Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL.

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.

HLA anchor optimization of the melan-A-HLA-A2 epitope within a long peptide is required for efficient cross-priming of human tumor-reactive T cells.

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16-40 LP bearing the HLA-A2-restricted epitope 26-35 or its analog 26-35(A27L) and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26-35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16-40(A27L) LP, for the treatment of HLA-A2(+) melanoma patients.

Immunodominant fragments of myelin basic protein initiate T cell-dependent pain.

BACKGROUND: The myelin sheath provides electrical insulation of mechanosensory Aß-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of Aß-fibers is believed to activate the nociceptive circuitry in Aß-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia). The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI), are not well understood. METHODS AND RESULTS: Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP) generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats. CONCLUSIONS: These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1 day post-injury is critical to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state.

Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses.

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide-major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8(+) T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8(+) T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI-CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function.

A comparative study of HLA binding affinity and ligand diversity: implications for generating immunodominant CD8+ T cell responses.

Conventional CD8(+) T cell responses against intracellular infectious agents are initiated upon recognition of pathogen-derived peptides presented at the cell surface of infected cells in the context of MHC class I molecules. Among the major MHC class I loci, HLA-B is the swiftest evolving and the most polymorphic locus. Additionally, responses restricted by HLA-B molecules tend to be dominant, and most associations with susceptibility or protection against infectious diseases have been assigned to HLA-B alleles. To assess whether the differences in responses mediated via two major HLA class I loci, HLA-B and HLA-A, may already begin at the Ag presentation level, we have analyzed the diversity and binding affinity of their peptide repertoire by making use of curated pathogen-derived epitope data retrieved from the Immune Epitope Database and Analysis Resource, as well as in silico predicted epitopes. In contrast to our expectations, HLA-B alleles were found to have a less diverse peptide repertoire, which points toward a more restricted binding motif, and the respective average peptide binding affinity was shown to be lower than that of HLA-A-restricted epitopes. This unexpected observation gives rise to new hypotheses concerning the mechanisms underlying immunodominance of CD8(+) T cell responses.

Impact of intrinsic cooperative thermodynamics of peptide-MHC complexes on antiviral activity of HIV-specific CTL.

The antiviral activity of HIV-specific CTL is not equally potent but rather is dependent on their specificity. But what characteristic of targeted peptides influences CTL antiviral activity remains elusive. We addressed this issue based on HLA-B35-restricted CTLs specific for two overlapping immunodominant Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). VY8-specific CTLs were more potently cytotoxic toward HIV-infected primary CD4(+) cells than RY11-specific CTLs. Reconstruction of their TCR revealed no substantial difference in their functional avidity toward cognate Ags. Instead, the decay analysis of the peptide-MHC complex (pMHC) revealed that the VY8/HLA-B35 complex could maintain its capacity to sensitize T cells much longer than its RY11 counterpart. Corroboratively, the introduction of a mutation in the epitopes that substantially delayed pMHC decay rendered Nef-expressing target cells more susceptible to CTL killing. Moreover, by using differential scanning calorimetry and circular dichroism analyses, we found that the susceptible pMHC ligands for CTL killing showed interdependent and cooperative, rather than separate or sequential, transitions within their heterotrimer components under the thermally induced unfolding process. Collectively, our results highlight the significant effects of intrinsic peptide factors that support cooperative thermodynamics within pMHC on the efficient CTL killing of HIV-infected cells, thus providing us better insight into vaccine design.

Interferon gamma (IFN-gamma) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice.

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-gamma, plays a role in the development of EAMG, we immunized IFN-gamma knockout (IFN-gko) (-/-) mice and wild-type (WT) (+/+) mice of H-2(b) haplotype with AChR in CFA. We observed that AChR-primed lymph node cells from IFN-gko mice proliferated normally to AChR and to its dominant pathogenic alpha146-162 sequence when compared with these cells from the WT mice. However, the IFN-gko mice had no signs of muscle weakness and remained resistant to clinical EAMG at a time when the WT mice exhibited severe muscle weakness and some died. The resistance of IFN-gko mice was associated with greatly reduced levels of circulating anti-AChR antibody levels compared with those in the WT mice. Comparatively, immune sera from IFN-gko mice showed a dramatic reduction in mouse AChR-specific IgG1 and IgG2a antibodies. However, keyhole limpet hemocyanin (KLH)-priming of IFN-gko mice readily elicited both T cell and antibody responses, suggesting that IFN-gamma regulates the humoral immune response distinctly to self (AChR) versus foreign (KLH) antigens. We conclude that IFN-gamma is required for the generation of a pathogenic anti-AChR humoral immune response and for conferring susceptibility of mice to clinical EAMG.

Heterospecific CD4 help to rescue CD8 T cell killers.

Help from CD4 T cells may be required for optimal generation and maintenance of memory CD8 T cells and also for optimal Ag reactivation. We examined whether the helper cell and the CD8 killer cell need to have the same Ag specificity for help to be effective during interactions of memory T cells with mature APC. This is important because virus and tumor Ag-specific CD4 T cell responses are selectively impaired in several chronic viral infections and malignancies. We performed studies in vitro and in vivo and found that functional memory CD4 T cells generated from a distinct antigenic source (heterospecific helpers) could provide direct and effective help to memory CD8 T cells. Functional heterospecific memory CD4 T cells could also rescue secondary CD8 T cell responses in an experimental tumor model in which homospecific CD4 help was impaired. This could provide a rationale for immunotherapy strategies designed to bypass impaired homospecific help.

Dominance of E. coli phagocytosis over LPS in the inflammatory response of microglia.

CNS bacterial infections are prevalent in neonates, the immune-compromised and elderly. During peripheral infections, macrophages employ multiple pattern recognition receptors (PRRs) to respond to pathogens, but less is known about brain microglia. We assessed microglial expression of PRRs, compared responses to whole E. coli and LPS, and tested the hypothesis that bacteria modulate the response to LPS. LPS increased the microglial phagocytic capacity, and changed expression of CD14, CR3, Fcgr1, Fcgr3a, TLR4, MARCO, MHCII, NOD2, TLR9 and SR-A, differently from stimulation with whole E. coli. Importantly, when added with LPS, E. coli dominated the microglial responses for 11/13 genes examined.

Viral fitness implications of variation within an immunodominant CD8+ T-cell epitope of HIV-1.

Cytotoxic T-lymphocyte (CTL) epitopes within the HIV genome are subject to negative and positive selective pressures, the balance of which influences CTL escape at a given epitope. We investigated whether viral fitness requirements dictate conservation of the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Viral clones incorporating changes throughout the SL9 epitope region were compared to consensus SL9 virus in terms of replication kinetics and relative viral fitness. Constructs recapitulating in vivo SL9-CTL escape variants showed markedly little effect on replication and fitness, as did non-natural conservative mutations targeting immunologically relevant positions of the epitope. Although certain residues of the epitope were constrained by viral requirements, our research reveals that there are multiple SL9 variants that are well tolerated virologically but fail to arise in vivo. In light of this data, assumptions regarding the balance of immune and viral selective pressures on this immunodominant epitope sequence need to be reassessed.

The T cell activation molecule H4 and the CD28-like molecule ICOS are identical.

The recently cloned CD28-like molecule ICOS displays striking similarities with H4, characterized some years ago in the mouse and recently in humans. Both molecules are selectively expressed by activated and germinal center T cells, display similar structure, and display co-stimulatory activities. H4 displays lateral association with the CD3/TCR and is expressed by mature thymocytes. In the mouse, H4 is also expressed at high levels by thymic NKT cells that are resistant to negative selection. The aim of this work was to evaluate whether H4 and ICOS are the same molecule using the C398.4A (binding human and mouse H4) and F44 (binding human ICOS) monoclonal antibody (mAb) in parallel experiments on human T cells. ICOS and H4 displayed the same expression pattern in a panel of T cell lines and the same expression kinetics in phytohemagglutinin-activated T cells. C398.4A completely blocked cell staining by F44, whereas F44 partially blocked C398.4A. H4 and ICOS immunoprecipitates displayed identical SDS-PAGE patterns and H4 immunoprecipitation completely removed ICOS from cell lysates. Finally, the C398.4A mAb specifically stained cells transfected with the human or mouse ICOS. These data prove that H4 and ICOS are the same molecule and that F44 and C398.4A bind partially different epitopes.

The role of tumor rejection antigens in host antitumor defense mechanisms.

BACKGROUND: Normal cells undergo contact inhibition of growth when their surface molecules interact. Tumor cells, however, have undergone a mutation that prevents this arrest of growth upon contact inhibition and allows constant growth. Thus, growth inhibition fails to occur despite the interaction of surface molecules. In recent years a subgroup of these surface molecules has been of interest to cancer investigators. This subgroup has been termed the tumor rejection antigens (TRAs). As the name implies, these are specific to the tumor of origin and may direct the immune system of the host to target the tumor cells and kill them. METHODS: A literature search was carried out on TRAs to ascertain the current thinking on the subject. RESULTS: Initial studies of TRAs have revealed that some of them may be heat shock proteins (HSPs). In particular, grp96, a number of the HSP90 family, has been implicated. More recent studies, however, have shown that HSPs alone may not be immunogenic but may act as carrier proteins for tumor specific peptides. CONCLUSION: Such findings have led to speculation that HSPs or their associated peptides may have a role in the diagnosis and/or treatment of specific cancers. Immunotherapy and bispecific antibodies in particular are areas in which HSPs may prove to be useful.

The B cell immune response to an idiotype-inducing peptide epitope can be inhibited by immunodominance of a neighboring epitope.

Three synthetic peptides, EEYEYE (peptide 1), EEYEYEEEYEYE (peptide 2), and YEEEEY (peptide 3), were tested for their ability to induce common Id that had been previously characterized for murine antibodies specific to random synthetic polymers of glutamic acid, alanine, and tyrosine ((Glu,Ala,Tyr)n) (cGAT Id). Protein conjugates of either peptide 2 or peptide 3, but not peptide 1, induced cGAT Id. This unique approach directly identified two peptides capable of inducing cGAT Id antibodies. Previous immunization with peptide 1-protein conjugate inhibited the cGAT Id response to peptide 2 conjugated to a different protein carrier. Thus, a neighboring or overlapping epitope can be used to inhibit a cGAT Id-inducing epitope without the participation of carrier-specific Ts and without using anti-Id antibodies. In contrast, previous immunization with peptide 1-protein conjugate did not inhibit cGAT Id induction by peptide 3-protein conjugate or by (Glu,Ala,Tyr)n. This ruled out the participation of Id-specific Ts cells. The effectiveness of inhibition coincided with the avid binding of anti-peptide 1 antibodies to peptide 2, which was > 10 and 100 times stronger than the binding to peptide 3 and (Glu,Ala,Tyr)n, respectively. We hypothesize that the primed peptide 1-specific B cells capture and process peptide 2-protein efficiently and act as APC to Th cells specific to the protein of the challenging Ag, resulting in the selective and dominant activation of peptide 1-specific B cells. Thus, our data suggest that epitope priming can inhibit an Id response to a neighboring epitope by a mechanism of clonal dominance.

Interleukin 10-mediated immunosuppression by a variant CD4 T cell epitope of Plasmodium falciparum.

The immunodominant CD4 T cell epitope region, Th2R, of the circumsporozoite protein of Plasmodium falciparum is highly polymorphic. Such variation might be utilized by the parasite to escape from or interfere with CD4 T cell effector functions. Here, we show that costimulation with naturally occurring altered peptide ligands (APL) can induce a rapid change from IFNgamma production to the immunosuppressive mediator interleukin 10 (IL-10). This mechanism may contribute to the low levels of T cell responses observed to this pathogen in malaria-endemic areas.

The repetitive domain of Trypanosoma cruzi trans-sialidase enhances the immune response against the catalytic domain.

Trypanosoma cruzi trans-sialidase consists of a C-terminal domain composed essentially of immunodominant amino acid repeat units (SAPA-repeats) and an amino region responsible for the enzymatic activity (catalytic domain). To investigate the possible function(s) of SAPA-repeats, recombinant trans-sialidases either containing or lacking the C-terminal region were tested in mice. The presence of SAPA-repeats in the intravenously injected protein has two consequences. First, they enhance the persistence of the trans-sialidase activity in blood. Second, SAPA-repeats promoted the production of antibodies directed to the catalytic domain that inhibit trans-sialidase activity. These results suggest that SAPA-repeats modulate the trans-sialidase activity in blood.

Different functional capacities of latent and lytic antigen-specific CD8 T cells in murine gammaherpesvirus infection.

Gammaherpesviruses can persist in the host in the face of an aggressive immune response. T cells recognize Ags expressed in both the productive and latent phases of the virus life cycle, however little is known about their relative roles in the long-term control of the infection. In this study we used the murine gammaherpesvirus 68 model system to investigate the relative properties of CD8 T cells recognizing lytic and latent viral Ags. We report that the CD8 T cell response to lytic phase epitopes is maximal in the lungs of infected mice at approximately 10 days postinfection, and is of progressively lesser magnitude in the mediastinal lymph nodes and spleen. In contrast, the CD8 T cell response to the latent M2 protein is maximal at approximately 19 days postinfection and is most prominent in the spleen, then progressively less in the mediastinal lymph node and the lung. Latent and lytic Ag-specific CD8 T cells had markedly different cell surface phenotypes during chronic infection, with latent Ag-specific cells being predominantly CD62L(high) or CD43 (1B11)(high). Lytic Ag-specific T cells had significantly lower expression of these markers. Importantly, latent but not lytic Ag-specific T cells could kill target cells rapidly in vivo during the chronic infection. These two different sets of CD8 T cells also responded differentially to IL-7, a cytokine involved in T cell homeostasis and the maintenance of T cell memory. These data have important implications for our understanding of immunological control during chronic gammaherpesvirus infections.

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.

Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells.

The gp100 melanoma-associated tumor Ag was selected as a model system to study the diversity of human antitumor cytotoxic T cell responses. First, peptides corresponding to dominant gp100 HLA-A2.1-restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol that uses peptide-pulsed dendritic cells as APCs and IL-7 and IL-10 as immune-enhancing cytokines. High CTL activity toward both peptide-pulsed target cells and gp100+ melanoma cells was obtained with four out of five peptides tested. Second, HLA-A2.1-binding peptides from gp100 that do not appear to represent CTL epitopes in melanoma patients were also tested for their capacity to induce CTL using the in vitro priming protocol. Three of six peptides tested induced CTL in lymphocytes from normal volunteers. One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission. Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T cell response, they may be considered as typical "subdominant" epitopes. The results are discussed in the context of the usefulness of this approach to detail the immunologic potential of a given tumor-associated Ag and its relevance for the design of effective immune-based therapies.

Sequences that flank subdominant and cryptic epitopes influence the proteolytic generation of MHC class I-presented peptides.

The proteasome has been shown to make the proper C-terminal cleavage for the generation of several immunodominant class I-presented peptides whereas aminopeptidases generate their proper N termini. In this study, we show that these two distinct proteolytic processes are also involved in generating a subdominant OVA peptide KVVRFDKL (K-L). Moreover, proteasome inhibitors did not enhance the presentation of any K-L construct, suggesting that destruction of this peptide by proteasomes, if any, does not limit its presentation. We have further examined in intact cells the influence of residues flanking this epitope on these proteolytic processes. When the N-terminal flanking residues of K-L are fused to an immunodominant OVA peptide SIINFEKL (S-L), the presentation of S-L is reduced as compared with a construct with its natural flanking sequence and was not inhibited (or enhanced) by proteasome inhibitors. Similarly, a reduction in presentation was observed when the C-terminal flanking residues of the subdominant epitope were attached to S-L. A detailed analysis revealed that the Pro at the P1' position of K-L was responsible for this reduction, and presentation of these C-terminally extended constructs was sensitive to proteasome inhibitor. The study suggests that both the N- and C-terminal flanks of the subdominant peptide are suboptimal for Ag presentation. Moreover, three of four C-terminal residues that flank other subdominant or cryptic epitopes in OVA reduced the presentation of S-L. Therefore, the residues that flank the C termini of several subdominant and cryptic epitopes are often suboptimal for cleavage and may contribute to the phenomenon of immunodominance.