Cancer mucosa antigens as a novel immunotherapeutic class of tumor-associated antigen.

Colorectal cancer is a leading cause of cancer-related mortality worldwide. Surgery and chemoradiation exhibit incomplete efficacy and, ultimately, 50% of patients die of metastatic disease. In the context of that unmet clinical need, immunotherapeutic approaches have enjoyed limited success, partly because of a paucity of suitable antigen targets. However, exploitation of immune compartmentalization, employing antigens with expression restricted to normal intestinal mucosa and derivative colorectal tumors--cancer mucosa antigens (CMAs)--may represent a previously unrecognized class of immune targets supporting efficacious antitumor immunotherapy. Guanylyl cyclase C (GCC) is an intestine/colorectal cancer-restricted protein ideally suited as the first CMA for clinical evaluation.

The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein.

Human minor histocompatibility Ags (mHag) present significant barriers to successful bone marrow transplantation. However, the structure of human mHag and the basis for antigenic disparities are still largely unknown. Here we report the identification of the gene encoding the human mHag HA-2 as a previously unknown member of the class I myosin family, which we have designated MYO1G. The gene is located on the short arm of chromosome 7. Expression of this gene is limited to cells of hemopoietic origin, in keeping with the previously defined tissue expression of the HA-2 Ag. RT-PCR amplification of MYO1G from different individuals led to the identification of two genetic variants, designated MYO1G(V) and MYO1G(M). The former encodes the peptide sequence previously shown to be the HA-2 epitope (YIGEVLVSV), whereas the latter shows a single amino acid change in this peptide (YIGEVLVSM). This change has only a modest effect on peptide binding to the class I MHC-restricted element HLA-A*0201, and a minimal impact on recognition by T cells when added exogenously to target cells. Nonetheless, as detected using either T cells or mass spectrometry, this amino acid change results in a failure of the latter peptide to be presented at the surface of cells that express MYO1G(M) endogenously. These studies have thus identified a new mHag-encoding gene, and thereby provide additional information about both the genetic origins of human mHag as well as the underlying basis of an Ag-positive vs Ag-negative state.

B-Myb overexpression results in activation and increased Fas/Fas ligand-mediated cytotoxicity of T and NK cells.

The human B-myb gene encodes a transcriptional regulator that plays an important role in cell cycle progression, differentiation, and survival. To assess the in vivo role of B-myb, we investigated the phenotype of mouse transgenic lines in which B-Myb expression in lymphoid tissues was driven by the LCK proximal promoter. Overexpression of B-Myb had no measurable effect on the subsets of splenic and thymic lymphocytes, but was associated with increased expression of Fas ligand in NK and T cells. B-Myb-overexpressing splenocytes expressed higher IFN-gamma levels and contained higher percentages of cytokine-producing cells than wild-type (wt) splenocytes, as detected by Western blot analysis and ELISPOT assays, respectively. Ex vivo-cultured transgenic thymocytes and splenocytes had decreased survival compared with the corresponding cells from wt mice, possibly dependent on increased expression of Fas ligand. In addition, Fas ligand-dependent cytotoxicity of transgenic T and NK cells was significantly higher than that mediated by their wt counterparts. Together, these results indicate that B-Myb overexpression results in T and NK cell activation and increased cytotoxicity. Therefore, in addition to its well-established role in proliferation and differentiation, B-myb also appears to be involved in activation of NK and T cells and in their regulation of Fas/Fas ligand-mediated cytotoxicity

The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing.

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201-restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75-81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8-specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.

Quantitative analysis of adenovirus-specific CD4+ T-cell responses from healthy adults.

Although nearly all adults are seropositive for adenoviruses, little is known about the cellular immune responses to these ubiquitous pathogens. We have previously identified adenovirus-specific proliferative T-cell responses in peripheral blood mononuclear cells (PBMC) from healthy adults. In this study, memory T-cell responses to adenovirus were further evaluated in healthy adult donors using a short term, quantitative enzyme-linked immunospot assay (ELISPOT) assay. Adenovirus antigen induced specific secretion of interferon-gamma (IFN-gamma) from PBMC within 12 hours of incubation. PBMC from 20 of 22 healthy donors (90.9%) expressed IFN-y in response to adenovirus. Responder cells were identified as CD4+ T cells by immunomagnetic depletion methods. Interleukin-4 (IL-4) secretion was not detected, consistent with a TH1 response. There was a 10-fold variation in the frequencies of adenovirus-specific CD4+ T cells between donors (range, 34 to 294; median, 122 per million PBMC). Adenovirus-specific T cell frequencies remained stable over periods up to 2 years among individual donors, but there was an inverse correlation between frequency and donor age. These quantitative data suggest that most adults retain adenovirus-specific cellular memory after childhood exposure. This assay may be useful for the evaluation of adenovirus-specific CD4+ T-cell responses in patients treated with adenovirus gene therapy vectors and the identification of major T-cell epitopes.

Impaired assembly yet normal trafficking of MHC class I molecules in Tapasin mutant mice.

Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.

Differential role of p38 and c-Jun N-terminal kinase 1 mitogen-activated protein kinases in NK cell cytotoxicity.

The serine-threonine mitogen-activated protein kinase (MAPK) family includes extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases. In NK cells, spontaneous or Ab-mediated recognition of target cells leads to activation of an ERK-2 MAPK-dependent biochemical pathway(s) involved in the regulation of NK cell effector functions. Here we assessed the roles of p38 and JNK MAPK in NK cell-mediated cytotoxicity. Our data indicate that p38 is activated in primary human NK cells upon stimulation with immune complexes and interaction with NK-sensitive target cells. FcgammaRIIIA-induced granule exocytosis and both spontaneous and Ab-dependent cytotoxicity were reduced in a dose-dependent manner in cells pretreated with either of two specific inhibitors of this kinase. Target cell-induced IFN-gamma and FcgammaRIIIA-induced TNF-alpha mRNA accumulation was similarly affected under the same conditions. Lack of inhibition of NK cell cytotoxicity in cells overexpressing an inactive form of JNK1 indicates that this kinase, activated only upon FcgammaRIIIA ligation, does not play a significant role in cytotoxicity. These data underscore the involvement of p38, but not JNK1, in the molecular mechanisms regulating NK cell cytotoxicity.

Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma.

Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10(4)-2 x 10(7) plaque-forming units (PFU)/lesion; 10(4)-8 x 10(7) PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of > or =10(7) PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4+ and CD8+ lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14-21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti-beta-galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease free. One patient with only dermal metastases confined to the scalp achieved a complete remission. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression.

The induction of virus-specific CTL as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained.

The role of epitope expression levels in CD8+ T cell priming has been controversial. Yet this parameter is of great importance in the design of rational approaches to optimize CTL responses to a variety of pathogens. In this paper we examine the influence of epitope production on CD8+ T cell priming by exploiting a system that allows a 200-fold range of cell surface epitope expression in vitro with a fixed dose of vaccinia virus. Our results demonstrate that, with the exception of a notable decline at the highest level of epitope, the magnitude of the responding CTL population generated in vivo following equivalent viral infections is essentially proportional to epitope density.

Dependence of both spontaneous and antibody-dependent, granule exocytosis-mediated NK cell cytotoxicity on extracellular signal-regulated kinases.

Extracellular signal-regulated kinases (ERK, also known as mitogen-activated protein kinases) are serine-threonine kinases transducing signals elicited upon ligand binding to several tyrosine kinase-associated receptors. We have reported that ERK2 phosphorylation and activation follows engagement of the low affinity receptor for the Fc portion of IgG (CD16) on NK cells, and is necessary for CD16-induced TNF-alpha mRNA expression. Here, we analyzed the involvement of ERK in NK cell-mediated cytotoxicity and IFN-gamma expression induced upon stimulation with targets cells, coated or not with Abs. Our data indicate that, as with immune complexes, ERK2 phosphorylation occurs in human primary NK cells upon interaction with target cells sensitive to granule exocytosis-mediated spontaneous cytotoxicity, and that this regulates both target cell- and immune complex-induced cytotoxicity and IFN-gamma mRNA expression. A specific inhibitor of mitogen-activated protein kinase kinase reduced both spontaneous and Ab-dependent cytotoxicity in a dose-dependent manner involving, at least in part, inhibition of granule exocytosis without affecting effector/target cell interaction and rearrangement of the cytoskeleton proteins actin and tubulin. Involvement of ERK in the regulation of Ca2+-dependent cell-mediated cytotoxicity was confirmed, using a genetic approach, in primary NK cells infected with a recombinant vaccinia virus encoding an ERK inactive mutant. These data indicate that the biochemical pathways elicited in NK cells upon engagement of receptors responsible for either spontaneous or Ab-dependent recognition of target cells, although distinct, utilize ERK as one of their downstream molecules to regulate effector functions.

Antigen processing of two H2-IEd-restricted epitopes is differentially influenced by the structural changes in a viral glycoprotein.

The factors that influence the intracellular location(s) of MHC class II-restricted epitope loading remain poorly understood. We present evidence that two I-Ed-restricted epitopes of the influenza hemagglutinin (HA) molecule, termed site 1 (S1; encompassing amino acid residues 107-119) and site 3 (S3; encompassing amino acid residues 302-313), are generated in distinct endocytic compartments. By means of an epitope-specific mAb, we show that S1 becomes detectable in late endocytic/lysosomal vesicles; using a mutant cell line, we also show that the presentation of S1 is dependent upon H2-DM expression. In contrast, S3; presentation is H2-DM-independent and appears in early endosomes as a result of acid-induced structural changes in HA. Presentation of both epitopes can be made H2-DM-independent by denaturing HA and made H2-DM-dependent by preventing the acid-induced conformational changes from occurring. These findings indicate that the structural context of a given epitope can determine where it is processed.

Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.

The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the Kd-restricted nonamer influenza nucleoprotein residues 147-155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O-permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.

Dinitrophenyl-modified autologous melanoma vaccine induces a T cell response to hapten-modified, melanoma peptides.

Active specific immunotherapy with dinitrophenyl (DNP)-modified autologous melanoma vaccine elicits inflammatory responses in metastatic tumor sites. Postsurgical adjuvant immunotherapy with this vaccine prolongs survival in stage III melanoma patients. We have reported that, after administration of DNP-modified melanoma vaccine, T cell responses to DNP-modified autologous tumor cells are demonstrable in vivo and in vitro. These responses are hapten specific and MHC restricted. To elucidate this phenomenon, we investigated the immune response to DNP-modified peptides eluted from autologous cells. Short peptides were extracted from DNP-modified and unmodified autologous melanoma cells by an acid elution technique and HPLC fractionation. Peptides were also extracted from DNP-modified and unmodified, EB virus-transformed, autologous B lymphoblasts. These various peptide fractions were loaded onto autologous B lymphoblasts and tested for ability to elicit a response by a DNP-specific T cell line as measured by IFN-gamma production. Unexpectedly, stimulatory activity of peptides from DNP-modified melanoma cells was confined to a single HPLC fraction. Spectrometric analysis of this fraction confirmed modification of peptides with DNP. A weaker T cell response was observed to a single HPLC fraction of DNP-modified peptides from the patient's B lymphoblasts. No T cell response was elicited by corresponding fractions of peptides eluted from unmodified melanoma cells or B lymphoblasts. These findings demonstrate the human T cell response to DNP-modified autologous melanoma cells is mediated by hapten-modified, MHC-associated peptides. Further investigation of these peptides could lead to a new strategy for peptide-based cancer immunotherapy.

Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I-restricted cryptic epitope expression.

Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.

Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein.

CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.

MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL.

MHC class I-restricted T cell responses to viral proteins focus on a limited set of peptides. To better understand this phenomenon, we examined all of the 26 nonameric peptides encoded by the influenza virus A/Puerto Rico/8/34 (PR8) conforming to the canonical Kd binding motif. Ten peptides bound strongly to Kd as assessed by a cell surface stabilization assay. Five of these 10 induced in vitro secondary CD8+ T cell responses from splenocytes derived from PR8-immunized mice. The strongest responses were induced by the two previously defined antigenic peptides, which ranked only second and fifth in relative binding affinity. To examine the limiting factors in the immunogenicity of Kd-binding peptides, we produced recombinant vaccinia viruses (rVVs) expressing cytosolic or endoplasmic reticulum (ER)-targeted peptides. rVVs expressing ER-targeted versions of the 7 peptides with the highest relative affinities for Kd rescued Kd cell surface expression in T2 cells, while those expressing the 3 lowest affinity peptides did not. The immunogenicity of several, but not all, of the highest affinity peptides was greatly enhanced when expressed as VV-encoded cytosolic or ER-targeted peptides as compared with full length proteins. We conclude that limitations in the immunogenicity of class I binding peptides reflects, in order of decreasing importance, peptide liberation by cellular proteases, T cell repertoire, and TAP-mediated peptide transport. We also observed an additional important contributing factor: suppression of T cell responses to nondominant peptides by an immunodominant peptide located in the same protein.

Regulation of class I-restricted epitope processing by local or distal flanking sequence.

Influenza A/Puerto Rico/8/34 nucleoprotein (NP) contains an H-2Kd-restricted CD8+ T cell (T CD8+) epitope spanning amino acid residues 147-155. It was previously demonstrated that expression of NP147-155 and NP147-158 in isolation via "minigene"/recombinant vaccinia virus (vac) technology leads to sensitization of target cells for NP-specific killing while expression of 147-158 lacking the arginine at position 156 (termed here as 147-155TG) does not. The presentation block was overcome by placing this fragment into the context of full length NP. We show that addition of a single amino acid, Met159, to the C terminus of the blocked peptide (creating 147-155TGM) restores presentation. Presentation of 147-155TGM was not due to trimming in the exocytic compartment, consistent with severe limitations on C-terminal trimming activity in this location. Rescued presentation was also achieved when the blocked construct was extended in the N-terminal direction only, but in this case more than 55 amino acids of flanking sequence were required. The transition to presentation was abrupt, with 91-155TG and shorter constructs showing little or no detectable presentation and 90-155TG showing full level presentation. Presentation could not be attributed to acquisition of conventional targets for ubiquitination since mutation of all Lys residues, to which the ubiquitin moiety is conjugated, does not abrogate presentation. Rescued presentation was not inhibited by the peptide aldehyde N-acetyl-L-leucinyl-L-leucinal-L-norleucinal, suggesting that the added elements may be recruiting nonproteasomal activity. We have therefore identified and begun to characterize protease targeting of regulatory elements, both local and distal to an epitope, which strongly influence the ability of the epitope to be excised.

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames.

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules.