sin L than B cells. mRNA expression relative to donor-matched BCL (A) alently low class II MHC expression. It is likely that alternative and in a panel of clones derived from a single donor (B). C, Hydrolysis of mechanisms of class II MHC regulation such as complex traffick- the synthetic peptide substrates Z-Arg-Arg-AMC (CatB specific) and Z- ing and targeted degradation also contribute to expression levels Phe-Arg-AMC (CatB/L) in lysates treated with the CatB inhibitor and are differentially active in B cells and T cells. In fact, our data CA074Me (100 nM) or with control amounts of DMSO. suggest that reduction of class II MHC expression in T cells The Journal of Immunology 951 postactivation is predominantly controlled by a mechanism other Disclosures than CatS down-regulation (Fig. 5). 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