Immunology Letters 102 (2006) 132–140

Idiotypic T cells specific for Morbillivirus nucleocapsid protein processand present their TCR to cognate anti-idiotypic CD8+ T cells

Girdhari Lal, M.S. Shaila, Rabindranath Nayak∗Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

Received 10 April 2005; received in revised form 20 August 2005; accepted 20 August 2005Available online 12 September 2005

Abstract

CD8+ T cells are activated by the presentation of antigenic peptide through MHC class I molecules. Newly synthesized proteins formed asdefective ribosomal products (DRiPs) can act as a major source of antigenic peptides for MHC class I presentation pathway. Majority of thesepeptides are generated from the intracellular degradation of self antigens. In the present study, we have shown that newly synthesized T cell receptor(TCR) beta chains formed as DRiPs in T cells are ubiquitinated and degraded by the proteasomes. These TCR-DRiPs are processed and presentedby activated T cells to cognate anti-idiotypic CD8+ T cells. Presentation of TCR idiopeptide (peptide derived from the variable region of idiotypicT byT©

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CR) by activated T cells leads to Bcl-2 expression and cytokine secretion by anti-idiotypic CD8+ T cells. Presentation of intracellular antigencells may have important implications in immunoregulation, control of lymphotropic virus infection and autoimmune diseases.2005 Elsevier B.V. All rights reserved.

eywords: T-APC; Proteasome; DRiPs; Anti-idiotypic T cells

. Introduction

T cell immune responses are initiated by T cell receptorTCR) recognition of pMHCs on APCs[1]. In case of MHClass I molecules, peptides derived mostly from newly synthe-ized proteins are generated by the multicatalytic proteasome,nd transported through TAP-complex[2,3]. These peptidesan be subjected to further N-terminal trimming by as yetndefined peptidases resident in endoplasmic reticulum (ER)

o generate the 8 and 9 residue peptides required by class IHC molecules[4]. Proteasomes are abundant macromolecularssemblies present in the nucleus and cytosol and are used byll eukaryotic cells to degrade unwanted proteins, usually those

hat are damaged or misfolded. These include nuclear, cytosolicnd proteins in ER, which are re-exported into cytosol for degra-ation by proteasomes[5]. The major means of tagging proteins

or proteasomal destruction is covalent modification by multiplehains of ubiquitin (Ub) protein[6]. Proteasomes participate inhe generation of many peptides that are displayed on the sur-ace complexed with MHC class I molecules for surveillance by

the immune system[4]. Peptides are generated indiscriminafrom host gene products as well as gene products of viruseintracellular pathogens[4]. It has been shown that during tprocess of protein synthesis,∼30% newly synthesized proteiare formed as defective ribosomal products (DRiPs)[3]. TheseDRiPs act as a source of antigenic peptides to provide triggcognate T cells. Discrimination between self and non-selftides is performed at the level of the T cell receptor expreby CD8+ T lymphocytes. There is sufficient plasticity in the stem to enable T cell recognition of some self antigens, waccounts for the generation of well-defined T cell responstumors and autoimmune diseases. Apart from the endogantigen presentation to MHC class I molecules, exogenousgen can also be presented by class I either through cross-por cross-presentation. These pathways have been demonfor bacterial, viral and tumor antigens[7–10].

Endogenous self or non-self antigen presentations by dent cell types are well characterized as all nucleated cells exMHC class I molecules. Presentations of self antigen haveproposed to have deleterious effect and leads to generatautoimmune diseases. These autoimmune diseases can b

∗ Corresponding author. Tel.: +91 80 22932703; fax: +91 80 23602697.E-mail address: nayak@mcbl.iisc.ernet.in (R. Nayak).

trolled by the immunization of anti-idiotypic T cells[11–13].The variable region of the TCR peptide which gets recognizedby the self reactive T cells has been proposed to control the

165-2478/$ – see front matter © 2005 Elsevier B.V. All rights reserved.

oi:10.1016/j.imlet.2005.08.007

G. Lal et al. / Immunology Letters 102 (2006) 132–140 133

self reactive population in T cell vaccination[11]. However, thepathways by which these anti-idiotypic T cells are generated arenot well established. These idiotypic and anti-idiotypic T cellpopulations are known to be present in the healthy individual[14,15].

We have used rinderpest virus (RPV) nucleocapsid protein(N) as antigen for generating antigen-specific idiotypic T cellin mice and show that TCR of these cells are recognized byanti-idiotypic T cells. RPV is a lymphotropic virus belonging tomorbillivirus genus under paramyxoviridae family and causesa devastating disease in large ruminants[16] The nucleocapsidprotein is the most abundant protein in infected cells and inducesstrong T cell response in cattle as well as mice[16,17]. Theimmunodominant cytotoxic and helper T cell epitopes of thisprotein has been characterized[17].

In the present study, we demonstrate that during proteinsynthesis, DRiPs are formed in mouse T cells. The newly syn-thesized TCRs are part of these DRiPs, which are ubiquitinatedand degraded by the proteasome. The N antigen-specific TCR-DRiPs are processed and presented by activated idiotypic T cellsto cognate anti-idiotypic T cells. The presentation of idiopeptide(peptide derived from the variable region of idiotypic TCR) byactivated T cells leads to Bcl-2 expression and cytokine secretionby anti-idiotypic CD8+ T cells.

2. Materials and methods

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mediated lysis was performed using anti-mouse Ig, anti-mouseMHC class II (clone m5/115), anti-mouse CD11c and rabbitcomplement. Mononuclear cells were purified by density gradi-ent centrifugation on Histopaque®-1077 (Sigma, USA). Cellswere further panned on anti-mouse Ig antibody coated platefor 1 h in complete medium (RPMI-1640 supplemented with25 mM HEPES, 100�g/ml penicillin, 100�g/ml streptomycin,20�g/ml gentamicin and 10% FBS). Unbound cells were har-vested and the purity of T cells was monitored by FACS andfound to be 99% pure.

2.4. Radiolabeling and immunoprecipitation

Radiolabeling and immunoprecipitations were performed asdescribed earlier[20]. Before radiolabeling, purified T cellswere incubated for 1 h in cysteine-methionine-deficient RPMI-1640 (Sigma) supplemented with 5% dialyzed FBS and 200unit IL-2. The cells were then pelleted and re-suspended inthe same medium at 4× 107 cells/ml and metabolically labeledby pulse labeling for 20 min with 200�Ci/ml [35S] methion-ine protein labeling mix (Perkin-Elmer, USA). After the pulse,the cells were pelleted, unincorporated radiolabel removed bywashing, and the cells were resuspended at 2× 106 cells/ml inpre-warmed RPMI medium containing 5% FCS supplementedwith 200 unit IL-2. Cells were then incubated for 0, 15, 30 or60 min with or without lactacystin (5 mM) in complete RPMIm

ed in0 m-p cleia atesw useI dia)o CR� pre-c une-c mMN re-s min.T using1 ried.T ands

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.1. Animals

Balb/c mice were bred and maintained in the Centralal Facility, Indian Institute of Science, Bangalore, India.

o eight weeks old male mice were used in all experimentsowing institutional ethical use of animal protocols.

.2. Reagents

The following antibodies: purified anti-mouse I-A/I-E (clo5/114), anti-mouse CD11c (clone HL3), anti-mouse IFN�-iotin (XMG1.2), purified recombinant mouse IL-2 and isotontrol antibodies were purchased from PharMingen, Unti-mouse TCR�-Cy5PE (H57.597) and Streptavidin-Cy5ere from e-Bioscience, USA. Donkey anti-mouse IgG-Fnd donkey anti-rabbit IgG-FITC were obtained from Jack

mmunoResearch Laboratories, USA. Purified anti-mouse/� antibody (H57.597) is a generous gift from Dr. P. MarraHMI, USA. Recombinant rinderpest virus nucleocapsid

ein (N protein) and phosphoprotein (P protein) were expren E. coli and purified as described earlier[17,18].

.3. Purification of T cells

T cells were purified as described earlier[19]. Briefly, miceere killed by cervical dislocation. Inguinal, posterior axilla

nternal axillary and popliteal lymph nodes were removedingle cell suspensions were prepared. Cells were panneh in fetal bovine serum (FBS) (Gibco BRL, USA) coalate (Falcon). Unbound cells were harvested and complem

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edium at 37◦C in a 5% CO2 atmosphere.After the pulse–chase, the cells were pelleted and lys

.5% Triton X-100, 300 mM NaC1, 50 mM Tris, pH 7.4, colete protease inhibitor cocktail (PharMingen, USA). Nund debris were eliminated by centrifugation. The cell lysere pre-cleared first, by an incubation with rabbit anti-mo

gG (Sigma) and Protein-A agarose (Bangalore Genie, Invernight at 4◦C. Lysates were incubated with anti-mouse T/� antibody (H57.597) for 2 h. Immune complexes wereipitated using Protein-G agarose (Sigma). Aggregate immomplexes were washed three times in 0.5% NP40, 30aC1, 50 mM Tris and pH 7.4. The beads were thenuspended in Laemmli sample buffer and boiled for 3he immunoprecipitates were analyzed by electrophoresis2% SDS-PAGE. The gels were treated with salicilate, and dhe gels were exposed to phosphoimager plate for 16 hcanned.

.5. Trichloroacetic acid (TCA) precipitation

TCA precipitations performed as described earlier[3].riefly, aliquot of cell lysates were spotted onto WhatmF/C filters, washed with 10% (w/v) TCA and twice more w0% ethanol then dried. The incorporated radioactivity wasured in a scintillation spectrometer.

.6. Western blotting

Purified mouse T cells (4× 106 cells/sample) were culturen complete RPMI medium in presence or absence of laystin (5 mM) for 3 h. Cells were lysed in lysis buffer (50 m

134 G. Lal et al. / Immunology Letters 102 (2006) 132–140

Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 5 mM EDTA, 1%NP-40 and protease inhibitor cocktail). Lysates were preclearedwith Protein A-agarose bead (Bangalore Genie) and immuno-precipitated with anti-TCR�/� antibody (H57.597) and ProteinG-sepharose bead (Sigma). The beads were washed four timeswith washing buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl,1 mM MgCl2, and 0.5% NP-40) and re-suspended in incubationbuffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol and0.01% bromophenol blue) and boiled for 5 min and run on 12%SDS-PAGE. The protein was transferred to nitrocellulose mem-brane and immuno-stained using rabbit anti-ubiqitin antibody(FL-76; Santa Cruz, USA)/anti-rabbit IgG-HRP conjugated anti-body and visualized with ECL Plus (Roche, Germany).

2.7. Confocal microscopy

Naive mouse T cells were cultured in presence or absenceof lactacystin (5 mM) in complete RPMI medium with 200 Urecombinant mouse IL-2 for 3 h. Cells were washed with PBS,intracellular staining was performed for ubiquitin and TCR�,using anti-mouse TCR�-Cy5PE (H57.597) and rabbit anti-mouse ubiquitin antibody (P4D1; Santa Cruz). Cell were washedand stained with anti-rabbit IgG-FITC conjugated antibody.Cells were visualized under a confocal microscope with 63×objective (Karl Zeiss, Germany).

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dendritic cells were used as antigen presenting T cells for invitro proliferation assay or intracellular cytokine secretion byanti-idiotypic T cells.

2.11. Purification of CD4+ and CD8+ T cells

CD4+ or CD8+ T cells were purified by complement-mediated lysis as described earlier[21]. Briefly, purified T cellswere incubated with anti-mouse CD4 antibody (GK1.5) or anti-mouse CD8 antibody (3.155) on ice for 30 min. Cells werewashed and treated with rabbit complement for 20 min on ice andpurified by using Histopaque®-1077 density gradient centrifu-gation. Depletion of CD4+ T cells and CD8+ T cells was tested byFACS using anti-mouse CD4 (H129.19)-FITC and anti-mouseCD8a (53–6.7)-FITC antibody and purity was 99%.

2.12. Cell adhesion assay

Purified idiotypic T cells were treated in vitro withPMA/ionomycin for 6 h in absence or presence of lactacystin(5 mM) in complete medium. Cells were harvested, washedand stained with corboxyfluorescein diacetate succinimidylester (CFSE; Molecular Probe, USA) as described earlier[22].Briefly, purified anti-idiotypic T cells (5× 106 cells) were sus-pended in 1 ml of PBS (pH 7.4). CFSE were added to a final

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.8. In vitro activation of T cells

Purified T cells (4× 106) were activated with Phorbo2-myristate 13-acetate (PMA) (500 ng/ml) and ionomy50 ng/ml) for 6 h in complete medium in a final volume.5 ml in each 24 well plate. Cells were harvested and waith Hank’s balanced salt solution (Sigma).

.9. Generation of idiotypic T cells

Mice were subcutaneously immunized with N protein (50�g)n Freund’s complete adjuvant, boosted after 3 weeks wit

rotein (50�g) in Freund’s incomplete adjuvant. After 1 wef booster, draining lymph node cells were harvested and T

rom these mice were purified as described above. The anpecificity of T cells was detected by in vitro proliferation ass

.10. Generation of anti-idiotypic T cells

Purified irradiated idiotypic T cells (20× 106 cells) werenjected intraperitoneally into syngenic Balb/c mice. Afteeeks, mice were boosted with same number of irradiated

ypic T cells. Lymph node T cells from immunized mice weurified as described above and used as anti-idiotypic T che specificity of anti-idiotypic T cells were checked by usendritic cells pulsed with apoptotic idiotypic T cells, and th

Adhesion efficiency (%)=

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concentration of 5�M and incubated for 10 min at 37◦C by mix-ing twice in between. Cells were washed with PBS contai20% FBS followed by complete medium. CFSE labeled aidiotypic T cells (1× 105 cells) were incubated with un-labelidiotypic T cells (4× 105 cells) or control T cells in a final voume of 200�l complete medium for 30 min with intermitteshaking at 37◦C. Cells were disturbed by tapping and transfeto a glass slide and visualized under a fluorescence micro(Lieca, Germany). Total number of cell clumps (clumps haat least 1 CFSE labeled cells with >3 unlabeled cells) prein a field were counted. More than eight random fields wexamined. Adhesion efficiency were calculated as given be

∑Total number of cell clumps with CFSE label

Total number of cell clumps with or without CFSE label× 100

2.13. Cytokine assay

Rinderpest virus nucleocapsid protein specific idiotypcells and anti-idiotypic T cells were purified as described abIdiotypic T cells were further fractionated into CD4+ or CD8+ Tcells using complement-mediated lysis as described abovetypic T cells were activated in vitro with PMA/ionomycin for 6and fixed with 0.2% paraformaldehyde and used as antigesenting T cells. Anti-idiotypic CD8+ T cells (1× 105 cells/well)were cultured with fixed idiotypic T cells (4× 105 cells/well)in 200�l of complete RPMI medium in U bottom 96 weplates. Cells were incubated for 48 h and supernatantscollected and secretion of IL-2 was detected using ELISA(e-Bioscience, USA).

G. Lal et al. / Immunology Letters 102 (2006) 132–140 135

2.14. Bcl-2 expression

Idiotypic T cells were in vitro activated with PMA andionomycin in presence or absence of lactacystin (5 mM) for12 h. Cells were washed and fixed with 0.2% paraformalde-hyde for 5 min on ice. Cells were washed and used as anti-gen presenting cells (T-APCs). Anti-idiotypic CD8+ T cells(1.0× 105 cells/well) were cultured with un-activated or acti-vated idiotypic T cells (3.0× 105 cells/well) in 200�l completemedium in 96 well U bottom plates. Plates were incubatedfor 8 h at 37◦C in 5% CO2 incubator. Brefeldin-A (10�g/ml)was added in last 6 h of incubation. Cells were harvested andintracellular staining for Bcl-2 was performed and IFN-� wereperformed as described earlier[23].

3. Results

3.1. DRiPs are formed in T cells

DRiPs are the major source of antigenic peptides for MHCclass I molecules. Proteasomal activity can be blocked by lac-tacystin leading to the accumulation of poly-ubiquitinated pro-teins[24,25]. Increased protein degradation is associated withenhanced generation of antigenic peptides[26].

To show that newly synthesized proteins are formed as DRiPsi 35

m ystiC ther as aft prot ch isi parto s. Ith andp titute∼

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3.2. DRiPs formation from newly synthesized TCRmolecules

To detect the formation of TCR as DRiPs in naive T cells, cellswere metabolically labeled with35S-methionine and chased fordifferent time points. TCR was immunoprecipitated using anti-TCR �/� antibody and immunoprecipitates were analyzed bySDS-PAGE. Results shown inFig. 2A and B demonstrate thatin lactacystin treated T cells degradation of TCRs is inhibited,indicating that TCR beta chain is formed as DRiPs in T cellsduring normal protein synthesis. In order to see if these DRiPsare ubiquitinated, TCR molecules from lactacystin treated cellswere immunoprecipitated using anti-TCR� chain antibody andWestern blotted using anti-ubiquitin antibody. Results givenin Fig. 2C show that as time increases, ubiquitinated TCRmolecules accumulate in T cells. We further studied the local-ization of ubiqitinated TCR proteins by confocal microscopyin naıve T cells treated with lactacystin.Fig. 2D shows co-localization of TCR with ubiquitin molecules inside the cells.Our results also correlate with previously reported result for co-localization of ubiquitin with viral and cellular proteins in HeLacells or human osteosarcoma cells[27] or mouse langerhanscells and dendritic cells[2].

3.3. T cell–T cell interaction

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n T cells, naive T cells were metabolically labeled withS-ethionine and chased in presence or absence of lactacell lysates were precipitated with trichloroacetic acid and

adioactivity in precipitated total proteins was measuredunction of time after treatment with lactacystin.Fig. 1 revealshat newly synthesized protein of T cells are degraded viaeasome (about 25% of total synthesized proteins), whinhibited by lactacystin. Thus, this experiment confirms thatf newly synthesized T cell proteins are formed as DRiPas been reported earlier that in dendritic cells, HeLa cellsooled lymph node cells, newly synthesized proteins cons30% as DRiPs[3].

ig. 1. Detection of cellular DRiPs in T cells. Purified naıve mouse T cellere metabolically labeled with35S-methonine and chased for upto 120

n presence or absence of lactacystin (5 mM). The radiolabeled proteinsecovered from cell lysate by TCA precipitation. (�) Represents the proterom cells treated with lactacystin and (�) represents proteins from cells withoactacystin.

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To provide evidence for the processing and presentatioCR by T cells to cognate T cells, cell adhesion assay

diotypic and anti-idiotypic T cells was performed. In this expment, N specific anti-idiotypic T cells were labeled withuorescent dye, CFSE and mixed with unlabeled N specificypic T cells. The specific adhesion using two types of T cere monitored under a fluorescence microscope. The riven in Table 1show that activated idiotypic T cells exhiighest adhesion efficiency (17%) in comparison to non-specells (7%), activated idiotypic T cells treated with lactacy

9%) or cycloheximide (6%). The specificity of adhesionested using irrelevant idiotypic T cells (specific for rinderpirus P protein) in conjunction with N protein-specific andiotypic T cells, which did not show significant cell adhesion

able 1ell adhesion of idiotypic T cells with anti-idiotypic CD8+ T cells

nti-idiotypic CD8+ T cells formingdhesion with

Adhesion efficiency (%

aive control T cells 7.0± 1.8n vitro activated control T cells 7.2± 2.2d T cells 9.0± 1.2n vitro activated Id T cells 17.4± 2.6n vitro activated Id T cells + lactacystin 9.3± 2.0n vitro activated Id T cells + cycloheximide 6.4± 2.9

FSE labeled anti-idiotypic CD8+ T cells were mixed with idiotypic T cells ihe ratio of 1:4, and incubated at 37◦C for 30 min. The idiotypic T cells wereitro activated with PMA/ionomycin in presence or absence lactacystin (5r cycloheximide (40�M). The cells in eight randomly selected fields wounted and scored for the presence of cell adhesion. Cell adhesion effi%) was calculated as described in Section2. The data are representative of tndependent experiments.

136 G. Lal et al. / Immunology Letters 102 (2006) 132–140

Fig. 2. Detection of TCR-DRiPs in T cells. (A) Purified naıve mouse T cells were metabolically labeled with35S-methonine and chased for up to 60 min in presence orabsence of lactacystin. TCR proteins was were immunoprecipitated from cell lysate using anti-mouse TCR�/� antibody. Immune complexes were resolved on SDS-PAGE and fluorographed. (B) Quantification of the35S-labeled proteins in the molecular weight 26–118 kD range plotted as phosphoImager units. (�) Representsthe protein from cells treated with lactacystin and (�) represents proteins from cells without lactacystin. (C) Immunoprecipitated proteins from lactacystin treatedsample were resolved on SDS-PAGE and transferred on nitrocellulose membrane. Ubiquitinated proteins were detected using rabbit anti-ubiquitin antibody andanti-rabbit IgG-HRP conjugated antibody. Western blot was developed using ECL kit. (D) Naıve mouse T cells were cultured in complete medium with 200 U rIL-2for 3 h in presence of lactacystin (5 mM). Cells were fixed with paraformaldehyde and intracellular staining for TCR and ubiquitin was performed using hamsteranti-mouse TCR�-Cy5PE antibody and rabbit anti-ubiquitin antibody. Cells were probed with anti-rabbit IgG-FITC antibody as secondary antibody. Cells visualizedunder confocal microscope using 63× objective.

is interesting to note that only activated T cells form efficient Tcell–T cell adhesion. It has been shown earlier that for efficientinteraction of T cells with APC, certain accessory moleculesare required[28]. These accessory molecules on T cells areexpressed in the activated stage[29,30]. It is possible that lackof expression or low level of expression of accessory moleculesin antigen-specific T cells may decrease the efficient interactionwith anti-idiotypic T cells.

3.4. Presentation of TCR molecules by T cells to cognateCD8+ T cells

MHC class I molecules are expressed in a wide variety ofcells and present antigen to cognate CD8+ T cells [31]. Allmouse T cells express MHC class I molecules on their sur-face as shown by FACS (Fig. 3A). Presentations of internallysynthesized antibody by B lymphocytes are well characterized[32,33]. We have tested the presentation of TCR molecules byT lymphocytes. Rinderpest virus nucleocapsid protein specificT cells (idiotypic T cells) were generated by immunization ofBalb/c mice with nucleocapsid protein, followed by in vitroenrichment of antigen-specific T cells by culturing the lymphnode cells for 2 weeks in presence of nucleocapsid protein.

Clonal repertoire analysis of TCR by FACS using different V�specific monoclonal antibody after enrichment shows that V�3and V�13 populations are 31 and 53%, respectively (data notshown). These idiotypic T cells were activated in vitro in pres-ence or absence of lactacystin and used as antigen presentingcells (T-APCs). Responder T cells have been generated in mouseby injecting irradiated, antigen-specific idiotypic T cells intosyngenic mice. Immunization with idiotypic T cells have beenshown to generate anti-idiotypic T cells reactive against TCRof idiotypic T cells[12,13]. CD8+ T cells from idiotypic T cellimmunized mice were purified and used as responder cells forin vitro stimulation assay. Anti-idiotypic T cells were culturedwith fixed idiotypic cells for 48 h. IL-2 in culture supernatantwas measured. The results show that activated T cells processand present their TCR to cognate CD8+ T cells (Fig. 3B). Thispresentation is sensitive to lactacystin and cycloheximide. Thesecretions of cytokines are restricted to in vitro activated antigen-specific T cells (Fig. 3B).

3.5. Bcl-2 and IFN-γ expression

It has been shown that antigen presentation by naıve or restingT cells leads to generation of anergy or apoptosis[34]. Results

G. Lal et al. / Immunology Letters 102 (2006) 132–140 137

Fig. 3. T cells present TCR molecules to cognate CD8+ T cells. (A) Expression of MHC class I molecules (H2Dd) on mouse lymph node T cells was detected byFACS after staining with anti-mouse CD3-PE and anti-mouse H2Dd-FITC antibody. (B) Purified N antigen-specific anti-idiotypic CD8+ T cells were cultured within vitro activated and fixed idiotypic T cells. Fixed control T cells or N specific idiotypic T cells activated in presence of lactacystin or cycloheximide were usedas control antigen presenting cells. Purified anti-idiotypic CD8+ T cells (responder T cells) (1× 105 cells/well) were co-cultured with fixed antigen presenting cells(T-APCs; 4× 105 cells/well) in U bottom 96 well plate for 48 h in 200�l of complete medium. Supernatants were collected and IL-2 secretion was measured byELISA. Data shown is the mean± S.E.M. of triplicates.

Fig. 4. Bcl-2 and IFN-� expressions in anti-idiotypic CD8+ T cells. Purified N specific idiotypic T cells were activated in vitro with PMA/ionomycin for 12 hin presence or absence of lactacystin (5 mM) and used as T-APCs. Purified anti-idiotypic CD8+ T cells (1× 105 cells/well) were cultured with idiotypic T cells(3.0× 105 cells/well) in 96 well U bottom plates for 8 h at 37◦C. Brefeldin-A (10�g/ml) were added in the last 6 h of culture. Intracellular staining of with anti-mouseIFN-�-biotin/streptavidin-Cy5PE and anti-mouse Bcl-2/anti-mouse-IgG-FITC were performed. Cells were acquired on FACS and data were analyzed using WinMDI2.8 software. Quadrants were set according to isotype control staining.

given inFig. 4show that a fraction of CD8+ anti-idiotypic T cellsexpress Bcl-2 and IFN-� secretion, upon triggering by activatedidiotypic T cells. In vitro stimulation of anti-idiotypic CD8+ Tcells with idiotypic T cells without in vitro activation does notlead to either expression of Bcl-2 or cytokine secretion. Thisobservation correlates with the earlier published work wherepresentation of antigen by naıve or resting T cells has been shownto induce apoptosis[35].

4. Discussion

We have studied the fate of newly synthesized TCR pro-tein and class I restricted presentation of ubiquitinated TCRbeta chain molecules. Intracellular presentation of cellular pro-tein and antibody molecules has been shown in B-lymphocytes[32,33]. Like antibody molecules, TCR molecules are also syn-thesized de novo and form DRiPs during normal protein syn-

138 G. Lal et al. / Immunology Letters 102 (2006) 132–140

thesis process. Selective proteolysis is the final step in theelaborate network of proof reading and editing processes thathave evolved to protect eukaryotic cells against the potentiallydeleterious consequences of errors, which could occur duringtranscription, and subsequent translation of mRNAs. Selectivedegradation is also required to eliminate unassembled subunitof hetero-oligomeric plasma membrane complexes, such as theheptameric TCR[35]. Eukaryotic cells contain two major prote-olytic systems: proteasomes, which are present in the cytoplasmand nucleus, and lysosomes. In contrast to the lysosome medi-ated disposal of mature or partially assembled TCR oligomers,the rapid, non-lysosomal degradation of un-assembled TCR�subunits had suggested the existence of a unique degradationsystem associated with the endoplasmic reticulum[36]. How-ever, several studies have shown that some misfolded protein inER can be degraded by cytoplasmic proteasomes following theirreverse translocation from the ER[37–39]. We demonstrate inmouse T cells that newly synthesized TCR beta chain proteinsare formed as DRiPs and this extends the earlier reported forma-tion of DRiPs and their degradation in other cell types[2,3,27].In HeLa cells transfected with cDNA of TCR� chain, more than80% of newly synthesized TCR beta chain was retained in ERand rapidly degraded in ER[40]. HeLa cells do not expressCD3 molecules. CD3 molecules require transportation of TCRpolypeptide from ER to plasma membrane as well as inhibitionof the degradation of TCR in pre-Golgi compartment[41]. Thei tranf ulea focm atios

resip haaT ratioh s ap lthh Rp pye fa isn tioo n,a nea ner atii enu sinia tiono atht rg regu owr pat

ways[50,51]. These degraded peptides can be presented throughMHC class II molecules. Activated T cells have been shown toexpress MHC class II molecules[52–56]. In the present work,we have shown that newly synthesized TCR also form DRiPs,which are processed via proteasomal degradation machinery.TCR-DRiPs are presented by MHC class I molecules to cognateanti-idiotypic CD8+ T cells. We have also shown T cell–T cellinteractions by cell adhesion assay, which are idiotypic TCRspecific, and which depend on the processing and presentationof idiotypic TCR. It is interesting to note that only in vitro acti-vated idiotypic T cells are able to induce Bcl-2 expression andcytokine secretion, which is in contrast to earlier reports wherepresentation of antigen by T cells to other T cells leads to theinduction of tolerance and anergy[34,57]. This is probably dueto lack of co-stimulatory molecules on idiotypic T cells. Acti-vated T cells are known to express co-stimulatory molecules[29,58].

Bcl-2 is an anti-apoptotic molecule, which helps in the sur-vival of cells [59]. We have shown that antigen presentationby activated mouse T cells to cognate anti-idiotypic CD8+ Tcells leads to expression of Bcl-2. It has been reported that Bcl-2 expression increase as cells get survival signal from MHCmolecules[60,61]. This observation also explains why antigenpresentation by naıve T cells to CD8+ T cells leads to apoptosis[34] while antigen presentation by in vitro activated T cells leadsto cytokine secretion.

inauickckcellsineryesstionanti-toari-tion

califiedD.nce,odyarlallore,foropeogy,is a

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ncreased retention and degradation of TCR beta chain inected HeLa cells is probably due to lack of CD3 molecnd this does not happen in T cells. Recently, using conicroscopy, it has been demonstrated that TCR ubiquitin

tart at the immunological synapse[42].We have detected that mouse T cells process and p

ntracellular self-TCR beta chain to cognate CD8+ T cells. Theresentation of antigen by T cells has been proposed ton important role in the control of autoimmune diseases[43].his control has been proposed to be due to the genef anti-idiotypic T cells and T cell–T cell interaction[12]. Itas been demonstrated that anti-idiotypic regulatory T cellart of normal T cell repertoire and can be identified in heauman subjects[14,15]. The rational design of modified TCeptides have been used for T cell mediated immunotheraxperimental autoimmune encephalitis[44]. The generation onti-idiotypic T cells after immunization of idiotypic T cellsot well defined. We hypothesize two pathways for generaf anti-idiotypic T cells. (1) During infection or immunizatiobout 90% antigen-specific T cells (idiotypic T cells) are geted in the body[45]. During the contraction phase of immuesponse, most of these cells die by apoptosis or activnduced cell death (AICD)[46]. These dead cells can be takp by the phagocytic cells, which are capable of proces

diotypic TCR and present to anti-idiotypic CD4+ as well asnti-idiotypic CD8+ T cells. It has been shown that presentaf antigen from apoptotic cells leads to immune response r

han immuno-suppression[47–49]. (2) Another pathway foeneration of anti-idiotypic T cells is by processing of downlated surface TCR by activated T cells. Activated T cells degulate their TCR and degrade them by the lysosomal

s-saln

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-nh-

Antigen presentation by T cells may play a key rolecontrolling infection by lymphotropic viruses[62]. Perhapslymphotropic virus upon infection of T cells generates a qresponse bringing in both CD4+ help and perhaps CTL attaon infected T cells. Therefore, antigen presentation by Tprovides an ideal mechanism to protect immune machinvolving T cells. We have shown that idiotypic T cells procand present their TCR to anti-idiotypic T cells via degradaby proteosomal machinery. Presentation of idiopeptide byidiotypic T cells for their functional activity is probably duepresence of peptidomimic in the anti-idiotypic TCR hyper vable region[63], and perhaps this contributes to the perpetuaand maintenance of T cell memory in absence of antigen.

Acknowledgements

We thank Dr. P. Marrack, HHMI, National Jewish Mediand Research Center, USA for generously providing puranti-mouse TCR� antibody (clone H57-597). We thank Dr.Nandi, Department of Biochemistry, Indian Institute of ScieBangalore, for the generous gift of anti-mouse CD8 antib(clone 3.155). We thank Dr. Ranga Uday Kumar, JawahNehru Center for Advance and Scientific Research, Bangafor anti-mouse CD4 antibody (GK 1.5) and Dr. Omana Joyhelp in FACS analysis. The FACS and confocal microscfacilities are supported by the Department of BiotechnolGovernment of India, under program support grants. GLsenior research fellow of Council of Scientific and IndusResearch, India. This work is supported in part by the InCouncil of Medical Research, Government of India.

G. Lal et al. / Immunology Letters 102 (2006) 132–140 139

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