Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8(+) CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.     

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.     

DC-expressed MHC class I single-chain trimer-based vaccines prime cytotoxic T lymphocytes against exogenous but not endogenous antigens

The poor immunogenicity of many tumors can be partly explained by the inefficiency of the MHC class I peptide presentation pathway. MHC-I-based single-chain trimers (SCT) represent a new class of molecules with the potential to overcome this limitation. We here evaluated the ability of SCT presenting a melanoma antigen peptide (TRP-2) to prime cytotoxic T lymphocyte (CTL) responses in mice when given as DNA vaccines via Gene Gun or when expressed by dendritic cells. The SCT was unable to induce detectable priming or significant anti-tumor activity of CTL using either vaccination strategy, whereas control SCT (with an exogenous peptide) primed strong responses. This study thus provides the first data related to the use of SCT in combination with DC and their application toward self antigens and suggest this potent technology, alone, is insufficient to overcome self tolerance.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.     

Efficient and qualitatively distinct MHC class I-restricted presentation of antigen targeted to the endoplasmic reticulum

For most nascent glycoprotein Ags, the MHC class I-restricted processing pathway begins in the endoplasmic reticulum (ER). From this location, they are translocated to the cytosol for degradation by the proteasome. A reasonable assumption is that processing of exocytic Ags is less efficient than that of cytosolic Ags, due to the requirement for additional handling, but that the processing pathways for the two types of proteins are otherwise similar. To test this, we compared the presentation of three epitopes within influenza nucleoprotein (NP) when this Ag is targeted to the cytosol or the ER. Surprisingly, under conditions of limited Ag expression, presentation of two proteasome-dependent epitopes is comparable when NP is targeted to the ER while presentation of a third is negatively impacted. Furthermore, presentation of the third epitope is unaffected by the addition of proteasome inhibitor when cytosolic NP is expressed but is significantly enhanced when exocytic NP is expressed. These results indicate that delivery of Ag to the ER need not preclude efficient presentation and that processing of cytosolic and ER-targeted Ag is qualitatively distinct.     

MHC class I-restricted presentation of exogenous antigen by thymic antigen-presenting cells in vitro and in vivo.

We have used a T-T hybridoma, RF33.70, to detect the MHC class I-restricted presentation of exogenous native OVA by thymic APC in vitro. Presentation of OVA with class I molecules by thymic APC requires intracellular processing. Phenotypic analyses indicated that low bouyant density, MHC class II+, FcR+ cells are capable of using this presentation pathway. In order to determine whether thymic APC have this function in vivo, thymic APC were isolated from mice after i.v. injection of native OVA. We find that OVA is presented in association with MHC class I, but not class II, molecules in the thymus. This is in contrast to splenic APC, which present exogenous OVA with both class I and class II molecules under these conditions. Our findings have implications for the repertoire of self-peptides that might be presented by thymic APC to developing T lymphocytes.     

Linkage of bacterial protein synthesis and presentation of MHC class I-restricted Listeria monocytogenes-derived antigenic peptides

The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217-225 and p60 449-457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.     

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.     

T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed.     

Tat-mediated protein delivery can facilitate MHC class I presentation of antigens

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.     

Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals

Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor. III. MHC class I-restricted CTL recognizes the processed form(s) of idiotype.

The purpose of this work was to determine the molecular nature of the idiotypic Ig of a B cell tumor, 2C3, involved in the induction of anti-idiotypic cytotoxic T-lymphocytes (CTL). We previously reported that hyperimmunization of mice with irradiated 2C3 cells provides effective tumor protection by inducing MHC class I-restricted CTL. Due to the enormous heterogeneity of the splenic CTL further study could not be undertaken on the idiotype (Id)-CTL interaction. Subsequently an anti-idiotypic CTL line, A102, and a highly Id-specific CTL clone, 102.F5, have been developed. In the present investigation we report that the processed forms of idiotypic determinants are responsible for induction and activation of these specific effector CTL. Inhibition studies using anti-TcR and anti-MHC class I mAbs showed that the TcR-CD3 complex of the anti-idiotypic CTL recognized 2C3 Id in the context of MHC class I antigens. The cytotoxicity of these CTL could not be inhibited with affinity-purified 2C3 Ig used as such or after pulsing with splenic antigen-presenting cells (APC). Furthermore, using brefeldin A (BFA) and chloroquine (CLQ), which are specific inhibitors of cytosolic and endosomal antigen processing pathways, respectively, it has also been observed that exposure of 2C3 to BFA but not CLQ prevents its cytolysis by both anti-idiotypic CTL line and clone. These results clearly indicate that endogenously produced idiotypic determinants of 2C3 Ig are processed in pre-Golgi vesicle, possibly in the ER, along with MHC class I antigens and then are transported to the membrane. Treatment of 2C3 with BFA, however, did not exert any effect on the expression of membrane-associated Ig of 2C3 cells. Therefore, it is the processed form rather than the bona fide receptor Ig on the cell surface that is recognized by the Id-specific CTL.     

Neutrophils process exogenous bacteria via an alternate class I MHC processing pathway for presentation of peptides to T lymphocytes

Peptides that are presented by class I MHC (MHC-I) molecules derive from cytosolic Ags processed via the conventional MHC-I pathway or exogenous Ags processed via alternate MHC-I processing mechanisms. Alternate MHC-I processing by macrophages and dendritic cells allows presentation of peptides from particulate Ags, including bacteria. Despite the established phagocytic activity of neutrophils, MHC-I processing and presentation of phagocytosed Ags by neutrophils has not been investigated. Murine neutrophils from peritoneal exudates were shown to express MHC-I molecules and tested for the ability to process HB101.Crl-OVA, Escherichia coli transfected to express a fusion protein containing the 257-264 epitope of OVA. Neutrophils were found to process HB101.Crl-OVA and present OVA(257-264)-K(b) complexes to CD8OVA T hybridoma cells via a pathway that was resistant to brefeldin A, an inhibitor of anterograde endoplasmic reticulum-Golgi transport, and lactacystin, a proteasome inhibitor. These results suggest that neutrophils process phagocytosed bacteria via a vacuolar alternate MHC-I pathway that does not involve cytosolic processing. In addition, neutrophils were found to secrete or "regurgitate" processed peptide that was subsequently presented by neighboring prefixed macrophages or dendritic cells. Thus, neutrophils may influence T cell responses to bacteria, either by directly presenting peptide-MHC-I complexes or by delivering peptides to other APCs for presentation. Hypothetically, neutrophils may directly present peptide to effector T cells in vivo at sites of inflammation, inducing cytokine production, whereas dendritic cells in receipt of neutrophil-derived antigenic peptides may migrate to lymphoid organs to initiate T cell responses.     

Re-evaluating the generation of a "proteasome-independent" MHC class I-restricted CD8 T cell epitope

The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP(147-155) of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently "proteasome-independent" epitopes remain poorly defined. We have examined the generation of NP(147-155) and a second proteasome-dependent NP epitope, NP(50-57), using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP(147-155), 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP(50-57) or NP(147-155), and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP(147-155) epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.     

CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.     

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.     

Expression of MHC class II antigens in human lung cancer cells.

Surgical specimens of lung cancers were examined immunopathologically for the expression of major histocompatibility complex class II (MHC-II) antigens in the tumor cells and their relationship to the lymphocytic infiltration. A lymphocytic infiltrate was frequently observed in the tumor tissue, though its intensity differed among the various histological types. MHC-II antigens were often demonstrated in tumors with a lymphocytic infiltrate. They were detected predominantly in the cytoplasm of tumor cells and to a lesser extent on the cell membranes. The emergence of the MHC-II-positive tumor cells was closely related to a local infiltration by lymphocytes including interferon-gamma (IFN-gamma)-producing T-cells. On the basis of the histological findings, an in vitro experiment was carried out. Four types of lung cancer cells were incubated with recombinant IFN-gamma in order to induce MHC-II antigens. MHC-II antigens (HLA-DR as well as HLA-DQ and HLA-DP antigens) were elicited in three cancer cell lines depending on the concentration of IFN-gamma. Immunoelectron microscopic study revealed that they were expressed on the surface of the cell membrane, though to a lesser extent than in the cytoplasm. It was considered that MHC-II antigens could be induced in some tumor cells in the immunological environment where IFN-gamma was secreted from T-cells and concentrated locally.     

Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation

Liposome-encapsulated protein Ag were used to dissect the roles of various subcellular compartments in Ag processing for class I and class II MHC-restricted presentation. Macrophages exhibited efficient processing of Ag encapsulated in acid-resistant dioleoylphosphatidylcholine/dioleoylphosphatidylserine liposomes, which sequester their contents from potential endosomal processing events and release them only after delivery to lysosomes. Lysosomal processing was demonstrated for all four Ag studied (OVA, murine hemoglobin, bovine ribonuclease A, and hen egg lysozyme), establishing the recycling of immunogenic peptides from lysosomes after Ag processing. These acid-resistant liposomes did not engender class I processing. Ag encapsulated within acid-sensitive dioleoylphosphatidylethanolamine/palmitoylhomocysteine liposomes were also processed via the class II pathway. Of the four Ag encapsulated in liposomes, one, OVA, was tested for ability to stimulate a class I-specific response. OVA in acid-resistant liposomes did not engender a class I-specific response. In contrast, OVA encapsulated in acid-sensitive liposomes was presented by class I molecules, albeit less efficiently than it was presented by class II molecules. We interpret this to be the result of the release of a minor portion of the encapsulated Ag into the cytosol.     

Differential stability of antigenic MHC class I-restricted synthetic peptides

Various synthetic peptides recognized as Ag by CTL in the context of MHC class I molecules were tested for stability in vitro and in vivo. Peptide inactivation in vitro was quantitated by titrating the amount of peptide required to sensitize target cells for lysis by specific CTL clones. The degree of inactivation after overnight incubation at 37 degrees C varied widely among a series of antigenic peptides. Some were nearly unaffected, whereas others lost activity by more than 100-fold or even 10,000-fold. However, no correlation was found between susceptibility to serum inactivation and antigenic potency as measured in short term cytolytic assays. No inactivation occurred at 4 degrees C, or at 37 degrees C in the absence of serum, under the conditions used. Serum inactivation most likely involved proteolysis because it could be inhibited by protease inhibitors. Moreover, presumed cleavage products of a radiolabeled susceptible peptide could be visualized by TLC. In vivo, the persistence of the antigenic activity of the injected peptides, either in extracellular fluids or on tumor target cells growing in an ascites form, correlated with the degree of stability found for the peptides in vitro. The differential stability of synthetic peptides may have important consequences for attempts to manipulate the development of an immune response in vivo.