Vaccine 24 (2006) 1149–1158

Booster immunization of antigen primed mice with anti-idiotypicT cells generates antigen-specific memory T cell response

Girdhari Lal, M.S. Shaila, Rabindranath Nayak∗

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

Received 15 June 2005; received in revised form 19 August 2005; accepted 26 August 2005Available online 13 September 2005

Abstract

Immunological memory is characterized by quick and enhanced immune response after re-exposure to the same antigen. In the presentwork, we have shown that anti-idiotypic T cells are generated in mice after immunization with idiotypic T cell clone or polyclonal T cellspecific for nucleocapsid protein of Rinderpest virus. Further, we have shown that N specific idiotypic T cell receptors from apoptotic idiotypicT cells are processed by bone marrow derived dendritic cells and presented to cognate anti-idiotypic T cells. Evidence has been provided forthe existence of antigen specific T cell idiotypic network in the body. Boosting with antigen specific anti-idiotypic T cells generates memoryr©

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esponse in the antigen-primed mice.2005 Elsevier Ltd. All rights reserved.

eywords: T cell memory; Anti-idiotypic T cells; Idiopeptide; Peptidomimics; Relay hypothesis

. Introduction

Immunological memory is characterized by quick andnhanced antigen specific immune response after re-exposure

o the same antigen. Maintenance of T cell memory has beenroposed to be due to triggering of the memory cells byross-reactive environmental antigen[1], self-antigen[2] orytokines[3]. The requirement for maintenance of effectiventigen specific memory is still debated[4,5]. A hypothe-is (relay hypothesis) has been proposed[6], which explainserpetuation of immunological memory without the need

or persisting antigen, cross reactive antigen or long livingemory cells. This hypothesis considers both B and T cellemory and the key event responsible for generation of mem-ry response is the interaction of idiotypic (antigen specificells or Burnet cells) and anti-idiotypic cells (cells reactiveo idiotypic determinant, Jerne cells). The anti-idiotypic cells

Abbreviations: APC, antigen presenting cells; DCs, bone marrowerived dendritic cells; Id T cells, idiotypic T cells; Anti-Id T cells, anti-

diotypic T cells

carry the peptidomimic, which drives the memory respoby triggering idiotypic memory T cells. A role for seruimmunoglobulin in the perpetuation of immunological meory has been proposed[7]. Experimental evidence in suppof this hypothesis has been provided for B cells[8–10].Anti-idiotypic T cells are thought to play a crucial rolethe maintenance and regulation of T cell memory whcarry peptidomimic in their antigenic determinants as oinally proposed in the relay hypothesis. The presencpeptidomimics for many antigens is known[11].

It has been established that anti-idiotypic T cells areof normal T lymphocyte repertoire and can be identiin healthy human individuals[12,13]. One of the peripheral regulatory mechanisms has been postulated to inrecognition of internal image by the idiotypic determinaof specific antibodies or T lymphocytes, which regulateimmune response to both foreign and self-antigens[6,14,15].

In the present work, we have used rinderpest v(RPV) nucleocapsid protein as a model antigen. RPVlymphotropic virus belonging to morbillivirus genus unParamyxoviridae family and causes a devastating disea

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

large ruminants[17]. The nucleocapsid protein is the mostabundant protein in infected cells and induces strong T cell

264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2005.08.099

1150 G. Lal et al. / Vaccine 24 (2006) 1149–1158

response in cattle as well as mice[16,17]. The immunodom-inant cytotoxic and helper T cell epitopes of this protein havebeen characterized[17]. We demonstrate that anti-idiotypicT cells are generated after immunization with antigen, anti-gen specific-idiotypic polyclonal T cells or cloned T cells. Wealso show that an idiotypic–anti-idiotypic network for T cellsexists in the body. Furthermore, boosting of antigen-primedanimals with anti-idiotypic T cells generates antigen specificmemory T cell response.

2. Materials and methods

2.1. Animals

Balb/c mice were bred and maintained in the CentralAnimal Facility, Indian Institute of Science, Bangalore, inaccordance with institutional guidelines. Six- to eight-week-old male mice were used in all the experiments.

2.2. Antigens and reagents

Recombinant nucleocapsid (N) protein and phosphopro-tein (P) of Rinderpest virus were expressed inE. coli andpurified as described earlier[17,18].

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cytes (5× 105) in 96 well plates in presence of IL-2 in a totalvolume of 200�l for 7 days. To obtain T cell clone, limit-ing dilution of cultured cells (0.5 cells/well) was performedin presence of irradiated (3000 rad) syngenic splenocytes(5× 105 cells/well) and IL-2 in a 96 well flat bottom plate ina final volume of 200�l. Single cells from a growing colonywere selected and expanded for testing antigen specificityand surface markers. Further studies were carried out usingone of the clones named 2C5.

To test the antigen specificity and MHC restriction of T cellclone (2C5), 2C5 cells (2× 106 cells) were co-cultured withirradiated (3000 rad) syngenic mouse splenocytes (4× 106

cells) and N protein (50�g/ml) with or without purified anti-mouse I-A/I-E antibody (10�g/ml; clone 2G9, PharMingen,USA) in 24 well plate with complete RPMI medium (2 mlfinal volume). Cells were incubated at 37◦C for 36 h. Cul-ture supernatant was harvested and IL-2 was measured usingIL-2 ELISA kit according to manufacturer’s instruction (e-Bioscience, USA). Generated N specific T cell clone (2C5)is CD4+ in nature and recognizes epitope of N protein, whichbelongs to the C terminal region (427–457 amino acids). ThisT cell clone uses V�3 TCR variable segment as detectedby FACS using TCR beta screening kit (PharMingen, USA)(unpublished results).

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.3. Culture medium and cytokines

RPMI-1640 medium containing 25 mM HEPES (SigmSA) supplemented with 100�g/ml penicillin, 100�g/mltreptomycin, 20�g/ml gentamicin and 10% FBS (Life Tecology, USA) (complete medium) were used. Recombiurine IL-4 was purchased from PharMingen, San Di63.OIL-2 cell culture supernatant (30%) was used as sof IL-2 supplement. L929 cell culture supernatant (30%)sed as source of GM-CSF.

.4. Generation of T cell clone

T cell clones reactive to N protein were generated accng to the procedure described earlier[19]. Briefly, Balb/c

ice were subcutaneously injected with purified recomant N protein (50�g/mouse) along with Freund’s compledjuvant. After 3 weeks, animals were boosted with 50�g of

he same antigen in Freund’s incomplete adjuvant. One-ater, inguinal lymph nodes were collected and single cellensions were made by crushing the lymph nodes bet

wo rough surfaced frosted glass slides. The lymph nells (2× 106) were co-cultured with irradiated (2000 rayngenic normal mouse splenocytes (6× 106) in 1.5 ml com-lete medium containing 50�g/ml N protein in each wef 24 well tissue culture plates. Plates were incubatedays in humidified 5% CO2 incubator at 37◦C. Cells werearvested and purified by centrifugation using Histopaq®-077 (Sigma, USA). Cells from above culture (3× 103) weregain co-cultured with irradiated (3000 rad) syngenic spl

.5. Bone marrow-derived dendritic cells (DCs)

DCs are generated as described earlier[20] with someodification. Briefly, femurs and tibiae were removed

he marrow was flushed out with RPMI-1640 using a syrith a 0.45 gauge needle. Clusters within the marrowension were disassociated by vigorous pipetting and fil

hrough a 70-�m cell strainer. Red blood cells were lysed wCK lysing buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mMDTA). Bone marrow cells were suspended in compedia. Cells were adjusted to 2× 105 cells/ml and plated o-well plates. They were cultured for up to 5 days inresence of L929 supernatant (30%) and 300 U recomburine IL-4 at 37◦C, 5% CO2. On days 4 and 7 of cultur

ame amount of L929 supernatant and IL-4 was addhe cells. Cells were harvested and purity of dendritic cas 83% as tested by FACS after staining with anti-moD11c-PE (PharMingen, San Diego, CA).

.6. In vitro generation of antigen specific Id T cells

Rinderpest virus N and P protein specific Id T cere generated from the antigen-primed mice. Mice w

mmunized with N protein (50�g) or P protein (50�g) inreund’s complete adjuvant, and boosted with N pro50�g) or P protein (50�g) in Freund’s incomplete adjuvafter 3 weeks of primary immunization. Lymph node c4× 106 cells/well) from immune mice were cultured foeek with irradiated normal mouse splenocytes (2× 106) andntigen (50�g/ml) in complete medium in 24 well plateells were harvested and purified by Histopaque®-1077

G. Lal et al. / Vaccine 24 (2006) 1149–1158 1151

density gradient centrifugation. These cells were culturedand maintained in complete medium supplemented with 30%X63.OIL-2 culture supernatant as source of IL-2. N pro-tein specific Id T cells were used as specific Id T cells,whereas P protein specific Id T cells were used as controlT cells.

2.7. Generation of P815 cell line stably expressing Nprotein

P815 cells (H-2d murine mastocytoma cell line) weremaintained in complete medium. P815 cells were seeded in60-mm tissue culture Petri plates and transfected with 10�gof the pSRN6 plasmid DNA (full length N gene cloned inpCDNA3 vector)[17], in serum-free medium using lipofec-tamine (Life Technology, USA). After 6 h, the transfectionmedium was removed and replaced with complete mediumfor 72 h. Cells were selected in presence of 400–1000�g/mlG418 containing medium. The resistant cells were screenedfor expression of the N protein after 3 weeks of antibioticselection. The clones were selected by single-cell cloningin the presence of the antibiotic. The N protein expressionfrom these cells was checked by FACS after staining withmouse monoclonal antibody made against N protein. Thepositive clones were maintained in complete medium con-taining 400�g/ml of G418.

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2.10. Anti-idiotypic T cell proliferation assay

Balb/c male mice were injected with 2C5 (2× 107)intraperitonially. After 3 weeks, mice were boosted with samenumber of apoptotic 2C5 cells. One week later, spleen cellsand lymph node cells were harvested and in vitro proliferationassay was performed using apoptotic 2C5 cells pulsed bonemarrow derived dendritic cells (DCs) as stimulator cells. Asa control, apoptotic P specific Id T cells pulsed DCs or DCsalone were used. Splenocytes or lymph node cells (5× 105)from 2C5 T cell immunized mice were co-cultured with vary-ing number of antigen presenting cells in complete RPMImedium in a 96 well flat bottom plate for 72 h.3H-thymidine(1�Ci/well) was added for the last 16 h of incubation. Cellswere harvested on glass fiber filters using S.E.M.i-automatedcell harvester (Nunc).3H-thymidine incorporation was mea-sured in a liquid scintillation spectrophotometer.

2.11. Generation of anti-idiotypic T cells after antigenimmunization

Mice were immunized with N antigen (50�g/ml) inFreund’s complete adjuvant (FCA) and booster dose wasgiven after 3 weeks in Freund’s incomplete adjuvant (FIA).One week later, animals were gamma irradiated (700 rad).Twenty-four hours later, mice were reconstituted with nor-m .O ouslyw na tiona icew mo-s enta

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.8. Antigen presenting cells for in vitro proliferation ofnti-idiotypic T cells

2C5 T cells or antigen specific polyclonal cells or crol T cells were irradiated (3000 rad) and cultured2 h in complete medium. Apoptosis was monitoredACS after staining with propidium iodide and annex-FITC and result shows that all the cells are apoptdata not shown). Naıve mice were immunized with thPV-P protein. Bone marrow derived cells were puith apoptotic T cells (1:5 ratio). After 12–16 h of cultu

ng, un-engulfed cells were removed by washing. In conxperiment, 4 h after adding apoptotic cells, 40 mM chluine (Sigma) or 10�g/ml brefeldin-A (Sigma) were addend incubated for the same period of time. The adheells were used as antigen presenting cells after irradi4000 rad).

.9. Engulfment of apoptotic T cells

2C5 T cells were gamma irradiated (3000 rad) and iated for 12 h in RPMI-1640 medium supplemented w5 mM HEPES, 10% FBS and antibiotics. Cells were wasnd labeled with PKH 26 dye (Sigma) according to manu

urer instructions. These labeled apoptotic cells were addn vitro cultured bone marrow derived dendritic cells at a rf 1:5 and incubated for 3 h. Unbound cells were removeashing with PBS. Cells were visualized under fluorescicroscope (Lieca, Germany).

al mouse bone marrow cells (20× 106) through tail-veinne set of reconstituted mice were injected subcutaneith in vitro generated DCs (1× 106). After 14 days, spleend lymph node cells were harvested and in vitro proliferassay was performed for anti-idiotypic T cells. These mere maintained in animal isolator under gnotobiotic atphere throughout the experiment with vitamin supplemnd antibiotics cover given through drinking water.

.12. In vitro activation of anti-idiotypic CD8+T cells

Lymph node cells were collected from immunized micaive mice, and CD4+ T cells were depleted by complemeediated lysis. CD4+ T cell from lymph node was deplets described earlier[21]. The CD4+ depleted lymph nodells (5× 106) were co-cultured in vitro with P815 ce0.2× 106) stably expressing nucleocapsid protein or wontrol P815 cells for 5 h in complete medium at 37◦C in4 well cell culture plates. Cells were harvested and sutained with anti-mouse CD3-PE and anti-mouse CDiotin/streptavidin-FITC. Cells were analyzed by FACesults were plotted as relative MFI (Mean Fluoresce

ntensity) of CD3+CD69+ double positive cells, which waalculated as MFI of experimental sample divided by MFontrol sample.

.13. Intracellular cytokine staining

Lymph node cells from N specific Id T cells immunizice were co-cultured with DCs or DCs pulsed with apopt

1152 G. Lal et al. / Vaccine 24 (2006) 1149–1158

N specific Id T cells or DC pulsed with apoptotic P specific IdT cells in complete medium at 37◦C in 5% CO2 incubator for4 h. Brefeldin-A was added after 4 h at a final concentration of10�g/ml. Six hours later, cells were harvested and intracellu-lar staining was performed as described earlier[22]. Briefly,cells were washed and incubated with 2.4G2 hybridomasupernatant for 10 min on ice. Cells were surface stained withanti-mouse CD8-FITC antibody for 30 min on ice and washedin washing buffer (PBS containing 0.1% BSA and 0.1 NaNs).Cells were fixed with 4% para-formaldehyde for 15 min onice and permeablized with 0.5% saponin containing washbuffer. Cells were stained with anti-mouse IFN-�-biotin con-jugated antibody (PharMingen, San Diego, CA). Counterstaining was performed with streptavidin-PE (PharMingen,San Diego, CA). Cells were washed with 0.2% saponin-containing buffer and fixed with 2% para-formaldehyde.

3. Results

3.1. Generation of anti-idiotypic T cells after adoptivetransfer of CD4+ T cell clone (2C5)

To demonstrate that anti-idiotypic T cells are generatedin the syngenic animal, 2C5 cells were adoptively trans-ferred into mice and booster doses were given after 3 weeks.A andt tod c Tc riticc l the2 llsb sidet dyei pro-c odies[ per-v ree h cap-t l bet rec-o inv nicm areao inet di-c d toa nta-t byc erao IdT n ofo oneo cyte

Fig. 1. Generation of anti-idiotypic T cells. (a) In vitro cultured bone marrowderived dendritic cells were pulsed with PHK26 dye labeled apoptotic T cells.Engulfment of apoptotic cells was detected by fluorescence microscopy; (b)2C5 cells (2× 107) were adoptively transferred into a syngenic mouse andboosted after 3 weeks, intraperitoneally. In vitro proliferation assay wasperformed using lymph node cells (4× 105 cells/well). DCs pulsed withapoptotic 2C5 T cells (�) or apoptotic P specific Id T cells (�) or apoptotic2C5 T cells in presence of 40 mM chloroquine () or apoptotic 2C5 cellsin presence of 10�g/ml brefeldin-A (©) or DCs alone (�) were used asantigen presenting cell for proliferation assay at different input cell num-bers. The data shown is the mean± S.E.M. of triplicates. Results shown arerepresentatives of three independent experiments.

repertoire has not encountered before (Fig. 1b). Immuniza-tion of mice with recombinant TCR beta chain protein derivedfrom the DO.11.10 T cell hybridoma, did not show any background proliferation indicating anti-idiotypic specificity ofthe response (data not shown).

3.2. Anti-idiotypic T cells generation after adoptivetransfer of poly-clonal idiotypic T cells

In order to test the generation of anti-idiotypic T cellsby adoptive transfer of N specific Id T cells (polyclonal)in syngenic mice, antigen specific T cells were generatedfrom antigen primed mouse lymph node cells and enrichedin vitro by culturing in presence of irradiated splenocytesand N protein. The V� screening of these N specific enrichedT cells shows that they use V�3 (31%) and V�13 (50%).These cells were adoptively transferred to syngenic naivemice intraperitoneally. After 1 week of booster, in vitro pro-liferations of splenocytes from recipient mice were examinedand the results are shown inFig. 2. DCs pulsed with apoptoticN specific Id T cells were able to re-stimulate anti-idiotypicT cells but not DCs pulsed with apoptotic P specific Id T cells(Fig. 2a). Furthermore, T cell proliferation was inhibited by

fter 1 week, T cells from lymph nodes were isolatedheir in vitro proliferation ability were tested in responseendritic cells pulsed with apoptotic 2C5 cells (idiotypiells). The engulfment of apoptotic 2C5 cells by dendells was demonstrated employing PKH26 dye to labeC5 cells.Fig. 1a shows the engulfment of labeled T cey dendritic cells. These apoptotic cells were digested in

he DCs with passage of time as revealed by fading ofnside the cells (data not shown). DCs are known toess and present antigens derived from the apoptotic b23–25]. The idiopeptides (peptide generated from the hyariable region of TCR) derived from idiotypic T cells axpected to be processed and presented by DCs (whicure the apoptotic idiotypic T cells). These peptides wilreated as non-self and APCs presenting them will begnized by anti-idiotypic T cells, which are generatedivo by adoptive transfer of idiotypic T cells in syngeice. The results show that DC pulsed with 2C5 cellsble to activate anti-idiotypic T cells (Fig. 1b). Inhibitionf processing of apoptotic bodies in DCs by chloroqu

reatment prevents the activation of anti-idiotypic T cells inating that idiopeptides of idiotypic T cells are presententi-idiotypic T cells. Further, the processing and prese

ion of the idiopeptide generated in the DC was inhibitedhloroquine and brefeldin-A (Fig. 1b), as if happens in othntigen presenting cells[26]. The low level of proliferationbserved in DC pulsed with control T cells (P specificcells) is probably due to processing and presentatio

ther cellular protein derived from the apoptotic T cell clr perhaps apoptosis related proteins which the lympho

G. Lal et al. / Vaccine 24 (2006) 1149–1158 1153

Fig. 2. Generation of anti-Id T cells after adoptive transfer of N specific poly-clonal Id T cells. (a) In vitro enriched N specific poly-clonal Id T cells(2× 107)were adoptively transferred to syngenic mice intraperitoneally. After 3 weeks, mice were boosted with the same number of N specific polyclonal Id T cells.After 1 week, splenocytes (4× 105) were used for in vitro proliferation in response to the following APCs (8000 cells/well), DCs alone (Bar 1), DCs pulsedwith apoptotic P specific Id T cells (Bar 2), DCs pulsed with apoptotic N specific Id T cells (Bar 3), DCs pulsed with apoptotic N specific Id T cells in presenceof 10�g/ml brefeldin-A (Bar 4), DCs pulsed with apoptotic N specific Id T cells in presence of 40 mM chloroquine (Bar 5). Data shown are the mean± S.E.M.of triplicates. The result is representative of three independent experiments; (b) splenocytes (10× 105 cells/well) from mouse adoptively transferred with Nspecific Id T cells were in vitro stimulated with DCs cells alone (i) or DCs pulsed with apoptotic P specific Id T cells (ii) or apoptotic N specific Id T cells(iii)for 4 h. Brefeldin-A (10�g/ml) was added and incubated for 6 h. Cells were surface stained for CD8 and intra-cellular IFN-�, analyzed by FACS. Quadrantswere set in accordance to isotype control antibodies staining. Cells were gated according to FSC Vs SSC profile; (c) dendritic cells (5× 105) or splenocytes(5× 106 cells) were pulsed with N protein (100�g/ml) in 24 well plate or DCs cells pulsed with apoptotic cells as described inFig. 1b. After 10 h of incubation,these cells were irradiated (4000 rad) and used as antigen-presenting cells with N primed T cells generated by N immunized mice. Results shown are meanS.E.M. of triplicates. Bar 1 represents DCs pulsed with N, Bar 2 represents splenocytes pulsed with N protein and Bar 3 represents DCs pulsed with apoptotic Nspecific Id T cells; (d) naıve Balb/c mice were gamma irradiated (700 rad) and N specific Id T cells (20× 106 cells) were adoptively transferred intravenously.After 20 days, lymph node T cells from these were isolated and injected into another naive mouse. After 3 weeks, mice were boosted with same apoptotic Tcells. In vitro proliferation reaction for anti-Id T cells was performed from the lymph node cells using DCs pulsed with apoptotic N specific Id T cells (�), DCspulsed with apoptotic P specific Id T cells (�) or DCs pulsed with apoptotic N specific Id T cells in presence of 40 mM chloroquine (�). Results shown aremean S.E.M. of triplicates.

chloroquine and brefeldin-A, which confirms that presenta-tion of peptides from apoptotic cells has taken place. Thecross-presentation of antigenic peptide from apoptotic bodywas tested using apoptotic idiotypic T cells pulsed DCs asAPCs. In vitro proliferation and IFN-� secretion by anti-idiotypic CD8+ T cells were monitored. The result given inFig. 2b shows that anti-idiotypic CD8+ T cells are generatedin the animal after adoptive transfer of polyclonal idiotypicT cells.

To further confirm that anti-idiotypic T cell response gen-erated in vivo is not due to antigen carryover by the transferredidiotypic T cells, two different approaches were employed.In the first approach, N antigen primed T cells were stim-ulated in vitro with DCs presenting the idiopeptides fromapoptotic idiotypic T cells or antigen pulsed APCs, and pro-liferation responses were measured. The data given inFig. 2cshow that antigen primed T cells proliferate in response toN protein pulsed splenocytes or DC but not with DC pulsed

with apoptotic idiotypic T cells. This result demonstrates thatthe conclusion drawn on the data shown inFig. 2a and bare based on specific anti-idiotypic T cell response and notdue to carryover of N antigen. In the second approach, toexclude the possibility of carryover of antigen, N specificId T cells (40× 106) were transferred to a set of irradiatedsyngenic mice. After 20 days, lymph node T cells were har-vested and used for immunizing another set of syngenic miceand boosted with equal number of same cells. After 1 week ofbooster, lymph node cells were harvested and in vitro prolifer-ation of anti-idiotypic T cells was measured after stimulationwith DCs presenting idiopeptides of idiotypic T cells. Theresults are shown inFig. 2d. In the irradiated mouse, adop-tively transferred CFSE labeled T cells had shown extensivein vivo proliferation as the CFSE label was undetectable byFACS at the time of re-harvesting of T cells (data not shown).It is known that in the lymphopenic mice, adoptively trans-ferred T cells proliferate extensively[27]. This proliferation

1154 G. Lal et al. / Vaccine 24 (2006) 1149–1158

Fig. 3. Generation of anti-idiotypic T cells after antigen immunization: (a) experimental strategy; (b) mice were immunized with N protein (50�g) in Freund’scomplete adjuvant and boosted after 3 weeks with N protein (50�g) in Freund’s incomplete adjuvant. The animals were lethally irradiated (700 rad) after 7days. The animals were reconstituted with normal mouse bone marrow cells (107 cells) through tail vein with or without DCs (1× 106) intraperitonealy. After14 days, splenocytes were collected and in vitro proliferation of anti-Id T cells was carried out. DCs alone (Bar 1), DCs pulsed with apoptotic P specific Id Tcells (Bar 2), DCs pulsed with apoptotic N specific Id T cell (Bar 3), DCs pulsed with apoptotic N specific Id T cells in presence of chloroquine (Bar 4), wereused as antigen presenting cells (8000 cells/well) for proliferation assay. Data shown is the mean± S.E.M. of triplicates. Results are shown for one of the threeindependent experiments.

would result in elimination of carryover of antigen if any,which may be present at the time of immunization.

3.3. Anti-idiotypic T cell generation after antigenimmunization

We hypothesize that anti-idiotypic T cells will be gen-erated after antigen immunization at the contraction phaseof immune response. The major pathway for the genera-tion of anti-idiotypic T cells is through processing and pre-sentation of apoptotic idiotypic T cells by phagocytic cells(macrophages or dendritic cells). The experiment is schemat-ically shown inFig. 3a. Mice were immunized with N proteinand antigen specific idiotypic T cells response was monitoredat different time points. A peak T cell response was observedafter 1 week of booster. These animals, at peak immuneresponse were lethally irradiated to kill all the lymphocytes invivo. After 24 h, the animals were given intravenous injectionof syngenic bone morrow cells with or without in vitro gener-ated bone marrow derived dendritic cells. These bone marrowcells restore the haematopoitic system of mice as detected bytotal splenocytes and lymph node cell count (data not shown).In vitro generated dendritic cells were injected into the ani-mals to enable them to take-up, process and present apoptoticidiotypic T cell proteins to anti-idiotypic T cells. The gener-ation of anti-idiotypic T cells from these mice was detectedb ent-i riticc cellr cT assI pop-t nlyb c T

cell response demonstrating that the presence of DCs has animportant role in processing of apoptotic cells to induce anti-idiotypic T cells (data not shown). This result demonstratesthat anti-idiotypic T cells are generated in vivo, after antigenimmunization.

3.4. Demonstration of T cell idiotypic network afteradoptive transfer of idiotypic CD4+T cells (2C5 cells)

To test the generation of idiotypic network in the bodyafter immunization with 2C5 T cells, lymph node cells fromimmunized mice were re-stimulated in vitro with P815-Ncells or dendritic cells pulsed with apoptotic 2C5 T cells.P815-N cells (N transfected stable cell line) express N proteinendogenously as shown inFig. 4a, and reactivation of T cellsor IFN-� secretion was monitored. The generation of N pro-tein specific CD8+ T cells after adoptive transfer of antigenspecific CD4+ T cells is probably due to idiotypic network. Nexpressing P815 cells were used as antigen presenting cellsfor monitoring the generation of idiotypic CD8+ T cells.Dendritic cell pulsed with apoptotic 2C5 cells were usedto monitor anti-idiotypic T cells. The results are presentedin Fig. 4b. Antigen specific idiotypic CD8+ T cells weregenerated in 2C5 (N specific Id T cells) immunized mousebut not in control T cells (P specific Id T cells) immunizedmouse. The presence of idiotypic CD8+ and anti-idiotypicC ringC onw -i lt oft 4T ratei toticd d the

y in vitro proliferation assay using DC as antigen presng cells. The results show that bone marrow and dendells-reconstituted mouse had generated anti-idiotypic Tesponse (Fig. 3b). The in vitro proliferation of anti-idiotypicells is inhibited by chloroquine showing that MHC cl

I mediated pathway of processing and presentation of aotic Id T cells is inhibited. The mice which received oone marrow cells did not show significant anti-idiotypi

D8+ T cells in the same mice was detected by monitoD3+CD69+ (double positive) cells on in vitro re-stimulatiith different APCs (Fig. 4c). The CD8+ idiotypic and anti

diotypic T cells are generated in vivo, probably as a resuhe operation of idiotypic–anti-idiotypic network. The CD+

cells (2C5) used for adoptive transfer do not prolifen vivo as they are irradiated and they undergo apopeath as tested by annexin-V staining. We have monitore

G. Lal et al. / Vaccine 24 (2006) 1149–1158 1155

Fig. 4. Generation of idiotypic CD8+ and anti-idiotypic CD8+ T cells after adoptive transfer of 2C5 cells: (a) intracellular staining for N protein expressionin P815 cell line was performed using anti-N mouse monoclonal antibody and anti-mouse IgG-FITC conjugate. Cells were analyzed by FACS. (i) representsvector transfected P815 cells, and (ii) represents N gene containing plasmid transfected P815 cells. Filled histogram represents control antibodystaining andopen histogram represents cells stained with anti-N antibody; (b) Balb/c mice were injected with 2C5 cells (20× 106 cells) intraperitoneally and 3 weeks later,boosted with same number of irradiated 2C5 cells. Draining lymph node cells from these mice were harvested after 1 week of booster. These cells (4× 106)were in vitro re-stimulated with P815 cells stably expressing N protein (P815-N) or vector transfected P815 cells (P815) in a 24 well plate for 2 h. Brefeldin-A(10�g/ml) was added and incubated for another 6 h. Intracellular cytokine staining for IFN-� was performed and analyzed by FACS. Cells were gated accordingto FSC Vs SSC profile and quadrants were set according to isotype control antibody staining; (c) lymph node cells from 2C5 immunized mice were depletedfor CD4+ T cells using complement mediated lysis and stimulated with different APCs for 8 h. Cells were surface stained for CD3 and CD69. Cells wereanalyzed by FACS and plotted as relative MFI of CD3+CD69+. (1) Represents reactivation with P815, (2) with P815-N, (3) with DC cells alone, (4) DCspulsed with apoptotic P specific Id T cells, and (5) DCs pulsed with 2C5 T cells; (d) antigen-specific in vitro proliferation assay was performed with 2C5immune mice splenocytes (5× 105 cells/well). (1) N-protein, (specific antigen; 16�g/well), (2) P-protein (non-specific antigen; 16�g/well). Data shown is themean± S.E.M. of triplicates.

persistence of adoptively transferred viable 2C5 cells in themice by transferring same number of CFSE labeled 2C5 cells.Different lymphoid organs were collected and CFSE positivecells were analyzed. Result shows that less than 1.5% cellswere present in any of the lymphoid organs at the time of anti-idiotypic T cell monitoring (data not shown). The presenceof persisting immunized T cells are monitored and prolifera-tion response is shown inFig. 4d. Thus, in the same animal,idiotypic CD4+ T cells (persisting), idiotypic CD8+ T cellsand anti-idiotypic CD8+ T cells have been shown to co-exist,demonstrating the operation of idiotypic and anti-idiotypic Tcell network.

3.5. Generation of T cell memory after boosting withanti-idiotypic T cells

To evaluate the role of anti-idiotypic T cells, in main-tenance of T cell memory, mice were immunized withN-protein in Freund’s complete adjuvant, and after 72days, boosted with original antigen (N protein), irradiatedpolyclonal N specific Id T cells or anti-idiotypic T cells.The memory recall response was monitored after 1 weekof booster, using splenocytes of these mice by in vitroproliferation assay. The results show that splenocytes form

anti-idiotypic T cell-boosted mice exhibit secondary immuneresponse similar to those mice boosted with antigen (Fig. 5a).CD8+ T cells from lymph node cells of boosted mice secreteIFN-� after in vitro re-stimulation with P815-N cells.Boosting of antigen immunized mice with anti-idiotypic Tcell gave comparable secondary response as antigen boosting(Fig. 5b). We have also detected a weak antigen (N) specificimmune response after immunization of P immune mice withN specific anti-idiotypic T cells. Boosting with irrelevantantigen specific (P protein) anti-idiotypic T cells did notresult in a secondary response in animals demonstrating thespecificity of recall response (data not shown).

4. Discussion

The existence of anti-idiotypic T cells has been shownearlier only in the autoimmune model systems where auto-antigen was continuously present in the body providing atrigger for the idiotypic T cells[15,28–30]. Operation of idio-typic network was postulated in the control of autoimmunedisease in animal models and patients[25,31–33]. However,the exact mechanism of generation of anti-idiotypic T cells,which requires presentation of TCR peptides by MHC, was

1156 G. Lal et al. / Vaccine 24 (2006) 1149–1158

Fig. 5. Generation of memory response after boosting with anti-idiotypic Tcells: (a) mice were immunized with N protein (50�g) in Freund’s completeadjuvant subcutaneously. After 72 days, a booster dose was given with N pro-tein (50�g in Freund’s incomplete adjuvant), irradiated N specific Id T cells(2× 107), anti-Id T cells (2× 107) or P specific Id T cells (2× 107), PBS inFreund’s incomplete adjuvant or with out any booster (none). After 1 week,in vitro proliferations of splenocytes (5× 105 cells/well) were performed inpresence of with N protein (50�g/ml). Data shown is the mean± S.E.M. oftriplicates. The result shown is representative of three independent experi-ments; (b) antigen primed mice were boosted after 72 days with antigen ordifferent cells as mentioned above and draining lymph node cells were col-lected after 1 week of booster. Lymph node cells (4× 106 cells/well) werein vitro re-stimulated with P815 cells expressing N protein or P815 cells(5× 106 cells/well) transfected with vector alone in 24 well plate for 8 hin complete medium. Brefeldin-A was added in the last 6 h. Intracellularcytokine staining for IFN-� was performed and analyzed by FACS. Resultshown is plotted as percentage of CD8+ IFN-� double positive cells.

not defined[34]. For generation of anti-idiotypic T cells, wepostulate two possible mutually non-exclusive pathways: (1)It is known that∼90% antigen specific T cells (idiotypicT cells) die at the contraction phase of immune response[35,36]. These dead cells are removed by phagocytic cells(e.g., macrophages and dendritic cells). These phagocyticcells can process TCRs and present the idiopeptide (pep-tide derived from the variable region of TCR) through MHCclass II or by cross presentation by MHC class I pathway.Presentation of idiopeptide can trigger anti-idiotypic T cells.It is now clear that presentation of antigen from apoptoticcells leads to immune response rather than immunosuppres-sion[20,37–39]. (2) Another possible way for generation ofanti-idiotypic T cells is through T cells functioning as anti-gen presenting cells. Activated T cells are known to presentantigen to other T cells[40,41]. It is reported that∼30% ofnewly synthesized proteins are formed as DRiPs (defectiveribosomal products) during normal protein synthesis process[42,43]. Thus, it is possible that TCR may also be synthesizedas DRiPs in the cells which could be processed and presentedby MHC class I molecules to provide trigger for anti-idiotypic

CD8+ T cells. Another pathway which includes down regula-tion of surface TCRs by activated T cells[44,45]. It is likelythat down regulated TCR molecules can go to phagolyso-some[46] to get processed into peptides for presentation viaMHC class II pathway[47], and thus stimulate anti-idiotypicCD4+ T cells.

The anti-idiotypic T cells have been proposed to act asregulatory T cells in the control of autoimmune diseases[48,49]. The true anti-idiotypic T regulators would, by def-inition, recognize a peptide from the domain of TCR that isunique to the clone that expresses that TCR; such clonallyunique T cells would include the CDR3 region. However,peptides from non-clonally restricted domains of the TCR,both within the TCR variable segments (CDR1/2) and inframework region of the TCR have been found to be tar-geted by Treg [50]. Thus, not all TCR-directed Tregsare anti-idiotypic. The T cell directed regulation that is not idiotypichas been explained as anti-ergotypic (anti-erg) regulation[49]. There are various auto-immune diseases where pep-tides derived from the framework 3 and CDR1/2 regions ofTCR�8.2 chain have been used to control antigen inducedexperimental autoimmune encephalitis (EAE) in both rats,mice and human[50–52]. Since TCR peptides belong to theconserved region of the TCR and do not significantly inter-act with the antigenic peptides, the regulation of autoimmunedisease is probably due to the deletion of the entire particu-l . Ins minoaa di notr lls.

thata sfero on-a areg avesa ypicC ellsu y att tedb anda m ad tingw em-o ughd idesb lls.T anti-g ichi c Tc vac-c tralh tidesf ate

ar V�8.2 T cells rather than specific autoimmune T cellstudy where CDR-3 peptides have been used, only 8–9 acid peptides that bind mostly to MHC class I molecules[53]nd trigger anti-idiotypic CD8+ T cells are probably involve

n the regulation of autoimmune population. Thus, it doeseflect the natural functional activity of anti-idiotypic T ce

In the present work, we have tested our hypothesisnti-idiotypic T cells are generated after adoptive tranf idiotypic T cells. For the first time, we have used a nutoimmune model and shown that anti-idiotypic T cellsenerated in the body after injection of antigen. We hhown that idiotypic CD8+ and anti-idiotypic CD8+ T cellsre generated in the body after transfer of cloned idiotD4+ T cells in syngenic mice. We observed that 2C5 csed for primary immunization also persist in the bod

he time of detection of anti-idiotypic T cells as detecy CFSE labelled 2C5 T cells. This shows that idiotypicnti-idiotypic T cells can co-exist in the body and may forynamic system of T cell idiotypic network. Further, boosith anti-idiotypic T cells generates antigen specific mry T cell immune response. This could be operative throirect T cell-T cell interactions or presentation of idiopepty phagocytic APCs after phagocytosis of idiotypic T cehe generated anti-idiotypic T cells are directed againsten reactive T cells after immunization with antigen, wh

mplies that the presence of idiotypic T cell-anti-idiotypiell cascade is natural to the system after infection orination. Although phagocytic cells which form the cenub for processing and presentation of TCR derived pep

rom the idiotypic T cells, idiotypic T cells in activated st

G. Lal et al. / Vaccine 24 (2006) 1149–1158 1157

process and present down-regulated surface TCR as well asTCR-DRiPs, leading to direct induction of anti-idiotypic Tcells (Lal et al., submitted elsewhere). The results providea mechanism for maintenance of both idiotypic and anti-idiotypic T cells, which are generated in a cyclic fashion ashas been proposed for B cell memory[6]. This also meansthat T cell memory of longer duration could arise due totriggering of idiotypic cascade. Based on the structural infor-mation and recent study of altered peptide ligands (APLs)[54,55], we propose that peptidomimics will be generated inthe TCR of anti-idiotypic T cells which may not be com-pletely homologous to original antigen peptide but carry thestructural complementarities[56]. If these structural comple-mentarities exist in the CDR3 region of the T cell receptor,then peptide derived from the CDR3 region will trigger idio-typic T cells. The memory response is self-sustaining andthe duration of the memory is dependent on how effectivelythe idiotypic and anti-idiotypic TCRs are processed, idiopep-tide (peptidomimics) generated and presented to cognateT cells.

Acknowledgements

We thank Dr. P. Marrack (HHMI, National Jewish Medicaland Research Center, Denver, USA) for providing X63.OIL-2 ry,I Dr.A re)f rlalN Ban-g .K.K R.M uteo . GLa rch,G Thisw ncea turef )i t ofI

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can4.iol

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Crit

log-5.

[7] Nayak R, Lal G, Shaila MS. Perpetuation of immunological mem-ory: role of serum antibodies and accessory cells. Microbes Infect2005;7:1276–83.

[8] Mitra-Kaushik S, Shaila MS, Karande A, Nayak R. Idiotypic–anti-idiotypic B cell interactions generated against a protective antigenof a morbillivirus in mice. Cell Immunol 2001;209:10–8.

[9] Mitra-Kaushik S, Shaila MS, Karande A, Nayak R. Idiotypeand antigen-specific T cell responses in mice on immunizationwith antigen, antibody, and anti-idiotypic antibody. Cell Immunol2001;209:109–19.

[10] Mitra-Kaushik S, Shaila MS, Karande A, Nayak R. Idiotype and anti-idiotype specific T cell responses on transplantation with hybridomasreactive to viral hemagglutinin and human tumor antigen. ImmunolLett 2002;80:81–7.

[11] Monzavi-Karbassi B, Cunto-Amesty G, Luo P, Kieber-Emmons T.Peptide mimotopes as surrogate antigens of carbohydrates in vaccinediscovery. Trends Biotechnol 2002;20:207–14.

[12] Saruhan-Direskeneli G, Weber F, Meinl E, Pette M, Giegerich G,Hinkkanen A, et al. T cell auto-immunity against myelin basicprotein; CD4+ cells recognizing epitope of the T cells receptorbeta-chain from a myelin basic protein specific T cell clone. EurJ Immunol 1993;23:530.

[13] Zipp F, Kerschensteiner M, Dornmair K, Malotka J, Schmidt S,Bender A, et al. Diversity of anti-TCR immune response and itsimplications for T cell vaccination therapy of multiple sclerosis.Brain 1998;121:1395–407.

[14] Jerne NK. Towards the network theory of the immune system. AnnImmunol (Inst Pasture) 1974;125c:373–89.

[15] Hong J, Zang YC, Tejada-Simon MV, Li S, Rivera VM, KillianJ, et al. Reactivity and regulatory properties of human anti-idiotypicantibodies induced by T cell vaccination. J Immunol 2000;165:6858–

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cell line, Dr. D. Nandi (Department of Biochemistndian Institute of Science, Bangalore) for L929 cell line,. Sarin (National Center for Biological Science, Bangalo

or P815 cell line and Dr. Ranga Uday Kumar (Jawahaehru Center for Advance and Scientific Research,alore) for GK1.5 hybridoma cell line. We thank Dr. Sar (Jawaharlal Nehru University, New Delhi) and Dr.anjunath (Department of Biochemistry, Indian Institf Science, Bangalore) for their valuable suggestionscknowledges Council of Scientific and Industrial Reseaovernment of India, for a Senior Research Fellowship.ork was supported in part by the Department of Sciend Technology, Government of India. The infrastruc

acility of division of Biological Sciences (FACS facilitys funded by Department of Biotechnology, Governmenndia.

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