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[CANCER RESEARCH 54, 1055-1058, Fcbruary 15, 1994] CD4 § T-Cells from Mice Immunized to Syngeneic Sarcomas Recognize Distinct, Non-Shared Tumor Antigens P e t e r A . C o h e n , 1 P h i l i p J. C o h e n , S t e v e n A . R o s e n b e r g , a n d J a m e s J. M u l ~ Branches of Surgery [P A. C., S. A. R.] and Dermatology [P J. C.], National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20814, and Systemics, Palo Alto, CA 94304 [J. J. M.] ABSTRACT MATERIALS We have utilized a newly developed culture system to study the properties of antitumor CD4 + T-cells relevant to the rejection of syngeneic methylcholanthrene sarcomas. Fresh syngeneic dendritic cells prepared from spleen, then pulsed with crude lysates of methylcholanthrene sarcomas, evoke antigen-specific proliferation by CD4 + but not by CD8 § T-cells from tumor-immune mice. Unfractionated splenocytes display similar antigen presenting capacity if they are not irradiated before the pulse with tumor lysate. CD4 + T-cells from mice immunized to individual methylcholanthrene sarcomas proliferate cross-reactively to dendritic cells pulsed with fresh tumor digests, but not to dendritic cells pulsed with cultured tumor cells. This apparent shared recognition of sarcoma lysates was demonstrated to be a result of sensitization to bacterial collagenase during the immunization procedure. Therefore, the murine CD4 § T-cell response to tumor immunization is similar to the CD8 § response in that sensitization occurs predominantly to tumor specific transplantation antigens rather than to shared tumor antigens. Strategies to avoid artefactual tumor cross-recognition by CD4 § T-cells are discussed. Mice. Female C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA) and National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). Animals were used between ages 8 and 12 weeks. Murine Tumors. All tumors were of C57BL/6 origin; the MC sarcomas 203 and 207 and adenocarcinoma MC38 were maintained in vivo by serial s.c. transplantation; the sarcomas were serially passed 9 times before reinitiating with early passage cryopreserved tumor mince. WP4 is a cultured clone from the MC sarcoma 205, subsequently passaged in mice. The Lewis lung carcinoma was obtained from the Frederick Cell Repository. All tumors were demonstrated free of viral pathogens and Mycoplasma by the National Cancer Institute-Frederick Cancer Research and Development Center. Culture Medium. All assays and cultures were performed in CM consisting of RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% heatinactivated fetal calf serum (Biofluids), 100 units/ml pencillin, 100 ~g/ml streptomycin, 50 txg/ml gentamicin sulfate, 2 ~g/ml Fungizone, 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1 p~g/ml indomethacin. Tumor Digestion and Cell Line Derivation. Serially passed s.c. tumors were aseptically harvested and finely minced, then enzymatically digested in 50 ml of Hanks' balanced salt solution containing DNase type IV (0.1 mg/ml), collagenase type IV (1 mg/ml), and hyaluronidase type V (2.5 units/ml) (Sigma, St. Louis, MO), with constant stirring over 2 h at 25~ After nylon mesh filtering and 2 washes in Hanks' balanced salt solution, the resulting single cell suspensions were used either for immunizations or as a source of tumor antigen. Murine Tumor Immunizations. Syngeneic mice were immunized to weakly immunogenic MC sarcomas as described previously (1, 2). Naive mice were first inoculated intradermally with 106 viable syngeneic sarcoma cells mixed with 100/xg formalin-killed Corynebacterium parvum; all mice developed small tumors which rapidly regressed. Three weeks following the initial inoculum, all mice received intradermal challenges with 2 • 105 viable tumor cells (without C. parvum); 10-60% of mice rejected these challenges dependent on a particular sarcoma's immunogenicity. Three weeks following this challenge, tumor-free mice received 106 viable tumor cells intradermally; mice which rejected these challenges (generally 100%) were designated hyperimmune, and used 3 weeks after the last tumor challenge as a source of immune spleens. Tumor Antigen. Freshly digested tumor cell suspensions were either used directly as a source of antigen or first cultured for 1 to 4 weeks in CM without indomethacin. To prepare tumor antigen, freshly digested or cultured tumor cells were snap-freeze-thawed (in CM without dimethyl sulfoxide) twice in rapid succession, then refrozen and stored in liquid nitrogen for later use. No viable tumor cells were detectable in these FT tumor lysates after the final thaw. Dendritic Cell Preparation. Splenic DC were freshly prepared from naive C57BL/6 mice following the method of Girolomoni et aL (7). After partial purification with DNase treatment and Percoll gradient fractionation, DC were resuspended in CM and placed in 25-cm e Falcon 3013 flasks, 3 spleen equivalents per flask. Nonadherent cells were discarded 2 h later, leaving partially purified adherent DC in culture. FT syngeneic tumor cell lysates were immediately added (1 • 107 tumor cell equivalents/flask), and antigen pulsing allowed to proceed for 18 h. DC were then harvested, having detached from the flasks during overnight incubation. Each group was counted, irradiated (1000 R) (8), then cocultured with CD4 § T-cells in proliferation assays. T Cell Subset Preparation. While DC were in preparation, fresh CD4 + T-cells were prepared from the spleens of naive or tumor-immune mice. INTRODUCTION It has long been recognized that mice vaccinated to w e a k l y i m m u nogenic M C 2 sarcomas can develop specific resistance to subsequent challenge with the same sarcoma (1). This i m m u n e protection requires both CD4 § and CD8 § T effector cells (2). D e v e l o p m e n t of crossprotectivity to other syngeneic sarcomas is seldom observed or is incomplete, indicating that T S T A rather than tumor-associated antigens are primarily responsible for M C sarcoma rejection following immunization (3). CD8 § T-cells derived from mice i m m u n i z e d to syngeneic M C sarcomas often fail to show cross-recognition o f other sarcomas in 51Cr-release lytic assays or cytokine-release assays, and do not show cross-protection in adoptive therapy experiments (4). Similarly, CD8 § T-cells g r o w n from tumor digests (tumor infiltrating l y m p h o c y t e s ) often manifest a similar absence o f cross-recognition (5). In contrast, CD8 § T-cells cultured from the l y m p h nodes of mice bearing progressive syngeneic M C sarcomas often show cross-recognition of other sarcomas in vitro, as well as cross-protection in adoptive therapy (4, 6). Technical problems previously limited the study of t u m o r - i m m u n e CD4 + T-cells in vitro. In the present report, we describe a newly developed culture technique to demonstrate that, similarly to CD8 § T-cells, CD4 § T-cells derived f r o m t u m o r - i m m u n e mice predominantly recognize unshared tumor antigens. Apparent cross-reactive antigens on tumors are shown to be artifacts of tumor digestion in bacterial collagenase, giving rise during tumor vaccination to collagenase sensitization. Received 8/16/93; accepted 12/14/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Surgery Branch, National Cancer Institute, Building 10, Room 2B46, 9000 Rockville Pike, Bethesda, MD 20892. 3 The abbreviations used are: MC, methylcholanthrene; DC, dendritic cells; CM, culture medium; FT, freeze-thawed;TSTA, tumor specific transplantation antigens; PHA, phytohemagglutinin;APC, antigen presenting cells; pl, one-sided p-valve. AND METHODS 1055 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. MURINE ANTITUMOR ('D4' ('ELLS RE('O(;NIZI- NON-SItARED ANTIGENS Splenocytes were first depicted of red blood cells with ammonium chloridepotassium lysing buffer. Negative selection immunoaffinity columns (Pierce Chemical Co., Rockford, IL) were then utilized with a single modification: the provided anti-CD8 reagent or purified anti-CD4 (GK1.5; American Type Culture Collection, Rockville, MD) was diluted in CM conditioned by M5/114 hybridoma (anti-I-N'~ American Type Culture Collection) to facilitate quantitative depletion of la + cells (9). Proliferation Assays. Flat-blottomed 96-well microtiter plates (Costar) were utilized. Each well received 1 • 105 CD4 + T-cells. DC were added to achieve a final CD4:DC ratio of 20:1 or 40:1. Each well contained a final volume of 0.2 ml CM. Control groups routinely performed included: CD4 + T-cells cultured without DC; pulsed irradiated DC cultured without CD4 + T-cells; each CD4 + T-cell group with 3/xg/ml PHA (Sigma); each CD4 + T-cell group sham-pulsed (i.e., cultured in CM only); each DC group with fresh BALB/c (allogeneic) CD4 + T-cells; each DC group with fresh naive C57BL/6 (syngeneic) CD4 + T-cells. Cultures were continued for a total of 5 days, except for PHA control groups, which received only 3 days' culture. At 18 h prior to assay completion, tritiated deoxythymidine (1 p,Ci/well) (Amersham, Arlington Heights, IL) was added to each microtiter well. At completion, plates were harvested with a semiautomated LKB cell harvester, and measured by a LKB/ Wallac 1205 Betaplate liquid scintillation counter (Gaithersburg, MD). Responses were reported as mean cpm -+ SEM from triplicate samples, and analyzed by Wilcoxon rank sum analysis. Fluorescence-activated Cell Sorter Analysis. Cells were routinely analyzed on a Becton-Dickinson FACScan or FACStar Plus using conjugated direct antibodies and subclass matched controls obtained from Becton-Dickinson (Lincoln Park, N J) and Pharmingen (San Diego, CA). Cells were pretreated with unconjugated anti-CD32 (2.4G2) to block FcR binding by conjugated antibodies (Pharmingen). I r[ ] (A) Sham Pulsed DC alone (B) MC 203 Pulsed DC alone (C) Naive CD4+ alone (D) Immune CD4+ alone (E) AIIogeneic CD4+ alone (F) Sham Pulsed DC + AIIo CD4+ (G) MC 203 Pulsed DC + AIIo C D 4 + H (H) PHA + DC + Naive C D 4 + (I) PHA + DC + Immune C D 4 + (J) Sham Pulsed DC + Naive C D 4 + (K) MC 203 Pulsed DC + Naive C D 4 + (L) Sham Pulsed DC+ Immune C D 4 + (M) MC 203 Pulsed DC + Immune CD4+ 0 5 10 15 20 25 30 35 Thousands of C P M Fig. 1. Representative proliferation assay showing mandatory control groups as described in "Results." CD4 + T-cell splenocyte sources included naive C57BL/6 mice (Naive CD4+), naivc BALB/c mice (Allogeneic CD4 ~), and MC203-immunc C57BL/6 mice (Immune CD4 ' ). CD44 T-cells were cocultured with sham-pulsed or MC203-pulsed naive C57BL/6 DC (20:1 ratio) for 5 days, cxcept for PHA groups which were cultured for 3 days. PHA groups received 3 ~g/ml PHA (Sigma) and sham-pulsed DC. All DC groups were irradiated (1000 R)just prior to coculture; tritiated deoxythymidine was added 18 h prior to harvest. Bars, +_SEM. Naive CD4 alone (A) (B) Naive CD4 + Naive CD8 (C) (D) Naive CD4 + Immune CD8 (E) RESULTS Immune CD4 alone (G) (H) Immune C04 + Naive CD8 (I) Control Group Analysis of 2-Step CD4 § T-Cell Proliferation Assay. A representative experiment illustrating all routine control groups is s h o w n in Fig. 1. Irradiated D C alone s h o w e d negligible proliferation at Day 4 in culture, whether or not pulsed with FT MC-203 t u m o r antigen. C D 4 + T-cells alone also s h o w e d negligible proliferation. W h e n naive B A L B / c C D 4 + T-cells were cocultured with each group o f irradiated C 5 7 B L / 6 DC, strong proliferation was seen; this c o n f i r m e d that each D C group manifests both Class II expression and costimulatory function. Each CD4 + T-cell group was also cultured with s h a m - p u l s e d D C and P H A to confirm proliferation potential. Naive C D 4 + T-cells s h o w e d consistently low level proliferation w h e n cocultured with each irradiated D C group. Finally, C D 4 + T-cells from mice i m m u n e to the MC-203 t u m o r s h o w e d low level proliferation to s h a m - p u l s e d irradiated DC. Taken together, these control groups enabled the demonstration o f significant antigen-specific proliferative effects w h e n MC-203 i m m u n e C D 4 ~ T-cells were cocultured with M C - 2 0 3 pulsed, irradiated D C (Fig. 1, Group L versus Group M, p l = 0.0248). Tumor-immune CD4 § T-Ceils but Not CD8 § T-Cells Show Specific Proliferation to Tumor-pulsed D C . W h e n C D 4 + and C D 8 § T-cell subgroups were each prepared from both naive and tumori m m u n e mice, only t u m o r - i m m u n e C D 4 + T-cells d e m o n s t r a t e d specific proliferation w h e n cocultured with tumor-pulsed D C c o m p a r e d to s h a m - p u l s e d D C (Fig. 2, Group G versus Group H, p l = 0.0248). This proliferation can be blocked by adding anti-Class II or anti-CD4 antibody 12 h after initial coculture (data not shown). Mixing experim e n t s w h i c h c o m p a r e d adding either naive or t u m o r - i m m u n e CD8 + T-cells to t u m o r - i m m u n e C D 4 + T-cells d e m o n s t r a t e d nonspecific augmentation of proliferation (Fig. 1, Group J versus Group L, pl -0.4136). In other experiments not shown, irradiation of the tumori m m u n e C D 4 + T-cell subset completely abrogated the proliferative response of either added C D 8 + T-cell group (9). (J) ] I I --J Immune CD4 + Immune CD8 (K) (L) Naive CDS+ alone (M) (N) Immune CD8+ alone (0) (p) 0 10 20 30 40 50 Thousands of CPM I i SEM / ~ Sham Pulsed DC / E1 MC203-Pulsed DCI Fig. 2. Representative T-cell subset proliferation assay as described in "Results." CD4 § and CD8 § T-cell subsets were prepared from naive and MC203-immune C57BL/6 mice, thcn cocultured with sham-pulsed or MC203-pulsed naive C57BL/6 DC (40:1 ratio) for 5 days. All DC groups were irradiated (1000 R) just prior to coculturc; tritiated deoxythymidine was added 18 h prior to harvest. Nonirradiated but not Preirradiated Splenocytes Contain the Antigen-processing Capacity of Partially Purified DC. Several previously published efforts to expand antitumor C D 4 + T-cells in vitro had used preirradiated (3000 R) unfractionated splenocytes as a source of A P C for antitumor C D 4 + T-cells (10, 11). Even though a dendritic cell subpopulation is present in such unfractionated splenocytes, such A P C have not proved to be an effective way to present t u m o r lysates to freshly harvested i m m u n e C D 4 § T-cells (10). We c o m p a r e d the antigen-presenting function of such preirradiated (3000 R) unfractionated splenocytes to our standard preparation of tumorpulsed splenic DC in which DC are irradiated only after the lysatepulse period. In addition, in order to assess the effect of preirradiation on naive unfractionated splenocytes, the latter were either preirradiated with 3000 R, then pulsed for 8 h with F T t u m o r lysate, or first pulsed with t u m o r lysate, then irradiated. Each A P C group was then cocultured with C D 4 § T-cells. As s h o w n in Fig. 3, unfractionated splenocytes did have the capacity to process and present t u m o r lysate, but this proved to be a radiosensitive event. 1056 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. MURINE ANTITUMOR CD4 + CELLS RECOGNIZE MC-102. We o b s e r v e d similarly selective proliferation by C D 4 + T- SplC+MC203 (5:1 Pulse) cells from mice i m m u n e to M C - 2 0 7 , M C - 1 0 5 , and W P 4 w h e n cultured tumor cells were used as stimulators in vitro. Cross-Reactive Tumor-immune C D 4 § T-Cell P r o l i f e r a t i o n Is SplC+MC203 (10:1 Pulsed) Caused by Immunization to Collagenase. D C were pulsed for 18 h with either collagenase, hyaluronidase, or DNase, or pulsed with FT fresh tumor digests or cultured tumor cells, then cocultured with C D 4 § T-cells either from syngeneic mice i m m u n e to M C - 2 0 3 tumor, or from naive allogeneic ( B A L B / c ) mice. As seen in Fig. 5, D C pulsed I SplC+MC203 (60:1 Pulsed) ~] MC203 alone with collagenase, but not hyaluronidase or DNase, e v o k e d strong proliferation from M C - 2 0 3 i m m u n e C D 4 + T-cells, as did all D C groups pulsed with fresh collagenase-digested tumor preparations. D C pulsed with cultured tumor cells did not e v o k e strong proliferation except for the relevant tumor cell line, M C - 2 0 3 . Importantly, all D C groups e v o k e d strong proliferative responses b y naive B A L B / c C D 4 + T-cells, indicating adequate antigen-presenting function in all D C preparations. Additionally, all D C groups e v o k e d negligible proliferative responses from naive C 5 7 B L / 6 C D 4 + T-cells (latter data not DC, Sham Pulsed DC + MC203Pulsed ~`:~:~`:~`:`:`:`:`:~`:`:`:~`:i~`:~i`:`:~`:`:~`:`:`:`:`:~iii~`:~`:`:`:`:`:~`:`:ii~`:`:H 0 10 20 30 40 50 60 Thousands of CPM I T SEM / ANTIGENS geneic M C sarcomas, namely MC-207, M C - 1 0 5 , M C - 2 0 5 , W P 4 , and SplC, Sham Pulsed~ SplC+MC203 (30:1 Pulsed) NON-SHARED Q P u l s e d , then irradiated / I l r r a d i a t e d , then pulsed Fig. 3. Antigen-presenting function of unfractionated splenocytes versus prepared dendritic cells. DC were prepared, pulsed, and irradiated in the standard way described in "Materials and Methods." Unfractionated splenocytes (SC) were ammonium chloride lysing buffer-treated, washed, and then resuspended in CM at 5 X 106 SC/ml. At that time they were either irradiated (3000 R) and then pulsed with MC203 FT cells for 8 h, or first 203-pulsed and then irradiated. The SC:MC203 pulse ratios are shown on the axis labels. SC and DC groups were each then cocultured with 203-immune CD4 § T-cells. Each microwell received 1 • 105 CD4 + T-cells and either 5 x 103 pulsed DC, 5 x l0 s pulsed SC, or 1 X 105 FF tumor cells alone. Tritiated deoxythymidine was added 18 h prior to harvest. 203-Immune CD4 § T-cell proliferation following coculture with SC, DC, or FT tumor cells alone is shown. shown). DISCUSSION The absence o f cross-protection to other syngeneic sarcomas after mice are immunized to particular sarcomas has long suggested that recognition of T S T A often plays the central role in the rejection o f these tumors. Such rejections are dependent upon both C D 4 + and C D 8 + T-effector cells (2). Previous w o r k had demonstrated that antitumor C D 8 § T-cells taken from t u m o r - i m m u n e mice or g r o w n from tumor digests (tumor-infiltrating l y m p h o c y t e s ) often fail to recognize shared s a r c o m a antigens in vitro and fail to s h o w cross-reactive therapeutic effects w h e n used in adoptive transfer experiments (1, 3-5). D u e to technical obstacles, it w a s previously difficult to characterize the C D 4 § T-cells involved in M C sarcoma rejections. The present Thousands of C P M Sham pulsed DC 3LL Digest Pulsed DC MC 38 Digest Pulsed DC MC 203 Digest Pulsed DC MC 207 Digest Pulsed DC i 3LL Culture Pulsed DC i w o r k demonstrates that C D 4 § T-cells taken from t u m o r - i m m u n e mice also predominantly recognized unshared tumor antigens. It remains to be determined w h e t h e r both C D 4 § and C D 8 § T-cells recognize epi- : MC 38 Culture Pulsed DC Thousands of C P M MC 203 Culture Pulsed DC l MC 207 Culture Pulsed DC 100 m 80 60 40 Sham Pulsed DC 20 M C - 2 0 7 Immune 0 10 20 30 40 50 60 70 I I WP4 Digest Pulsed DC MC 207 Digest Pulsed DC MC 203 Digest Pulsed DC M C - 2 0 3 Immune Fig. 4. Proliferation of immune CD4 + T-cells when cocultured with DC previously pulsed with either fresh tumor digests or cultured tumor cells. Naive C57BL/6 DC were prepared and pulsed with FT tumor cells either freshly digested from tumor mince (Digest) or grown for 1 week in vitro (Culture) prior to freeze-thaw. Tumors included Lewis lung carcinoma (3LL), MC38 adenocarcinoma, and MC207 and MC203 sarcomas. Pulsed DC were then cocultured with either MC207-immune (left) or MC203-immune (right) CD4 § T-cells for 5 days. Tritiated deoxythymidine was added 18 h prior to harvest. (Stippled caps, +- SEM.) WP4 Culture Pulsed DC 3LL Culture Pulsed DC MC 203 Culture Pulsed DC "Triple enzyme" Pulsed DC DNAase Pulsed DC CD4 § T-Cells from Mice Immunized to Individual MC Sarcomas Proliferate Cross-Reactively to DC Pulsed with Fresh Tumor Digests, but not to DC Pulsed with Cultured Tumor Cells. As s h o w n in Fig. 4, C D 4 + T-cells obtained from mice immunized to M C - 2 0 3 or M C - 2 0 7 s h o w e d low level proliferation to sham-pulsed DC, but strong proliferation to D C pulsed with any syngeneic tumor tested, including nonsarcomas, w h e n fresh tumor digest w a s the source o f tumor antigen. In contrast, w h e n tumor digests w e r e cultured in vitro for 1 to 3 w e e k s prior to use as a source o f FT antigen, no cross-reactive proliferation w a s seen. In multiple experiments not shown, C D 4 + T-cells from M C - 2 0 3 i m m u n e mice consistently failed to proliferate cross-reactively to D C pulsed with other cultured syn- HYALURONIDASE Pulsed DC COLLAGENASE Pulsed DC 160 120 80 40 Natve AIIoaeneie CD4+ 0 40 80 120 Immune Svnaeneie CD4+ Fig. 5. Proliferation of 203-immune CD4 + T-cells when cocultured with DC previously pulsed with either Fq" tumor antigen or the enzymes used in tumor digestion. Naive C57BL/6 DC were prepared and pulsed with FT tumor cells which were either freshly digested from tumor mince (Digest) or grown for 1 week in vitro (Culture) prior to freeze-thaw. Tumors included Lewis lung carcinoma (3LL) and MC sarcomas WP4, 207, and 203. Alternatively, enzymes were added to DC instead of FT tumor antigen: DNase 1/.tg/ml, collagenase l0/xg/ml, hyaluronidase 25 milliunits/ml, or all 3 (Triple enzyme). These enzyme concentrations reflected a 1:100 dilution of those used during actual tumor digestions. MC203-immune CD4 § T-cells were cocultured with each DC group for 5 days. Tritiated deoxythymidine was added 18 h prior to harvest. (Stippled caps, +- SEM.) 1057 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. MURINE ANTITUMOR ('D4' ('ELLS RE('O(iNIZE NON-SttAREI) ANTIGENS topes derived from a single TSTA in the case of each M C sarcoma. It also remains to be determined w h e t h e r CD4 + T-cells from tumorbearing mice recognize shared tumor antigens in the pattern observed for CD8 + T-cells from tumor-bearing l y m p h nodes (4, 6). The results presented here underscore the danger that seemingly shared tumor antigens m a y often represent experimental artifact. Mice inoculated with tumor cells freshly digested in clostridial collagenase develop a prominent i m m u n e response to that enzyme. No such response has been observed for other digestive enzymes, which are bovine rather than bacterial in origin. Therefore, DC preparation methods w h i c h begin with collagenase digestions are likely to generate profound artifacts in this type o f tumor model. The repeated washing o f tumor cells following digestion does not fully remove the collagenase antigenic activity, but culturing digested tumor cells for several days eliminates the collagenase activity, as demonstrated in Fig. 5 by the loss o f cross-reactive tumor recognition. In other experiments not shown, w e have demonstrated that t u m o r - i m m u n e CD4 + T-cells can be expanded in culture to enrich for either tumorrecognizing or collagenase-recognizing subpopulations. Although tumor cells grown chronically in culture do not carry the risk o f generating collagenase sensitization w h e n used as a source o f tumor for vaccines, animals given repeated injections of cultured tumor cells are apt to develop sensitization to culture m e d i u m proteins. In this instance, CD4 + T-cells taken from mice i m m u n i z e d to tumor cultured cell lines show an abnormally high proliferative response to "sham-pulsed" dendritic cells prepared in the same culture m e d i u m ( e . g . , RPMI 1640 with 10% fetal calf serum) used to culture the tumor lines (data not shown). Our current strategy to prepare tumor cells for vaccination involves an initial tumor digestion (of necessity in collagenase) followed by several days' culture in RPMI 1640 supplemented with 0.5 to 1.0% non-heat-deactivated syngeneic m o u s e serum. This culture period facilitates catabolism of collagenase antigen, and adds no new foreign proteins to c o n f o u n d vaccinations. Either Langerhans cells or spleen DC pulsed with crude FT tumor preparations can be used to present specific tumor antigens to CD4 + T-cells (9). Fresh unfractionated splenocytes also possess significant antigen-presenting capacity, but this is impaired by irradiation prior to the antigen pulse period. Previous investigators studying antitumor CD4 + T-cells used unfractionated splenocytes as their source of APC; however, the splenocytes w e r e irradiated prior to antigen pulsing (10, 11). We believe that this partly explains the historical difficulty associated with growing specific antitumor CD4 + T-cells in vitro. In n u m e r o u s experiments, we have never observed tumor-specific CD8 + T-cell proliferation to be e v o k e d by tumor lysate-pulsed DC; this is true w h e t h e r or not recombinant interleukin-2 is added to the assay (data not shown). This suggests that DC process and present crude tumor antigen, in v i t r o at least, solely in a Class II context, and parallels the experience o f others w h o studied DC presentation of exogenous protein antigens such as ovalbumin (12). Since CD4 + T-cells can function effectively in v i v o against Class II nonexpressing tumors, including the M C sarcomas described in the present study (2, 11, 13), it is possible that A P C within solid tumors process and present exogenous tumor debris in a Class II context to CD4 + T-effector cells, triggering cytokine release which leads to accumulation and activation of CD8 + T-cells (14, 15) as well as accessory cells such as tumoricidal macrophages and lymphokine-activated killer cells (11, 16-18). Such accessory cells kill tumor targets independent o f tumor cell Class I and Class II expression (16, 17), and may explain how CD4 + T-cells achieve effectiveness in v i v o against Class 11 nonexpressing tumors (11, 13) as well as Class I nonexpressing tumors (17). We are currently using our culture system to expand specific antitumor CD4 + T-cells and investigate their m e c h a n i s m o f tumor rejection in v i v o (19). ACKNOWLEDGMENTS We would like to thank Stephen I. 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