Distinct modulation of allergic T cell responses by subcutaneous vs. sublingual allergen-specific immunotherapy.

BACKGROUND: Allergen-specific immunotherapy is the only curative treatment for type I allergy. It can be administered subcutaneously (SCIT) or sublingually (SLIT). The clinical efficacy of these two treatment modalities appears to be similar, but potential differences in the immunological mechanisms involved have not been fully explored. OBJECTIVE: To compare changes in the allergen-specific T cell response induced by subcutaneous vs. sublingual administration of allergen-specific immunotherapy (AIT). METHODS: Grass pollen-allergic patients were randomized into groups receiving either SCIT injections or SLIT tablets or neither. PBMCs were tested for Timothy grass (TG)-specific cytokine production by ELISPOT after in vitro expansion with TG-peptide pools. Phenotypic characterization of cytokine-producing cells was performed by FACS. RESULTS: In the SCIT group, decreased IL-5 production was observed starting 10 months after treatment commenced. At 24 months, T cell responses showed IL-5 levels significantly below the before-treatment baseline. No significant reduction of IL-5 was observed in the SLIT or untreated group. However, a significant transient increase in IL-10 production after 10 months of treatment compared to baseline was detected in both treatment groups. FACS analysis revealed that IL-10 production was associated with CD4(+) T cells that also produced IFNγ and therefore may be associated with an IL-10-secreting type 1 cell phenotype. CONCLUSION AND CLINICAL RELEVANCE: The most dominant immunological changes on a cellular level were a decrease in IL-5 in the SCIT group and a significant, transient increase of IL-10 observed after 10 months of treatment in both treated groups. The distinct routes of AIT administration may induce different immunomodulatory mechanisms at the cellular level.

Sequence conservation predicts T cell reactivity against ragweed allergens.

BACKGROUND: Ragweed is a major cause of seasonal allergy, affecting millions of people worldwide. Several allergens have been defined based on IgE reactivity, but their relative immunogenicity in terms of T cell responses has not been studied. OBJECTIVE: We comprehensively characterized T cell responses from atopic, ragweed-allergic subjects to Amb a 1, Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 8, Amb a 9, Amb a 10, Amb a 11, and Amb p 5 and examined their correlation with serological reactivity and sequence conservation in other allergens. METHODS: Peripheral blood mononuclear cells (PBMCs) from donors positive for IgE towards ragweed extracts after in vitro expansion for secretion of IL-5 (a representative Th2 cytokine) and IFN-γ (Th1) in response to a panel of overlapping peptides spanning the above-listed allergens were assessed. RESULTS: Three previously identified dominant T cell epitopes (Amb a 1 176-191, 200-215, and 344-359) were confirmed, and three novel dominant epitopes (Amb a 1 280-295, 304-319, and 320-335) were identified. Amb a 1, the dominant IgE allergen, was also the dominant T cell allergen, but dominance patterns for T cell and IgE responses for the other ragweed allergens did not correlate. Dominance for T cell responses correlated with conservation of ragweed epitopes with sequences of other well-known allergens. CONCLUSIONS AND CLINICAL RELEVANCE: These results provide the first assessment of the hierarchy of T cell reactivity in ragweed allergens, which is distinct from that observed for IgE reactivity and influenced by T cell epitope sequence conservation. The results suggest that ragweed allergens associated with lesser IgE reactivity and significant T cell reactivity may be targeted for T cell immunotherapy, and further support the development of immunotherapies against epitopes conserved across species to generate broad reactivity against many common allergens.

Lack of allergy to timothy grass pollen is not a passive phenomenon but associated with the allergen-specific modulation of immune reactivity.

BACKGROUND: Timothy grass (TG) pollen is a common seasonal airborne allergen associated with symptoms ranging from mild rhinitis to severe asthma. OBJECTIVE: The aim of this study was to characterize changes in TG-specific T cell responses as a function of seasonality. METHODS: Peripheral blood mononuclear cells (PBMCs) obtained from allergic individuals and non-allergic controls, either during the pollen season or out of season, were stimulated with either TG extract or a pool of previously identified immunodominant antigenic regions. RESULTS: PBMCs from allergic subjects exhibit higher IL-5 and IL-10 responses in season than when collected out of season. In the case of non-allergic subjects, as expected we observed lower IL-5 responses and robust production of IFN-γ compared to allergic individuals. Strikingly, non-allergic donors exhibited an opposing pattern, with decreased immune reactivity in season. The broad down-regulation in non-allergic donors indicates that healthy individuals are not oblivious to allergen exposure, but rather react with an active modulation of responses following the antigenic stimulus provided during the pollen season. Transcriptomic analysis of allergen-specific T cells defined genes modulated in concomitance with the allergen exposure and inhibition of responses in non-allergic donors. CONCLUSION AND CLINICAL RELEVANCE: Magnitude and functionality of T helper cell responses differ substantially in season vs. out of season in allergic and non-allergic subjects. The results indicate the specific and opposing modulation of immune responses following the antigenic stimulation during the pollen season. This seasonal modulation reflects the enactment of specific molecular programmes associated with health and allergic disease.

hERG1 channels drive tumour malignancy and may serve as prognostic factor in pancreatic ductal adenocarcinoma.

BACKGROUND: hERG1 channels are aberrantly expressed in human cancers. The expression, functional role and clinical significance of hERG1 channels in pancreatic ductal adenocarcinoma (PDAC) is lacking. METHODS: hERG1 expression was tested in PDAC primary samples assembled as tissue microarray by immunohistochemistry using an anti-hERG1 monoclonal antibody (α-hERG1-MoAb). The functional role of hERG1 was studied in PDAC cell lines and primary cultures. ERG1 expression during PDAC progression was studied in Pdx-1-Cre,LSL-Kras(G12D/+),LSL-Trp53(R175H/+) transgenic (KPC) mice. ERG1 expression in vivo was determined by optical imaging using Alexa-680-labelled α-hERG1-MoAb. RESULTS: (i) hERG1 was expressed at high levels in 59% of primary PDAC; (ii) hERG1 blockade decreased PDAC cell growth and migration; (iii) hERG1 was physically and functionally linked to the Epidermal Growth Factor-Receptor pathway; (iv) in transgenic mice, ERG1 was expressed in PanIN lesions, reaching high expression levels in PDAC; (v) PDAC patients whose primary tumour showed high hERG1 expression had a worse prognosis; (vi) the α-hERG1-MoAb could detect PDAC in vivo. CONCLUSIONS: hERG1 regulates PDAC malignancy and its expression, once validated in a larger cohort also comprising of late-stage, non-surgically resected cases, may be exploited for diagnostic and prognostic purposes in PDAC either ex vivo or in vivo.

Definition of a pool of epitopes that recapitulates the T cell reactivity against major house dust mite allergens.

BACKGROUND: Allergens from house dust mites (HDM) are a common cause of asthma. Der p and Der f from Dermatophagoides sp. are strong immunogens in humans. Allergen extracts are used to study T helper (Th2) cell responses to HDM, which are implicated in the development and regulation of allergic disease. OBJECTIVE: To define an epitope mixture that recapitulates, and might substitute for, HDM extract in terms of detecting and characterizing Th2 cell responses. METHODS: Peripheral blood mononuclear cells (PBMC) from 52 HDM allergic and 10 non-allergic individuals were stimulated with HDM extracts and assayed with a set of 178 peptides spanning mite allergens group Der p 1, 2, 23 and Der f group 1 and 2 allergens. A pool of the most dominant T cell epitopes identified in the present study and from published literature was assembled and tested for ex vivo T cell responses. Correlation with HDM-specific IgE titres was examined. RESULTS: Patterns of T cell reactivity to Der p and Der f - derived peptides revealed a large number of epitopes. Clear patterns of immunodominance were apparent, with HDM allergen group 1 and 2 dominant over group 23. Furthermore, within a given antigen, 6-11 epitopes accounted for the vast majority of responses. Based on these results and published data, a comprehensive dust mite pool (DMP) of epitopes was designed and found to allow detection of ex vivo T cell responses. DMP ex vivo reactivity correlated with HDM-specific IgE titres and was similar to that detected with commonly used HDM extracts. Ex vivo DMP stimulation was associated with a predominant Th2 response in allergic donors, and minor reactivity of T cells producing IFNγ, IL17 and IL10. CONCLUSIONS & CLINICAL RELEVANCE: A detailed map of Der p and Der f antigens defined a pool of epitopes that can be used to detect ex vivo HDM responses.

HLA-DQ allele-restricted activation of nitroso sulfamethoxazole-specific CD4-positive T lymphocytes from patients with cystic fibrosis.

BACKGROUND: For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied. OBJECTIVE: To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens. METHODS: T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells. RESULTS: A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. CONCLUSION AND CLINICAL RELEVANCE: HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides.

Different Bla-g T cell antigens dominate responses in asthma versus rhinitis subjects.

BACKGROUND AND OBJECTIVE: The allergenicity of several German cockroach (Bla-g) antigens at the level of IgE responses is well established. However, less is known about the specificity of CD4+ TH responses, and whether differences exist in associated magnitude or cytokine profiles as a function of disease severity. METHODS: Proteomic and transcriptomic techniques were used to identify novel antigens recognized by allergen-specific T cells. To characterize different TH functionalities of allergen-specific T cells, ELISPOT assays with sets of overlapping peptides covering the sequences of known allergens and novel antigens were employed to measure release of IL-5, IFNγ, IL-10, IL-17 and IL-21. RESULTS: Using these techniques, we characterized TH responses in a cohort of adult Bla-g-sensitized subjects, either with (n = 55) or without (n = 17) asthma, and nonsensitized controls (n = 20). T cell responses were detected for ten known Bla-g allergens and an additional ten novel Bla-g antigens, representing in total a 5-fold increase in the number of antigens demonstrated to be targeted by allergen-specific T cells. Responses of sensitized individuals regardless of asthma status were predominantly TH 2, but higher in patients with diagnosed asthma. In asthmatic subjects, Bla-g 5, 9 and 11 were immunodominant, while, in contrast, nonasthmatic-sensitized subjects responded mostly to Bla-g 5 and 4 and the novel antigen NBGA5. CONCLUSIONS: Asthmatic and nonasthmatic cockroach-sensitized individuals exhibit similar TH 2-polarized responses. Compared with nonasthmatics, however, asthmatic individuals have responses of higher magnitude and different allergen specificity.

Relation of HLA class I and II supertypes with spontaneous clearance of hepatitis C virus.

Human leukocyte antigen (HLA) genotype has been associated with the probability of spontaneous clearance of hepatitis C virus (HCV). However, no prior studies have examined whether this relationship may be further characterized by grouping HLA alleles according to their supertypes, defined by their binding capacities. There is debate regarding the most appropriate method to define supertypes. Therefore, previously reported HLA supertypes (46 class I and 25 class II) were assessed for their relation with HCV clearance in a population of 758 HCV-seropositive women. Two HLA class II supertypes were significant in multivariable models that included: (i) supertypes with significant or borderline associations with HCV clearance after adjustment for multiple tests, and (ii) individual HLA alleles not part of these supertypes, but associated with HCV clearance in our prior study in this population. Specifically, supertype DRB3 (prevalence ratio (PR)=0.4; P=0.004) was associated with HCV persistence, whereas DR8 (PR=1.8; P=0.01) was associated with HCV clearance. Two individual alleles (B*57:01 and C*01:02) associated with HCV clearance in our prior study became nonsignificant in analysis that included supertypes, whereas B*57:03 (PR=1.9; P=0.008) and DRB1*07:01 (PR=1.7; P=0.005) retained their significance. These data provide epidemiologic support for the significance of HLA supertypes in relation to HCV clearance.

Survey on infectious disease telephone hotlines in primary care: General practitioners' satisfaction and compliance with advice.

OBJECTIVES: Infectious disease (ID) advice is a major part of antimicrobial stewardship programs. The objective of this study was to assess general practitioners' (GPs)' opinions and compliance with advice given by ID hotlines. PATIENTS AND METHODS: This multicenter survey was based on the 7-day assessment of initial advice requested by GPs to a hotline set up by volunteer hospital ID teams to record advice for 3 years. The primary endpoint was the GPs' satisfaction with the advice given by ID specialists. RESULTS: Ten ID teams participated in the study and recorded 4138 requests for advice, of which 1325 requests included a proposal for antibiotic therapy and justified a follow-up call at seven days. Only 398 follow-up calls (30%) were carried out because many GPs were not reachable. GPs were very satisfied with ID hotlines: 58% considered them indispensable and 38% very useful. The recommendations provided by ID specialists were followed by GPs in more than 80% of cases. The two main motivations for GPs to call the hotline were to get quick advice (86%) and to receive help in managing a patient (76%). CONCLUSIONS: The ID telephone consultations and advice systems for GPs are highly appreciated and are effective in terms of following the recommendations.

Immunology in the Clinic Review Series; focus on host responses: T cell responses to herpes simplex viruses.

Herpes virus infections are chronic and co-exist with acquired immune responses that generally prevent severe damage to the host, while allowing periodic shedding of virus and maintenance of its transmission in the community. Herpes simplex viruses type 1 and 2 (HSV-1, HSV-2) are typical in this regard and are representative of the viral subfamily Alphaherpesvirinae, which has a tropism for neuronal and epithelial cells. This review will emphasize recent progress in decoding the physiologically important CD8(+) and CD4(+) T cell responses to HSV in humans. The expanding data set is discussed in the context of the search for an effective HSV vaccine as therapy for existing infections and to prevent new infections.

CD4+ T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes.

Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4(+) and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef.

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.

Pathways to post-traumatic growth in cancer patients: moderated mediation and single mediation analyses with resilience, personality, and coping strategies.

BACKGROUND: Cancer diagnosis is a potentially traumatic experience, which could generate significant long-lasting emotional distress, but also positive changes linked to post-traumatic growth (PTG). This study aimed to analyze the role of resilience, coping, and personality in determining PTG or post-traumatic symptoms, and to test a moderated mediation model and a single mediation model in a sample of individuals diagnosed with cancer. METHODS: A sample of 154 individuals diagnosed with cancer (Mage = 51.4, SD = 11.25) completed the Post-Traumatic Growth Inventory, Impact of Event Scale, Connor-Davidson Resilience Scale, Ten Item Personality Inventory, and Coping Orientation to Problems Experienced after providing written informed consent. RESULTS: Results showed that the impact of resilience in PTG is partially mediated by positive attitude, with a significant and negative moderating effect of openness on the relationship between resilience and positive attitude. Furthermore, resilience negatively predicted the impact of trauma, with a partial mediation of avoidance strategies. LIMITATIONS: The cross-sectional nature of the study, the use of only self-report measures, heterogeneity of the sample, and the risk of influence of unobserved prognostic variables should be kept in mind while interpreting the results. CONCLUSIONS: The findings showed that the level of resilience predicted PTG or post-traumatic symptoms, both directly and indirectly, with different coping strategies as mediators. Furthermore, the lower the level of openness reported by participants, the higher the resilience induced by positive attitude. These findings may significantly contribute toward tailoring interventions for improving the mental health of cancer patients.

Immune signatures underlying post-acute COVID-19 lung sequelae.

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)­specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

Binding of major histocompatibility complex class II to the invariant chain-derived peptide, CLIP, is regulated by allelic polymorphism in class II.

Major histocompatibility complex class II-associated invariant chain (Ii) provides several important functions that regulate class II expression and function. One of these is the ability to inhibit class II peptide loading early in biosynthesis. This allows for efficient class II folding and egress from the endoplasmic reticulum, and protects the class II peptide binding site from loading with peptides before entry into endosomal compartments. The ability of Ii to interact with class II and interfere with peptide loading has been mapped to Ii exon 3, which encodes amino acids 82-107. This same region of Ii has been described as a nested set of class II-associated Ii peptides (CLIPs) that are transiently associated with class II in normal cells and accumulate in human histocompatibility leukocyte antigen-DM-negative cell lines. Currently it is not clear how CLIP and the CLIP region of Ii blocks peptide binding. CLIP may bind directly to the class II peptide binding site, or may bind elsewhere on class II and modulate class II peptide binding allosterically. In this report, we show that CLIP can interact with many different murine and human class II molecules, but that the affinity of this interaction is controlled by polymorphic residues in the class II chains. Likewise, structural changes in CLIP also modulate class II binding in an allele-dependent manner. Finally, the specificity and kinetics of CLIP binding to class II molecule is similar to antigenic peptide binding to class II. These data indicate that CLIP binds to class II in an analogous fashion as conventional antigenic peptides, suggesting that the CLIP segment of Ii may actually occupy the class II peptide binding site.

Selective immunosuppression by administration of major histocompatibility complex (MHC) class II-binding peptides. I. Evidence for in vivo MHC blockade preventing T cell activation.

Draining lymph node cells (LNC) from mice immunized with hen egg white lysozyme (HEL) display at their surface antigen-MHC complexes able to stimulate, in the absence of any further antigen addition, HEL peptide-specific, class II-restricted T cell hybridomas. Chloroquine addition to these LNC cultures fails to inhibit antigen presentation, indicating that antigenic complexes of class II molecules and HEL peptides are formed in vivo. MHC class II restriction of antigen presentation by LNC from HEL-primed mice was verified by the use of anti-class II monoclonal antibodies. Coinjection of HEL and the I-Ak-binding peptide HEL 112-129 in mice of H-2k haplotype inhibits the ability of LNC to stimulate I-Ak-restricted, HEL 46-61-specific T cell hybridomas. Similar results are obtained in mice coinjected with the HEL peptides 46-61 and 112-129. Inhibition of T hybridoma activation can also be observed using as antigen-presenting cells irradiated, T cell-depleted LNC from mice coinjected with HEL 46-61 and HEL 112-129, ruling out the possible role of either specific or nonspecific suppressor T cells. Inhibition of T cell proliferation is associated with MHC-specific inhibition of antigen presentation and with occupancy by the competitor of class II binding sites, as measured by activation of peptide-specific T cell hybridomas. These results demonstrate that administration of MHC class II binding peptide competitors selectively inhibits antigen presentation to class II-restricted T cells, indicating competitive blockade of class II molecules in vivo.

Altered peptide ligands can control CD4 T lymphocyte differentiation in vivo.

Antigen priming of naive CD4 T cells can generate effector CD4 T cells that produce interleukin 4 (T helper [Th]2-like) or interferon-gamma (Th1-like). Using a system in which priming leads to responses dominated by one or the other of these cell types, we show that varying either the antigenic peptide or the major histocompatibility complex class II molecule can determine whether Th1-like or Th2-like responses are obtained. Our results show that peptide/major histocompatibility complex class II complexes that interact strongly with the T cell receptor favor generation of Th1-like cells, while those that bind weakly favor priming of Th2-like T cells. Thus, signals from the T cell receptor can influence the differentiation of CD4 T cells into specific types of effector cells.

Recognition of multiple peptide cores by a single T cell receptor.

We present evidence that a single T cell clone can recognize at least five different overlapping peptides, each with its distinct core structure, in the context of the same major histocompatibility complex (MHC) molecule. Distinct core residues are crucial for triggering the T cell receptor (TCR) in each case. These results suggest that the TCR (a) has multiple sets of contact residues for alternative peptide-MHC ligands, the binding to any one of which can trigger the cell; and/or (b) is able to attach to the peptide-MHC complex in more than one orientation. In this sense, the TCR is a multisubsite structure capable of being stimulated by a variety of peptide ligands associated with the same MHC molecules.

Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide.

Certain peptide analogs that carry substitutions at residues other than the main major histocompatibility complex anchors and are surprisingly much more antigenic than wild-type peptide (heteroclitic analogs). To date, it was unknown how frequently wild-type epitopes could be modified to obtain heteroclitic activity. In this study, we analyzed a large panel of analogs of two different human histocompatibility leukocyte antigen (HLA)-A2.1-restricted epitopes and found that heteroclitic analogs were associated with higher magnitude responses and increased (up to 10(7)-fold) sensitivity to antigen, and corresponded to conservative or semiconservative substitutions at odd-numbered positions in the middle of the peptide (positions 3, 5, or 7). These findings were validated by performing additional immunogenicity studies in murine and human systems with four additional epitopes. The biological relevance of heteroclitic analogs was underlined when predicted analogs of the p53.261 epitope was shown to induce cytotoxic T lymphocytes (CTLs) that recognize low concentrations of peptide (high avidity) in vivo and demonstrate in vitro antitumor recognition. Finally, in vitro immunization of human peripheral blood mononuclear cells with two heteroclitic analogs resulted in recruitment of more numerous CTLs which were associated with increased antigen sensitivity. In conclusion, heteroclitic analogs were identified in each of the six cases studied and structural features were defined which allow identification of such analogs. The strong CTL immunity elicited by heteroclitic epitopes suggest that they could be of significant value in vaccination against tolerant or weakly immunogenic tumor-associated and viral antigens.