TITLE: Epitope Specific T-Cell Immunity to Breast Cancer. PRINCIPAL INVESTIGATOR: Constantin G. Ioannides, Ph.D.

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1 AD GRANT NUMBER DAMD TITLE: Epitpe Specific T-Cell Immunity t Breast Cancer PRINCIPAL INVESTIGATOR: Cnstantin G. Iannides, Ph.D. CONTRACTING ORGANIZATION: M.D. Andersn Cancer Center Hustn, Texas REPORT DATE: August 1999 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland DISTRIBUTION STATEMENT: Apprved fr public release; distributin unlimited The views, pinins and/r findings cntained in this reprt are thse f the authr(s) and shuld nt be cnstrued as an fficial Department f the Army psitin, plicy r decisin unless s designated by ther dcumentatin

2 REPORT DOCUMENTATION PAGE Frm Apprved OMB N Public reprting burden fr this cllectin f infrmatin is estimated t average 1 hur per respnse, including the time fr reviewing instructins, searching existing data surces gathering and maintaining the data needed, and cmpleting and reviewing the cllectin f infrmatin. Send cmments regarding this burden estimate r any ther aspect f this cllectin f infrmatin, including suggestins fr reducing this burden, t Washingtn Headquarters Services, Directrate fr Infrmatin Operatins and Reprts, 1215 Jeffersn Davis Highway, Suite 1204, Arlingtn, VA , and t the Office f Management and Budget, Paperwrk Reductin Prject ( ), Washingtn, DC AGENCY USE ONLY (Leave blank) 2. REPORT DATE August TITLE AND SUBTITLE 3. REPORT TYPE AND DATES COVERED Annual (1 Aug Jul 99) 5. FUNDING NUMBERS Epitpe Specific T-Cell Immunity t Breast Cancer DAMD AUTHOR(S) Iannides, Cnstantin G., Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) M.D. Andersn Cancer Center Hustn, Texas PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland SPONSORING /MONITORING AGENCY REPORT NUMBER 11. SUPPLEMENTARY NOTES 12a. DISTRIBUTION / AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE Apprved fr public release; distributin unlimited 13. ABSTRACT (Maximum 200 wrds) Studies during the nging grant perid demnstrated that CD4 + cells specific fr the HER-2 helper epitpe G89 ( ) are present in an activated frm in healthy dnrs and cancer patients. G89-reactive cells, rapidly induce IL-12 frm antigen presenting cells (dendritic cells). DC induces CD40 upregulatin n DC. They are als necessary and required fr inductin f specific CTL activity t a + specific CTL epitpe. Hwever, G89-reactive CD4 + cells shw significantly weaker reactivity with G89 in the lymph ndes cntaining breast cancer cells. Simultaneus presentatin f a HER-2 CTL epitpe, and f a HER-2 helper epitpe leads t higher levels f IFN-y than presentatin f each epitpe alne. Since IFN-y is dwnmdulatr f HER-2 and effectr mlecule in inhibiting the grwth f HER-2 + breast cancer cells, its inductin in situ may have beneficial effects fr cntrlling the disease. 14. SUBJECT TERMS Breast Cancer 15. NUMBER OF PAGES PRICE CODE 17. SECURITY CLASSIFICATION OF REPORT Unclassified 18. SECURITY CLASSIFICATION OF THIS PAGE Unclassified 19. SECURITY CLASSIFICATION OF ABSTRACT Unclassified NSN Standard Frm 298 (Rev. 2-89) Prescribed by ANSI Std. Z LIMITATION OF ABSTRACT Unlimited USAPPCV1.00

3 FOREWORD Opinins, interpretatins, cnclusins and recmmendatins are thse f the authr and are nt necessarily endrsed by the U.S. Army. eft- 1 * k T- ^ Where cpyrighted material is quted, permissin has been btained t use such material. M /A Where material frm dcuments designated fr limited distributin is quted, permissin has been btained t use the material. Citatins f cmmercial rganizatins and trade names in this reprt d nt cnstitute an fficial Department f Army endrsement r apprval f the prducts r services f these rganizatins. '" In cnducting research using animals, the investigatr(s) adhered t the "Guide fr the Care and Use f Labratry Animals," prepared by the Cmmittee n Care and use f Labratry Animals f the Institute f Labratry Resurces, natinal Research Cuncil (NIH Publicatin N , Revised 1985). Fr the prtectin f human subjects, the investigatr(s) adhered t plicies f applicable Federal Law 45 CFR 46. In cnducting research utilizing recmbinant DNA technlgy, the investigatr(s) adhered t current guidelines prmulgated by the Natinal Institutes f Health. In the cnduct f research utilizing recmbinant DNA, the investigatr(s) adhered t the NIH Guidelines fr Research Invlving Recmbinant DNA Mlecules. \J In the cnduct f research invlving hazardus rganisms, the investigatr(s) adhered t the CDC-NIH Guide fr Bisafety in Micrbilgical and Bimedical Labratries. PI - Signature Date

4 TABLE OF CONTENTS A. INTRODUCTION 6 B. BODY. C. CONCLUSIONS 22 D. REFERENCES 24 E. APPENDICES 26

5 Abbreviatins used: Cyttxic T lymphcytes, CTL; Peripheral bld mnnuclear cells, PBMC; Antigen, Ag; T cell receptr, TCR; HER-2/neu prt-ncgene, HER-2; stimulatin index, S.I.; standard deviatin, SD; tetanus txid, TT; influenza hemaglutinin, HA; wild-type, w.t; lymph nde, LN, dendritic cells, DC, T helper 1, Thl, T helper 2, Th2, CD40, cluster f differentiatin 40, (surface antigen, gp39 receptr). CD40 ligand (gp39, CD 154), Mean Channel Flurescence, MCF.

6 INTRODUCTION The verall bjective f these studies is t develp nvel therapeutic appraches t breast cancer by understanding the requirements fr successful inductin f anti-tumr respnses using newly defined T cell epitpes. The use f specific epitpes can vercme limitatins in the use f whle self-prtein r biased invlvement f Th2 cells. On the ther hand, the whle prtein is nt sufficient fr activatin f immune cells (1). Breast tumrs release tumr Ag in the envirnment, while antigen presenting cells (APC) macrphages, M0, dendritic cells (DC) uptake and re-present tumr prteins. Hwever the disease prgresses indicating that this respnse is inefficient r suppressed. Our recent studies identified an immundminant CTL epitpe n HER-2(2). Furthermre, we identified immundminant epitpes n tw ther tumr prteins: Flate binding prtein/flate receptr cc(fbp/fr-<x) and the aminenhancer f split (AES) f the Ntch cmplex (3,4). These epitpes by themselves cannt induce a CTL respnse t the Ag and/r tumr. Fr this reasn we are currently using the Thl subset f CD4 + cells t induce and amplify a CTL respnse t tumr. The ratinale and the reasns fr the use f the helper epitpes have been described in detail in the previus reprt and are nt repeated here. Furthermre, studies during the previus year have identified a dminant HER-2 epitpe recgnized by CD4 + cells designated as G89 = HER-2 ( ) SPYVSRLL GICL. This epitpe induced prliferatin and type 1 cytkines frm PBMC f breast cancer patients, and healthy dnrs, suggesting that precursrs fr HER-2 specific Thl CD4 + cells exist (5). T address the rle f the CD4 + epitpe G89 and f its related epitpe F7 (HER-2, ) (5,6) in specific cell mediated ummunity (7), ur studies, during this perid fcussed first n characterizatin f the interactin between Thl cells and APC. This aimed t address whether interactin f Thl with APC induced expressin f B7 and/r f ther cstimulatry mlecules as well. A secnd bjective f the studies f the interactin between APC and Thl used was t determine whether this interactin induces predminantly inflamatry cytkines frm DC (i.e. Interleukin-12, IL-12), with reciprcal inductin f IFN-y frm T cells. Cnditining APC fr activatin (IL-12, IFN-y) versus tlerance/suppressin (n IL-12, but IL-10) (8,9,10,11) is an indicatin f the stimulatry ability f CD4 + cells, and f their functin as Thl cells. (7)

7 7 A third bjective f this study was t determine the ability f the newly identified immundminant CTL and Th epitpes t induce a CTL respnse r t enhance an existent CTL respnse. A furth bjective f this study was t determine whether HER-2 peptides with Thl activity can activate T cells during disease prgressin. We fund that G89 recgnitin f APC(DC) enhanced expressin f CD40, suggesting a rle fr the Thl cells interacting with DC in the inductin f IL-12 > IFN-y pathway. We als fund that interactin f Thl cells with DC during recgnitin f G89 induces IL-12 frm DC and IFN-y frm Thl cells. Thus recgnitin f Thl HER-2 peptides cnditined APC fr an inflamatry (autimmune respnse). A third finding was that G89 is required fr inductin f E75 specific CTL. Hwever, in lymph ndes with disease, stimulatin with G89 fllwed by culture in IL-2, and restimulatin f the resulting cells did nt induce G89-specific cells expansin. Our results suggest that the lack f G89 respnse during disease prgressin may be assciated with failure f an immune respnse t breast cancer. Breast cancer prgresses thrugh lymph ndes. Thus ur results identified a key pint where ptimizatin f the immune respnse t breast cancer shuld be targeted.

8 MATERIALS AND METHODS Cells. HLA-A2 + PBMC were btained frm healthy vlunteers frm the Bld Bank f M.D. Andersn Cancer Center. T2 cells, varian SKOV3, SKOV3.A2 cells, and indicatr tumrs frm varian ascites were described (2). Antibdies and Cytkines. mab t CD3, CD4, CD8 (Orth), CD13 and CD14 (Caltag Labratries, San Francisc, CA), B7.1 and B7.2 (CD80 and CD86, Calbichem), ICAM-1 (CD54, Calbichem), CD40L (Ancell, Bayprt, MN), HLA-A2 (clne BB7.2, ATCC), and MHC-II (L243, DAKO Crp., Carpintera, CA) were used as uncnjugated FITC r PE cnjugated. Anti-CTLA-4 was a kind gift frm Dr. Peter Linsley (Bristl-Myers). mab specific fr IL-12, IFN-y and istype cntrls were btained frm Pharmingen. The fllwing cytkines were used: GM-CSF (Immunex Crp., Washingtn, DC), specific activity 1.25 x 10 7 CFU/250 mg; TNF- (Cetus Crp., Emeryville, CA), specific activity 2.5 x 10 U/mg IL-4 (Bisurce Internatinal), specific activity, 2 x 10 6 U/mg; IL-2 (Cetus), specific activity 18 x 10 6 IU/mg; IL- 12 f specific activity 5 x 10 6 U/mg was a kind gift frm Dr. Stanley Wlf, Department f Immunlgy, Genetics Institute, Cambridge, MA. Synthetic peptides. The HER-2 peptides used were: E75 ( ); and the helper peptides: F7 (HER-2, ): GSPYVSRLLGICL, F6: (HER-2, ) GSPYVSRLLGICLTSTVQ. G89: (HER ):SPYVSRLLGICLT. The mdified Muc-1 peptides used were D125: (GVTSAKDTRV) and D132 (SLADPAHGV). The psitive cntrl CTL epitpe used was the influenza matrix peptide (58-66): GILGFVFTL, designated as Ml. Ml frms an immundminant epitpe recgnized by memry CTL in healthy dnrs (12). All peptides were prepared by the Synthetic Antigen Labratry f M.D. Andersn Cancer Center and purified by HPLC. Peptides were 95-97% pure by amin acid analysis. Peptides were disslved in PBS and stred frzen at -20 C in aliquts f 2 mg/ml. Immunflurescence. Antigen expressin by DC, T2 cells, and T cells was determined by FACS using a fiw-cytmeter (EPICS - Prfile Analyzer, Culter C, Hialeh FL). DC cells were defined by the presence f CD 13 and absence f CD 14 marker after culture in GM-CSF and IL-4. Fr phentype analysis, DC were incubated with PE-cnjugated anti CD 13 mab and FITC-cnjugated mab specific fr a surface Ag. Fr determinatin f the effects f cytkines,

9 9 peptides and T cells n surface antigen expressin, DC were incubated with the same amunts f cytkines and peptides as in T cell activatin assays fr 24 h, and the levels f Ag expressin were determined in the gated CD13 + ppulatin. Culture f PBMC-derived DC. CD13 + DC were generated frm freshly islated PBMC fllwing the established CD 14 methds (13, 14). Cmplete RPMI medium (cntaining 10% FCS) supplemented with 1000 U/ml GM-CSF r 500 U/ml IL-4 was added t each well cntaining plastic-adherent cells and maintained fr 7 days. T cells were btained frm the plastic nn-adherent PBMC by remval f CD16 + and CD56 + cells. CD8 + cells were islated by remving first the CD4 +, and then the CD16 + and CD56 + cells frm the nnadherent ppulatin using Dynabeads (Dynal, Osl, Nrway). CD8 + subppulatins were btained using anti- CD45RO mab and anti-cd45ra mab (UCHL-1, DAKO) as described (12). After depletin, the resulting cells were 97% CD8 + as determined by flw cytmetry. T cell stimulatin by peptide pulsed DC. We used CD13 + DC, as APC because such cells have been reprted t activate bth naive and memry CTL () and t present E75 (15, 16). After culture in GM-CSF+IL-4, DC frm all dnrs were >96% CD13 +, and CD14 - expressed high levels f MHC-I, MHC-II, CD54, CD40, and CD86, but lwer levels f CD80 in agreement with the described phentype f peripheral bld CD14 + derived DC (12). DC were plated at 1.2 x 10 5 cell/well in 24-well culture plates, and pulsed with peptides at 50 ug/ml in serum-free medium fr 4 h befre additin f respnders. TNF-c (50 U/ml) was added t DC fr the last hur t stimulate Ag uptake and presentatin (13). Autlgus, islated CD8 + cells r islated CD8 + cells (CD45RO + and CD45R0+cells depleted) in RPMI 1640 cntaining 10% HS were added t DC at 1.5 x 10 6 /ml, fllwed by IL-12. IL-2 was added h later t each well. Fr inhibitin studies, mab specific fr B7.1, B7.2, HLA-A2 and istype cntrl MOPC myelma were added t DC r tumr cells, 1 h befre respnders in amunts reprted t be inhibitry by the manufacturers. Anti CTLA-4 and CD40L were added t T cells 1 h befre they were added t cultures. The effects f peptides and cytkines n T cell survival were determined by cunting the numbers f recvered viable cells, and determinating the numbers f CD8 + and CD4 + cells in the sample by flw-cytmetry. Specific prliferative respnses t E75 in the presence r absence f cytkines were determined by measuring the incrprated radiactivity in equal cell numbers pulsed with 1 (ici f ( 3 H)-TdR (5, 6).

10 10 CTL and cytkine assays. Recgnitin f peptides used as immungens by CTL was perfrmed as described (2). Equal numbers f viable effectrs frm each well were used in all assays. Supernatants cllected at 6, 24, r 48 h were tested in duplicate fr the presence f IL-2, IL-4, IL-10, IL-12 and IFN-y using cytkine ELISA-kits -(Bisurce Internatinal, Camarill, CA) r R&D systems as described (6) with a sensitivity f 4-7 pg/ml. IL-12 was detected using an ELISA kit which recgnizes bth p40 and the natural heterdimeric mlecule. The amunt f cytkines was quantitated using standard plts f knwn cncentratins f cytkines determined in the same experiment. Prliferatin assays. Fr prliferatin assays 100 ul aliquts were remved frm each well f the 24-well plate f primary cultures after 4-6 days as described (6). Tetraplicate samples were cultured in a 96 well plate with 1 uci ph]-tdr in a final vlume f 200 ul. The cells were harvested 16 h later, and the radiactivity cunted in a Beckman LS3501 liquid scintillatin cunter (6). A psitive prliferative respnse was defined as psitive when differences in cpm values between cultures that received peptides cmpared with cultures which did nt receive peptides were significant by the unpaired Student's t-test (p < 0.05). Stimulatin indexes (S.I.) represented the rati between the mean c.p.m. f the cultures stimulated with peptide, and the mean c.p.m. f the cultures that have nt been stimulated with peptide (N.P.). Cytkine prductin. The ability f PBMC t secrete antigen-induced IFN-y, IL-4, and IL-10 was determined by culturing the PBMC with the crrespnding peptides. Supernatants were cllected at different times and stred frzen at -20 C. The cytkine cncentratins were measured by duble sandwich-elisa using the crrespnding kits prvided by BiSurce Internatinal (Camariy, CA). The cytkine assays were calibrated with human recmbinant IFN-y, IL-4, and IL-10 t detect each cytkine in the range f pg/ml. Statistical Methds. Differences in prliferative respnses were analyzed using Student's t-test fr unpaired samples. 10

11 11 RESULTS Characterizatin f recgnitin fthl peptide G89, presented n dendritic cells (DC) by T cells. T characterize the interactins between CD4 + cells and APC and their effects n surface Ag expressin and cytkine inductin we fcussed n peptide G89 (Tutle et. al., Clinical Cancer Res. 4: , 1998) (5). Our studies shw that G89 is recgnized by the majrity f HLA- DR4 + healthy dnrs and breast cancer patients with n evidence f disease (5). T identify the effects f G89 recgnitin n activatin f antigen presenting cells (APC) we first generated dendritic cells (DC). DC are cnsidered the mst efficient APC, fr activatin f immune respnses (13). Results frm several labratries indicate that DC have an excellent ptential fr cancer vaccinatin. We generated DC by culture f plastic-adherent PBMC in GM-CSF and IL- 4 (described in details in the Materials and Methds sectin) DC generated frm PBMC are characterized by the expressin f the CD 13 marker and dissappearance f the CD 14 (mncyte marker). T characterize the effects f G89 recgnitin n surface Ag expressin n DC, DC were pulsed with G89 and the expressin f CD13, CD40, MHC-I (W6/32), MHC-II, (HLA-DR) B7.1, and ICAM-1 was determined after cstimulatin in the presence r absence f freshly islated autlgus T cells. The results in Fig. 1 shw that incubatin f DC with autlgus T cells lead t upregulatin f CD40, MHC-1 and MHC-II antigens but nt f B7.1. This experiment was repeated with tw additinal dnrs and the results were cnfirmed. T address whether this is the result f recgnitin f G89, T cells (CD8 + depleted) frm the same dnr were primed with G89 (designated as G89P). In a separate experiment islated CD8 + cells were primed with E75, (E75P). Then autlgus DC were incubated in parallel with G89P cells, with G89P cells + G89, with E75P + E75, r with (G89P + G89) tgether with (E75P + E75). 20 h later (t determine the early respnse), supernatants were cllected and used fr quantitatin f the IFN-y secreted, while DC were cllected and the expressin f CD40 was determined in the gated CD13 + ppulatin using tw clrs FACS. analysis. The results in Fig. 2 shw that recgnitin f G89P cells induced lw levels f IFN-y. These levels increased by tw fld when G89 was added at 25 ug/ml. Similarly recgnitin f E75 n DC by E75P T cells induced IFN-y. Simultaneus stimulatin f G89P and E75P with DC pulsed with E75 + G89 11

12 12 demnstrated a synergistic effect f G89 and E75 in IFN-y inductin. Since IL-12, which is a cstimulatr f the IFN-y inductin in respnse t Ag (17), was nt used, these results demnstrate a synergy Thl + CD8 + in IFN-y inductin Furthermre, recgnitin f G89 by G89P increased CD40 expressin by mre than 2.5 fld, cnfirming the results in Fig. 1. E75 + E75P had a much weaker effect n CD40 inductin. Of interest when bth peptides (E75 + G89) and G89P + E75P cells were used in the same experiment, the increase in CD40 expressin was weaker than the increase induced by G89P alne. Hwever E75 + E75P induced nly a mdest increase in CD40 expressin, thus cmpared with the increase induced by the CTL epitpe alne, G89 + G89P enhanced CD40 levels n APC. These effects were fllwed by inductin f CTL activity at priming. This experiment is described here tgether with CD40 and IFN-y inductin fr imprved clarity. CD8 + cells frm the same dnr were stimulated with DC alne (DC) as a cntrl, DC pulsed with E75 (DC- E75), DC pulsed with G89 (DC-G89), r DC-E75-G89, in the absence, r presence f G89P cells (in the G89 grups). The results are shwn in Fig. 3. The CTL activity was determined in bth 5 h and 20 h assays t detect the presence f even few E75 reacting CTL. The results shw that E75 specific activity was detected nly in the DC-G89-E75 grup at 20h, suggesting that the E75-specific CTL were induced but the frequency f E75-specific cells was lw. N specific CTL activity was detected in the ther three grups, indicating that G89 is required fr HER-2 (E75) - specific CTL inductin. E75 and G89 als appear t synergize in T cell prliferatin and expansin. This is indicated by a parallel experiment where islated T cells frm the same dnr (CD4 and CD8 + ) were stimulated in the same cnditins with E75, G89, E75 + G89, r as cntrl E75 + D100. D100 is a HER-2 peptide with n prliferatin induced activity. The results in Fig. 4 shw that stimulatin f these cells with E75 + G89 has a strng synergistic effect in enhancing their expansin cmpared with stimulatin with E75 r G89 alne. HER-2 epitpes F7 and G89 are required fr inductin and/r expansin f CTL. 12

13 13 T address the questin whether CD4 + help is required fr inductin f CTL activity, we extended the studies presented abve t stimulatin f DC with T cell lines induced by stimulatin with HER-2 peptides F7 and F13. Because f their ptential fr crss-reactivity, F7 and F13 are mre suitable fr stimulatin f T cells frm dnrs f phentypes distinct frm HLA-DR4. A F7-reactive T cell line was induced frm a HLA-DR4 + healthy dnr. In this line CD4 + cells were 76%, while CD8 + cells were 18%. A CTL inductin experiment was established t test the feasibility f the apprach. DC were pulsed with E75 in the presence r absence f F7 in parallel grups. Autlgus plastic nn-adherent PBMC were used as respnders. The autlgus CD4 + line was added at a rati f helper t respnder (1:10, i.e CD4 + line cells, t 10 6 Aut-PBMC). T cntinue the investigatin f the nature f the helper effects by recgnitin f Th peptides IL-12 was added in a parallel experiment. The results summarized in Table II shw that El5 specific CTL activity was detected at priming when the CD4 + line was added but required either the presence f F7 r in the presence f IL-12. These results cnfirm previus results (p9, 10) regarding CD40 upregulatin and IFN-y inductin. Since the T cell dependent pathway fr IL-12 inductin invlve CD40-CD40L interactins (18, 19), these results suggest that CD4 + cells/line recgnitin f G89/F7 n APC, activates APC. The enhancing ability f HER-2 peptide G89 was als cnfirmed using as respnders breast TIL. Since the MHC-II phentype f these cells was nt knwn, we stimulated these TIL with E75 + F13, r E75 + F7 using E75 as a cntrl. The results in Fig. 5, shw that in this case, F13 had a strnger stimulatry activity than F7. The CTL activity f TIL stimulated by E75 + F13 increased 5 fld cmpared with the TIL stimulated with E75 alne. F7 in this case had n effect. Thus helper HER-2 peptides are required bth fr priming and maintenance and expansin f a CTL respnse. Since the preliminary experiments shw that (a) CTL activatin require CD4 + cell help, which is mediated by G89, and (b) CTL activity is induced by stimulatin with E75 + G89, we expanded n these experiments t establish the requirements fr G89 in CTL generatin. Fur grups were set in parellel: 13

14 14 (1) Priming: DC-G89 with G89P + IL-12 : Restimulatin : DC-575 E75P + IL-12 (2) Priming: DC-G89 with G89P + IL12 : Restimulatin : DC-E75-G89 E75P + G89P + IL- 12 (3) Priming: DC-G89 with G89P + IL-12 : Restimulatin : DC-NP NP-P + IL-12 (4) Priming: DC-NP with NP-P + IL-12 : Restimulatin : DC-E75-G89 + E75P + G89P + IL-12 As respnders plastic nn-adherent T cells were used since they cntained bth CD4 + and CD8 + cells. The results are shwn in Fig. 6. At this time highly specific CTL activity was bserved nly frm the grup (2) where G89P + G89 were included bth in the priming and restimulatin phases. The absence f G89 + G89P during priming (Grup 4) r f G89P + G89 during restimulatin (Grup 1) lead t nnspecific CTL activity. These results indicate that fr inductin f E75-epitpe specific CTL bth E75 and G89 are required at the priming and restimulatin phases. Since IL-12 was present in all grups, these results indicate that additinal factrs are invlved in the helper effect mediated by G89. Prliferative respnses t G89 are inhibited in tumr psitive lymph ndes. T address the questin whether G89 recgnitin is affected during disease prgressin, we cntinued ur investigatin f the respnses t G89 f the T cells frm tumr psitive lymph ndes (Turn + LN) and tumr negative lymph ndes (tum-ln). Preliminary data during the previus year with ne LN sample frm ne dnr shwed that prliferative respnses t G89 by LN+ cells decrease frm the distal t the diseased LN. We cntinued these experiments with 5 additinal pairs f lymph ndes and the results were cnfirmed. These results are summarized in Table III. Significant prliferative respnses were btained frm tumr-ln, but nly frm 2/4 tum+ln. T address the questin whether the respnding T cells frm nn prliferative tum+ln are anergic, accrding t the general definitin f this term, cells frm a pair f LN. (Tum + and Turn") were first cultured in IL-2, withut peptide stimulatin, then stimulated with G89, fllwed by culture in IL-2. Equal number f cells frm the primary stimulatin grups, cntrl (NP), G89, and F13, were restimulated with NP, G89 and F13. The results and the experimental settings are shwn in Fig. 7. These results shw that G89 primed cells frm Turn - cells prliferated better than cells stimulated with cntrl (NP) r with the cntrl peptide F13. In cntrast, the levels f G89 induced prliferatin were similar with the cntrl (NP) stimulated 14

15 15 cells in LN + dnr. These results indicate that the prliferative respnses t Ag f G89 reactive cells are inhibited in LN cntaining tumrs but this inhibitin it is nt a frm f classical anergy, because it is nt reverted by culture in IL-2, befre and after Ag stimulatin. T address the fine epitpe specificity f these cells they were stimulated in parallel in the same experiment with F7, G89, and F6. As reprted befre, F6 is a lnger epitpe frm the same area which includes bth the F7 and G89 sequences. The results are shwn in Fig. 8. In parallel we determined the G89 cncentratin dependent respnses f cells frm Turn - LN and Tum + LN t G89. The results in Fig. 8A shw that T cells frm Turn - LN respnded better t G89 than Tum + LN, suggesting that either the affinity r the frequency f G89-reactive cells is higher in Turn" than in Tum + LN. G89 induced prliferatin was always higher in respnse t G89 than t F7 and F6. (Fig. 8B). This indicate that the fine specificity f LN cells is clser t G89 than t F6 and F7. Mechanisms fg89 induced T cell help. T better understand the effects f G89 in the stimulatin f T cells by bth the helper and CTL epitpe we used the same DR4 + dnr and we stimulated its PBMC with E75 ver a range f cncentratins in the presence f a cnstant cncentratin f G89. The G89 induced CD4 + line (G89L) btained after tw stimulatins with G89 (1) was used as psitive cntrl. The results in Fig. 9 shw that in the presence f 20 ug G89 the amunt f IFN-y secreted increased dramatically and at 1 ug/ml E75 was higher than at 20 ug/ml in the absence f G89. Of interest the amunt f IL-2 present slightly decreased. This suggested that the IL-2 is cnsumed faster than is prduced. Even when 100 ug/ml E75 was added and G89 cells were present, they prduced lw amunts f IL-2. Recent studies using varius experimental mdels have cnfirmed the requirement fr help fr inductin f CTL. Hwever, the general perceptin was that the CD4 + help cnsists mainly in secretin f cytkines such as IL-2 and IFN-y/IL-4 which mdulate the CD8 + cells repnse. This raise the questin that if these cytkines can be prvided (exgenusly) why the CD4 + cells are needed. Furthermre, ifcd4 + cells will interact with APC and lead t inductin f IL-12 frm APC (DC, MO), why this IL-12 is needed. Additin f exgenus IL-12, r stimulatin with trimeric CD40L shuld be sufficient t induce IL

16 16 T address these questins we needed t set up a parallel system f stimulatin: E75- DC-CD8 + frm the same dnrs. We quantitated the amunts f IFN-y, IL-12, IL-2, (and in parallel IL-10, IL-4 and IP-10) induced at priming by E75. Our findings are summarized belw and in the attached manuscript (submitted). (Lee, T.V. et al) We investigated the ability f HER-2 peptide E75, t activate effectr functins in freshly islated CD8 + cells. IFN-y was rapidly induced by E75 within 20-24h in six f six healthy dnrs, in the presence f IL-12 and was detectable as early as 6h. The IFN-y levels were Ag-cncentratin dependent. Similar results were btained with peptides mapping CTL epitpes frm tw ther tumr Ag: flate binding prtein (FBP) and amin-enhancer f split f Ntch (AES). IFN-y was als detected, in respnse t HLA-A2 matched tumrs+il-12 but nt f IL-12 alne. The majr surce f IFN-y were CD45RO + CD8 + cells. Inductin f IFN-y and IL-2 frm CD8 + cells and f IL-12 frm dendritic cells (DC) by CD8 + cells reactive with E75 mirrred their inductin by the influenza matrix peptide (Ml: 58-66) in the same individual. Respnses t Ml are used t define the presence f activated memry cells in healthy individuals. Cmpared t Ml respnses E75 recgnitin induced 2-4 fld lwer levels f IL-12 frm the same APC and IFN-y and IL-2 frm CD8 + cells. At lwer Ag cncentratins the endgenus IL-12 induced by E75-reactive CD8 + cells did nt reach the threshld required t cstimulate fr IFN-y. cb7.1 synergized with E75 in increasing the verall levels f IL-2 induced within 24h. Priming f CD8 + cells with E75+IL-2+ CTLA-4/B7.1 prmted marginal prliferatin suggesting a functinal dichtmy in the activating effects f E75. The cnclusin f these studies is that activated CD8 + cells (f memry phentype) when present in healthy dnrs can induce type 1 cytkines, but their simulatry ability is just belw the threshld fr activatin f type 1 cytkine effectr functin. T reach this threshld we need t use very high cncentratins f CD8 + epitpes ( um, = ug/ml). Alternatively in the absence f Ag we need t use exgenus IL-12, CD40L r LPS-type bacterial prducts. Hwever, activatin f IFN-y inductin by T cells require Signal 1 = Ag + Signal 2 = IL-2 (cstimulatin). Thus in the absence f the Ag, IL-2, CD40L will either activate nn-specific effectrs, r they will activate a whle range f CTL with varius unrelated specificities. The utcme f escalating IL-12 shuld be suppressin. In cntrast, the presence 16

17 17 f the helper Ag (such as G89 epitpe) and its recgnitin by CD4+ cells shuld induce IL-12 frm DC nly at interactin with APC, and cnsequently lwer the threshld fr activatin f CTL. by inducing respnses when the Ag cncentratin is within feasible range (5-20 ^M). T address the questin whether G89 synergy with E75 in IFN-y inductin is because f the inductin f IL-12, DC were pulsed in the NP, E75, G89 and E75+G89 at cncentratins (50 fig/ml) where they bth can induce IFN-y.) The results are shwn in Fig. 10. Recgnitin f G89 lead t IL-12 inductin frm DC althugh at lwer levels than frm recgnitin f E75 alne. E75 + G89 had a ndest synergistic efect in enhancing the verall IL-12 levels. Hwever, this mdest synergistic effect lead t a strng synergistic effect in IFN-y inductin frm the E75+G89 grup. Cnsistent with the hypthesis that IFN-y is induced by Ag and IL-12 has a cstimulatry rle. Since exgenus IL-12 was nt added these results shw that simultaneus stimulatin with E75+G89 enhance IFN-y levels thrugh inductin f higher levels f IL-12. Thus, these studies identified an effect f CD4 + cell recgnitin f HER-2 helper peptide which is assciated with help fr CD8 + cells activatin. This effect is due t the ability f CD4 + cells t activate DC t prduce IL-12. The IL-12 induced by CD4 + cells t activate DC t prduce IL-12. The IL-12 induced by CD4 + cells cmpensate fr the required amunt f IL-12 by CD8 + cells t induce IFN-y. This IL-12 addresses the requirement fr exgenus IL-12 in CD8 + cells activatin. Our results als shw that there is an additinal helper effect mediated by CD4 + cells which is independent f IL-12. This is evident where exgenus IL-12 is prvided and appears nt t be dependent n IL-2 since exgenus IL-2 is als prvided. Since this effect is evident in inductin f cytlyte functin, ur studies will cntinue as planned t determine whether this is related t enhanced prliferatin, prtectin frm activatin induced cell death f CD8 + cells, by interactin with CD4 + cells. Furthermre, we will fcus n tlerance (anergy) reversal in the lymph ndes, as planned, t address whether reversal f cytkine mediated immunsuppressive restres the ability f CD4 + cells t expand and they can help in inductin f tumr specific CTL. 17

18 Table I. CD4 antigen expressin n T cell lines frm DR4 + dnr induced by stimulatin with HER-2 peptides. 18 % Psitive Cells CD4 CD8 G90* G F F F TT (Cntrl)* 93 4 * Indicates significant increase in CD4 Ag expressin cmpared t the ther lines, TT (tetanus txid) was used as psitive cntrl. Table II. Requirements fr help fr inductin f E75 specific CTL. CD4 + % Specific Lysis DC E75 Line F7 IL-12 E75 D Dendritic Cells (DC) were cultured in GM-CSF + IL-4. Respnder T-cells cnsisted f the plastic nn-adherent fractin f HLA-A2 + dnr's PBMC. The CD4 + line was maintained by stimulatin with HER-2 peptide F7. D132 is an unnatural mutated MUC-1 peptide: D132, S L ADPAHGV. CTL activity was determined at an E:T rati f 10:1. IL-12 was used at 300 pg/ml. Targets were T2 cells pulsed with E75.

19 19 Table III. Decreased prliferative respnse f T cells frm Tum + lymph ndes t HER-2 peptide G89. Prliferatin * Sample Turn" Tum + LN1 + - LN2 + + LN3 + N.D** LN4 + - LN5 + + Ttal respnding: +5/5 2/4 * A respnse was cnsidered psitive when the stimulatin index was > 1.6 and the difference between replicates were cnsidered significant (p < 0.05). ** The amunt f lymphcyte material was nt sufficient fr prliferatin assays. 19

20 20 Legends t the figures. Figure 1. Surface antigen expressin n dendritic cells frm a DR4 + dnr after stimulatin with G89 peptide alne ( ). r peptide plus islated autlgus T cells ( ). Nte the marked upregulatin f HLA-DR (4.5 fld), MHc-I (3.5) fllwed by CD40, but nt f B7.1. Results are expressed as MCF (mean channel flurescence). Surface antigen expressin was determined 24h after incubatin with T cells and peptides. Figure 2. Recgnitin f G89 n DC by G89 stimulated cells (g89p) upregulates CD40 Ag expressin. E75 and G89 synergize in upregulatin f IFN-y inductin. G89P, and E75P indicate that T cells frm DR4 + dnr were primed (P) with G89 and E75 respectively. Supernatants were cllected and the levels f IFN-y, (A) and f CD40 expressin (B) were determined 20h later. Figure 3. Cnditining f APC (DC) by the interactin between G89-primed cells and G89 presented n APC is required fr inductin f CTL activity recgnizing HER-2 peptide E75. DC were pulsed either with n peptide (DC), r with E75 (DC-E75) r bth G89 (DC-G89). DC and DC-E75 were then incubated with E75 primed cells. DC-G89 were incubated either with G89 primed cells alne, r with DC-G89+E75+E75 primed cells. G89 and E75 primed cells were stimulated nly nce with peptide. CTL activity was determined 5 days later, using as targets T2 pulsed with n peptide (T2-NP) r T2 pulsed with E75 (T2-E75). Inductin f specific CTL activity at restimulatin was detected nly in the DC-G89, - E75, +G89P + E75P grup. Figure 4. Increased prliferatin f DR4 + T cells at restimulatin with the cmbinatin f CTL epitpe (E75 = E) plus the helper epitpe (G89 = G). Equal number f cells f the DR4 + (E+G) T cell line were stimulated with E75 ( ), G89 ( ), E+G (E75 + G89) (O), r as cntrl E75+D100 (). D100 is anther HER-2 peptide which lacks stimulatry ability. Autlgus DC were used as APC. IL-2 was added in the cultures 48h after Ag stimulatin. The prliferatin index was determined by dividing the number f recvered live cells after 8 days f stimulatin with the number f live cells at the initiatin f stimulatin. Figure 5. Stimulatin f HLA-A2 + tumr infiltrating lymphcytes frm a breast cancer patient, with the CTL epitpe E75 tgether with the helper epitpe F13 augments recgnitin f E75 20

21 pulsed target T2 cells by CTL-TIL. E:T was 10:1. The effects are F13 specific since F7 des nt induce an increase in specific recgnitin. Results f a 5 h CTL assay. 21 Figure 6. Priming and restimulatin f T cells frm an HLA-A2 + HLA-DR4 + dnr with E75 + G89 leads t inductin f specific CTL activity detected in a 5 h CTL assay. E:T rati was 8:1. (**) = indicate significant differences (p < 0.05), NP and E75 indicate that 51 Cr-labelled T2 cells were pulsed either with medium cntrl (n peptide) r with E75. Details n stimulatin grups in pages Figure 7. Prliferatin f G89 reactive cells is impaired in breast tumr psitive lymph ndes, but nt in breast tumr negative lymph ndes. Culture in IL-2 and restimulatin f LN cells frm the tumr psitive LN with G89 des nt enhance their prliferatin suggesting that the respnders are anergized. This pssibly invlves different mechanisms than classic anergy. Figure 8A. Cncentratin dependent recgnitin f G89 by LN cells frm Turn - and Tum + LN. frm a cancer patient 8B. F6 and F7 are recgnized less than G89 by the same cells frm TunT and Tum + LN. F6 and F7 were used at 25 ug/ml. Figure 9. HER-2 helper peptide G89 enhanced E75 induced IFN-gamma secretin. IFN-y in supernatants cllected 24 hurs after stimulatin was measured with IFN-y specific ELISA. 2xG89 indicate that autlgus T cells stimulated twice with G89 (G89L) were added. Figure 10. The synergy E75 + G89 in IFN-y inductin crrelates with an additive effect f E75 + G89 in IL-12 inductin. DC were pulsed with NP, E75, G89, and E75 + G89. Autlgus plastic nnadherent PBMC were used as respnders. Supernatants were cllected 20 h later and used fr determinatin f IL-10, IFN-y and IL-12 in the same experiment. 21

22 22 Cnclusins (1) We fund that interactin f islated HLA-DR + cells with autlgus DC cells (APC) lead t upregulatin f HLA-DR, HLA-ABC, and CD40, but a lesser extent f B7.1. This effect was mediated by specific recgnitin f G89. since interactin f G89 cells alne with APC did nt lead t upregulatin f CD40. (2) We generated cell lines frm a HLA-DR4 + dnr by stimulatin with HER-2 peptides. These lines are predminantly 75-90% CD4 +. Interactin f these lines with APC (DC) presenting the helper epitpes F7 + F13 helps expressin f cytlytic functin by CTL stimulated with the CTL epitpe peptide E75. A similar effect was bserved when TAL frm a patient with breast cancer were stimulated with E75 + F13/F7. Thus, indeed these helper epitpes help in expressin f CTL functin. (3) Simultaneus stimulatin with the CTL + helper epitpe is necessary and required fr HER-2 specific CTL inductin. This was cnfirmed in tw independent dnr systems. Specific CTL activity was detected nly in cultures initiated with E75 + G89. The reasn fr this requirement appeared t be related t the fact that APC were required t present G89 and t interact with G89 primer cells. (4) We fund that recgnitin f G89 leads t activatin f DC and inductin f IL-12, but nt f IL-10. This indicate that activated (expressing CD40L) CD4 + cells exist in the healthy dnrs and patients. Similarly we fund that activated CD8 + memry cells exist in these individuals (in a parallel study. Recgnitin by CD8 + cells f the tumr Ag induces IL-12 but the levels f IL- 12 are just belw the levels required t cstimulate with Ag IFN-y inductin. Thus the additinal levels f IL-12 induced by CD4 + cells, recgnized G89, vercame the need fr exgenus IL-12 fr activatin f type 1 respnse. (5) We extended and cnfirmed ur previus findings n G89 recgnitin by lymph nde cells. G89-reactive cells are present predminantly in the lymph ndes withut disease. In the diseased lymph ndes, this respnse is suppressed. The mechanism respnsible fr this suppressin likely invlve clnal anergy, since the G89 primed cells frm tumr + LN, even after culture in IL-2 d nt respnd at restimulatin with G89. 22

23 23 In summary, studies dne this year demnstrated that the helper epitpe G89 (r HER-2 epitpes with CD4 + stimulating functin) are required fr priming and inductin f a CTL respnse t the immundminant HER-2 epitpe E75. We plan t reverse the tlerance bserved in the lymph ndes by cnditining f DC fr inflamatry respnses, and the use f antibdies t TH2 cytkines, and design f G89 analgs which may be mre ptent activatrs f a helper respnse fr CTL inductin. 23

24 24 REFERENCES 1. Disis ML, Gralw JR, Bernhard H, Hand SL, Rubin WD, Cheever MA. (1996) Peptidebased, but nt whle prtein, vaccines elicit immunity t HER-2/neu, an ncgenic selfprtein. J Immunl 156: Fisk, B., Blevins, T. L., Whartn, J. T., and Iannides C. G Identificatin f an immundminant peptide f HER-2/neu-prt-ncgene recgnized by varian tumr specific CTL lines. J.Exp. Med. 181: Peples, G. E., Andersn, B. W., Fisk, B., Kudelka, A. P., Whartn, J. T., and Iannides, C. G. Ovarian cancer-assciated lymphcyte recgnitin f flate binding prtein peptides. Ann. Surg. Oncl., 5: , Babcck, B., Andersn, B. W., Papayannpuls, I., Castilleja, A., Murray, J. L., Stifani, S., Kudelka, A. P., Whartn, J. T., and Iannides, C. G. Ovarian and breast cyttxic T lymphcytes can recgnize peptides frm the amin-enhancer f split prtein f the Ntch cmplex. Ml. Immunl., 35: , Tuttle, T. M., Andersn, B. W., Thmpsn, W. E., Lee, J. E., Sahin, A., Smith, T. L., Grabstein, K. H., Whartn, J. T., Iannides, C. G., Murray, J. L. Prliferative and cytkine respnses t class II HER-2/neu-assciated peptides in breast cancer patients. Clinical Cancer Research, 4: , Fisk, B., Hudsn, J. M., Kavanagh, J., Whartn, J.T., Murray, J.LO., Iannides, C.G. and Kudelka, A.P Existent prliferative respnses f peripheral bld mnnuclear cells frm healthy dnrs and varian cancer patients t HER-2 peptides. Anticancer Res. 77: Tes, R.E.M., Ossendrp, F. Offringa, R. Melief, C.J.M. CD4 T cells and their rle in antitumr immune respnses. J. Exp. Med. 189: , Salgame, P., Abrams, J. S., Clayberger, C., Gldstein, H., Cnvit, J., Mdlin, R. L., and Blm, B. R. Differing lymphkine prfiles f functinal subsets f human CD4 and CD8 T cell clnes. Science (Wash. DC) 254:219, Grux, H., Bigler, M., devries, J. E., and Rncarl, M-G. Interleukin-10 induces a lng-term antigen-specific anergic state in human CD4 + T cells. J. Exp. Med. 184:19-29, Deeths, M. J., Kedl, R. M., Mescher, M. F. CD8 + T cells becme nnrespnsive (anergic) fllwing activatin in the presence f cstimulatin. J. Immunl., 163: , Miller, C, Ragheb, J. A., and Schwartz, R. H. Anergy and cytkine-mediated suppressin as distinct superantigen-induced tlerance mechanisms in viv. J. Exp. Med., 190:53-64,

25 12. Lalvani, A., R. Brkes, S. Hambletn, W.J. Brittn, A.V.S. Hill, and A.J. McMichael. Rapid effectr functin in CD8 + memry T cells. J. Exp. Med., 186: , Sallust, F., and A. Lanzavecchia. Efficient presentatin f sluble antigen by cultured human dendritic cells is maintained by granulcyte/macrphage clny-stimulating factr plus interleukin 4 and dwnregulated by tumr necrsis factr a. J. Med. Exp., 179: , Pickl, W. F., O. Majdic, P. Khl, J. Stckl, E. Riedl, C. Scheinecker, C. Bell- Fernandez, and W. Knapp. Mlecular and functinal characteristics f dendritic cells generated frm highly purified CD14 + peripheral bld mncytes. J. Immunl, 157: , Brssart, P., G. Stuhler, T. Flad, S. Stevanvic, H-G Rammensee, L. Kanz, and W. Brugger. Her-2/neu-derived peptides are tumr-assciated antigens expressed by human renal cell and cln carcinma lines and are recgnized by in vitr induced specific cyttxic T lymphcytes. Cancer Res., 58: , Wng, C, M. Mrse, and S.K. Nair. Inductin f primary, human antigen-specific cyttxic T lymphcytes in vitr using dendritic cells pulsed with peptides. J. Immunther., 21:32-40, Barbulescu, K, Becker, C, Schlaak, J. F., Schmitt, E., zum Buschenfelde, K. M., and Neurath, M. F. Cutting Edge: IL-12 and IL-18 differentially regulate the transcriptinal activity f the human IFN-y prmter in primary CD4 + T lymphcytes. J. Immunl., 160: , DeKruyff, R. H., Gieni, R. S., and Umetsu, D. T. Antigen-driven but nt lipplysaccharide-driven IL-12 prductin in macrphages requires triggering f CD40. J. Immunl, 158: , Gately, M. K., Renzetti, L. M., Magram, J., Stern, A., Adrini, L., Gubler, U., and Presky, D. H. The interleukin-12/interleukin-12-receptr system: Rle in nrmal and pathlgic immune respnses. Annu. Rev. Immunl, 16: ,

26 26 APPENDICES Publicatins Resulting frm this Grant Lee, T.V., Andersn, B.W., Castilleja, A., Peples, G. E., Whartn, J. T., Murray, J. L., Iannides, C. G. Identificatin f activated tumr antigen-reactive CD8 + cells in healthy individuals. (Submitted) 26

27 Figure Ü CD13 CD40 MHC I MHC II B7.1 ICAM-1

28 a CO CO XI m w - a t m 09 C

29 % specific lysis OQ c (tl r-t 3 c O) r* O -l (/) en i" O) r* O -1 (/)

30 X <D u c c (0 <5 **- ummm. days Figure 4

31 <2 "55 > ü Q. E75+F13 E75+F7 Figure 5

32 50 <Z> 40 «a 0 s 30 NP E75 NP E75 NP E75 NP E Figure 6

33 P'l CPM K> W cn > H + OP c l-l

34 CPM *s> \ 00 OP c l-t n> - id Ol fd T3 e-k h^ a fd ^«-^ 1 * C/l c crq > 3 *^> 1 ^,, V + / H (^1 u> c () 3 c J 1 ^ -a ON X\\\\\\\\\\\\\\\^^ v\\\\\\\\\\\\\^ *1 +

35 WD U G E X Stim. G89 Peptide (ug/ml) Figure 9

36 Figure 10 AC\r\r\ - A: ^ UUU T / IL-10 ^ IFN-q W IL E DJ a L 1 <D C u a NP E75 G89 peptide E75+G89

37 Identificatin f activated tumr antigen-reactive CD8 + cells in healthy individuals. Tm V. Lee*, Brett W. Andersn*, Agapit Castilleja*, Gerge E. Peples*, J. Taylr Whartn*, James L. MurrayJ, and Cnstantin G. Iannides*'f Frm the departments f Gyneclgic Onclgy, %Biimmuntherapy, and ^Immunlgy, The University f Texas M.D. Andersn Cancer Center, Hustn, Texas Running Title: Activated HER-2 reactive memry CD8 Key wrds: CTL, HER-2, interfern-y, IL-12, IL-2, CTLA-4, B7 Abbreviatins used in this paper: x, anti; DC, dendritic cells; HER-2, HER-2/neu prtncgene, TAL, tumr assciated lymphcytes, HS, human serum; NP, n peptide; AES, aminenhancer f split; FBP, flate binding prtein. 2 Acknwledgements: We thank Mr. Luis J. Sule fr utstanding editrial assistance. We als thank Mr. David McCnkey fr fruitful discussins and advice. This wrk was supprted in part by grant DAMD Peptide synthesis was supprted in part by the cre grant CA ^Please address crrespndence t: Dr. Cnstantin G. Iannides, The University f Texas M.D. Andersn Cancer Center, Department f Gyneclgic Onclgy, 1515 Hlcmbe Blvd., Bx 67, Hustn, Texas 77030, Tel: , Fax:

38 Summary We investigated the ability f HER-2 peptide E75, which maps an immundminant CTL epitpe fr varian and breast tumr-assciated lymphcytes (TAL) 1, t activate effectr functins in freshly islated CD8 + cells. IFN-y was rapidly induced by E75 within 20-24h in six f six healthy dnrs, in the presence f IL-12 and was detectable as early as 6h. The IFN-y levels were Ag-cncentratin dependent. Similar results were btained with peptides mapping CTL epitpes frm tw ther tumr Ag: flate binding prtein (FBP) and amin-enhancer f split f Ntch (AES). IFN-y was als detected, in respnse t HLA-A2 matched tumrs+il-12 but nt f IL-12 alne. The majr surce f IFN-y were CD45RO + CD8 + cells. Inductin f IFN-y and IL-2 frm CD8 + cells and f IL-12 frm dendritic cells (DC) by CD8 + cells reactive with E75 mirrred their inductin by the influenza matrix peptide (Ml: 58-66) in the same individual. Respnses t Ml are used t define the presence f activated memry cells in healthy individuals. Cmpared t Ml respnses E75 recgnitin induced 2-4 fld lwer levels f IL-12 frm the same APC and IFN-/and IL-2 frm CD8 + cells. At lwer Ag cncentratins the endgenus IL-12 induced by E75-reactive CD8 + cells did nt reach the threshld required t c-stimulate fr IFN-y. cb7.1 synergized with E75 in increasing the verall levels f IL-2 induced within 24h. Priming f CD8 + cells with E75+IL-2+ CTLA-4/B7.1 prmted marginal prliferatin suggesting a functinal dichtmy in the activating effects f E75. The presence f tumr Ag-reactive activated CD8 + cells in unimmunized healthy individuals may imprve ur understanding f the mechanisms f immunsurveillance and regulatin f immune respnses by tumrs.

39 Intrductin The recent characterizatin f tumr Ag recgnized by CTL pened the pssibility f develpment f Ag and epitpe-specific cancer vaccines. Tumr Ag recgnized by melanma, varian, and breast CTL have been demnstrated t be self-prteins (1). The fact that in cancer patients, CTL-specific fr these self peptides c-exist with prgressive tumrs, suggest that such respnses can be primed in viv, but either CTL d nt expand t the numbers required t mediate an effective respnse, r they expand but they are nt functinal in their state in viv. This raises the questin whether: (a) such CD8 + cells are already present in healthy individuals; (b) represent naive (ignrant), activated r activated and tlerized effectrs; and (c) if they are ignrant and tlerized which mechanisms are invlved. Since tumr derived factrs such as IL-10 inhibit priming f type 1 respnses t tumr Ag (2) clarificatin f the presence and requirements fr activatin f these cells may be beneficial fr preventive cancer vaccinatin in high risk individuals r patients with n evidence f disease after chemtherapy. If the Ag targets naive cells, they will respnd if cstimulatry receptrs are present n APC and bind t their apprpriate ligands. An effectr respnse by cytkines will be bserved after prgressin thrugh the cell cycle and 2-4 divisins (i.e h). (3) This respnse will be inhibited by antibdies t cstimulatry mlecules such as B7.1/B7.2. "Ignrant" cells may require increased TCR signaling which can be achieved by Ag mdificatin (4). Hwever, the kinetics f IFN-y secretin and requirement fr B7-CD28 will nt change. If Ag induces tlerance, then naive cells may express a partial respnse at priming but they will nt develp a respnse at restimulatin (5). If activated effectrs are present and are tlerized/anergized by expsure t Ag in the absence f cstimulatin, they will be unable t respnd t the cgnate/crssreactive stimulus that was initially effective fr their activatin. Analysis f the cytkine respnse can distinguish whether Ag induces clnal anergy (characterized by minimal secretin) r cytkine mediated immunsuppressin due t high levels f IL-10 (6). In cntrast, if activated effectrs are present, they will immediately r rapidly respnd t Ag by cytkine secretin withut requiring divisin (7). In this case cstimulatin thrugh surface receptrs may have an enhancing/stabilizing effect n sme respnses (e.g. CD28 n IL-12 R) and a regulatry effect n ther respnses due t B7 ligatin