From animal studies into clinical trials: the relevance of animal models to develop vaccines and therapies to reduce disease severity and prevent hRSV infection.

INTRODUCTION: Human respiratory syncytial virus (hRSV) is an important cause of lower respiratory tract infections in the pediatric and the geriatric population worldwide. There is a substantial economic burden resulting from hRSV disease during winter. Although no vaccines have been approved for human use, prophylactic therapies are available for high-risk populations. Choosing the proper animal models to evaluate different vaccine prototypes or pharmacological treatments is essential for developing efficient therapies against hRSV. AREAS COVERED: This article describes the relevance of using different animal models to evaluate the effect of antiviral drugs, pharmacological molecules, vaccine prototypes, and antibodies in the protection against hRSV. The animal models covered are rodents, mustelids, bovines, and nonhuman primates. Animals included were chosen based on the available literature and their role in the development of the drugs discussed in this manuscript. EXPERT OPINION: Choosing the correct animal model is critical for exploring and testing treatments that could decrease the impact of hRSV in high-risk populations. Mice will continue to be the most used preclinical model to evaluate this. However, researchers must also explore the use of other models such as nonhuman primates, as they are more similar to humans, prior to escalating into clinical trials.

Oral vaccine based on a surface immunogenic protein mixed with alum promotes a decrease in Streptococcus agalactiae vaginal colonization in a mouse model.

The Surface Immunogenic Protein (SIP) of Group B Streptococcus (GBS) had been described as a good target for vaccine development. To date, SIP has been reported as a highly conserved protein, and in a mouse model it induces protection against lethal GBS challenge. Also, similar effects have been described by intranasal immunization with a SIP-based vaccine. In this study, we show the immune response induced by an oral SIP-based vaccine formulated on alum in a mouse model. Our vaccine can reduce vaginal GBS colonization and induce specific SIP-antibodies with opsonophagocytosis activities against GBS. Moreover, we observed the activation of T-cells producing IFN-γ, TNF-α, IL-10, IL-2, and increased expression of the transcription factor T-bet, suggesting a Th1-type humoral response. The oral SIP-based vaccine is a novel alternative in the development of a vaccine against GBS.

Modulation of Immunity and Inflammation by the Mineralocorticoid Receptor and Aldosterone.

The mineralocorticoid receptor (MR) is a ligand dependent transcription factor. MR has been traditionally associated with the control of water and electrolyte homeostasis in order to keep blood pressure through aldosterone activation. However, there is growing evidence indicating that MR expression is not restricted to vascular and renal tissues, as it can be also expressed by cells of the immune system, where it responds to stimulation or antagonism, controlling immune cell function. On the other hand, aldosterone also has been associated with proinflammatory immune effects, such as the release of proinflammatory cytokines, generating oxidative stress and inducing fibrosis. The inflammatory participation of MR and aldosterone in the cardiovascular disease suggests an association with alterations in the immune system. Hypertensive patients show higher levels of proinflammatory mediators that can be modulated by MR antagonism. Although these proinflammatory properties have been observed in other autoimmune and chronic inflammatory diseases, the cellular and molecular mechanisms that mediate these effects remain unknown. Here we review and discuss the scientific work aimed at determining the immunological role of MR and aldosterone in humans, as well as animal models.

Purification and characterization of saxitoxin from Mytilus chilensis of southern Chile.

In the current communication we describe an innovative method to purify saxitoxin (STX), a toxin presents in contaminated muscle of Mylitus chilensis extracted in the southern part of Chile, using a liquid chromatographic methodology based on ionic pairs. The STX was extracted using HCl and treated with ammonium sulfate following a treatment with trichloroacetic acid and hexane/diethyl ether (97/3). The samples were analyzed by a semi-preparative HPLC in order to collect pure fractions of STX and these fractions were eluted in solid-phase cationic interchange SCX extraction columns. The purified STX was stable and homogeneous and its identity was confirmed by LC-MS-MS, which demonstrated a high quality purification of STX, without presence of analogs such as neosaxitoxin (Neo) and decarbamoyl saxitoxin (dcSTX). The STX biological activity was analyzed in a bioassay in mice model and compared to the standard STX produced by the FDA and no significant differences were observed.

Carbon monoxide inhibits T cell activation in target organs during systemic lupus erythematosus.

Systemic lupus erythematosus is characterized by the presence of circulating anti-nuclear antibodies (ANA) and systemic damage that includes nephritis, haematological manifestations and pulmonary compromise, among others. Although major progress has been made in elucidating the molecular mechanisms responsible for autoimmunity, current therapies for lupus have not improved considerably. Because the exposure of carbon monoxide (CO) has been shown to display beneficial immunoregulatory properties in different immune-mediated diseases, we investigated whether CO therapy improves lupus-related kidney injury in lupus mice. MRL-Fas(lpr) lupus mice were exposed to CO and disease progression was evaluated. ANA, leucocyte-infiltrating populations in spleen, kidney and lung and kidney lesions, were measured. CO therapy significantly decreased the frequency of activated B220(+) CD4(-) CD8(-) T cells in kidneys and lungs, as well as serum levels of ANA. Furthermore, we observed that CO therapy reduced kidney injury by decreasing proliferative glomerular damage and immune complexes deposition, decreased proinflammatory cytokine production and finally delayed the impairment of kidney function. CO exposure ameliorates kidney and lung leucocyte infiltration and delays kidney disease in MRL-Fas(lpr) lupus mice. Our data support the notion that CO could be explored as a potential new therapy for lupus nephritis.

High sodium intake is associated with increased glucocorticoid production, insulin resistance and metabolic syndrome.

OBJECTIVE: High sodium (HS) diet is associated with hypertension (HT) and insulin resistance (IR). We evaluated whether HS diet was associated with a dysregulation of cortisol production and metabolic syndrome (MetS). PATIENTS AND MEASUREMENTS: We recruited 370 adults (18-85 years, BMI 29·3 ± 4·4 kg/m(2) , 70% women, 72% HT, 61% MetS). HS diet (urinary sodium >150 mEq/day) was observed in 70% of subjects. We measured plasma hormones, lipid profile, urinary free cortisol (UFC) and cortisol tetrahydrometabolites (THM). RESULTS: Urinary sodium was correlated with UFC (r = +0·45, P < 0·001), cortisol THM (r = +0·41, P < 0·001) and inversely with adiponectin, HDL and aldosterone, after adjusting by age, gender and BMI. Subjects with high, compared with adequate sodium intake (50-149 mEq/day) had higher UFC (P < 0·001), THM (P < 0·001), HOMA-IR (P = 0·04), HT (81% vs 50%, P < 0·001), MetS (69% vs 41%, P < 0·001) and lower adiponectin (P = 0·003). A multivariate predictive model adjusted by confounders showed a high discriminative capacity for MetS (ROC curve 0·878) using four clinical variables: HS intake [OR = 5·6 (CI 2·3-15·3)], HOMA-IR [OR 1·7 (1·3-2·2)] cortisol THM [OR 1·2 (1·1-1·4)] and adiponectin [OR = 0·9 (0·8-0·9)], the latter had a protective effect. CONCLUSIONS: High sodium diet was associated with increased urinary cortisol and its metabolites. Also, HS diet was associated with HT, insulin resistance, dyslipidaemia and hypoadiponectinaemia, even when adjusting by confounding variables. Further, we observed that high salt intake, IR and higher cortisol metabolites, alone or combined in a clinical simple model, accurately predicted MetS status, suggesting an additive mechanism in obesity-related metabolic disorders.

Immune system alterations by aldosterone during hypertension: from clinical observations to genomic and non-genomic mechanisms leading to vascular damage.

Hypertension is traditionally considered a disease in which elevated blood pressure contributes to inflammation and activation of the immune system, leading to cardiovascular injury and end-organ damage. Here, we discuss the effects of aldosterone on the immune system and aldosterone's contribution to vascular pathogenesis. Studies in human have suggested a broader role for aldosterone, beyond elevating blood pressure. Recent clinical data support the notion that aldosterone can directly alter the function of the immune system and cause vascular-damaging inflammation. Clinical observations have been reproduced in experimental models of hypertension, further supporting the idea that an aberrant immune response contributes to the onset of hypertension. Such studies have shown that myeloid cells are required to induce the disease and IL-17-producing CD4(+) T cells may contribute to maintaining aldosterone-mediated hypertension. In addition, regulatory T cells diminish the inflammatory damage caused by aldosterone during hypertension. This is a very active area of research that could lead to new therapeutic targets for treating hypertension.

Understanding respiratory syncytial virus infection to improve treatment and immunity.

Despite significant research since it was discovered more than 50 years ago, respiratory syncytial virus (RSV) continues to be the leading agent causing infant hospitalization and respiratory distress worldwide. Although RSV normally does not cause mortality, this virus is recognized as a major public health and economic burden around the globe. RSV can modulate host immunity leading to an inflammatory response that produces lung damage and virus dissemination in the host airways. Remarkably, infection with the virus elicits poor immunity that in most cases fails to protect against subsequent exposures. Here, we review advances made on the understanding of the lifecycle of the virus, some of the molecular mechanisms it has evolved to cause pathology and ineffective immunity during infection. Hopefully, ongoing research will contribute to developing new drugs and candidate vaccines that will decrease the health burden caused by this virus.

M-protein gene-type distribution and hyaluronic acid capsule in group A Streptococcus clinical isolates in Chile: association of emm gene markers with csrR alleles.

Streptococcus pyogenes causes a variety of infections because of virulence factors such as capsular hyaluronic acid and M protein. The aim of this study was to determine emm types and capsule phenotype in 110 isolates of S. pyogenes from patients with invasive (sterile sites) and non-invasive (mainly pharyngitis) infections in Chile, and the relationship between both virulence factors. The most abundant types found were emm12, emm1, emm4 and emm28 and their distribution was similar to that seen in Latin America and developed countries, but very different from that in Asia and Pacific Island countries. Ten of 16 emm types identified in pharyngeal isolates were found in sterile-site isolates, and three of nine emm types of sterile-site isolates occurred in pharyngeal isolates; three emm subtypes were novel. The amount of hyaluronic acid was significantly higher in sterile-site isolates but did not differ substantially among emm types. Only three isolates were markedly capsulate and two of them had mutations in the csrR gene that codes for a repressor of capsule synthesis genes. We found a non-random association between emm types and csrR gene alleles suggesting that horizontal gene transfer is not freely occurring in the population.

Evasion of host immunity by virulent Salmonella: implications for vaccine design.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) capable of linking innate and adaptive immunity during infection. After recognition of pathogen-associated molecular patterns (PAMPs), DCs can engulf, process and present bacteria-derived antigens on MHC molecules to T cells. Because of the key role that DCs play on the initiation of innate and adaptive immunity, alterations in their function could render the host susceptible to bacterial dissemination. Consistent with this notion, is the observation that several pathogenic bacteria have evolved mechanisms to impair the DC capacity to prime naïve T cells. One of such bacteria is Salmonella enterica serovar Typhimurium, which causes a typhoid-like disease in mice and gastroenteritis in humans. Recent studies have shown that virulent Salmonella can use intestinal DCs to spread inside the host, evading T cell priming. The avoidance of T cell recognition by Salmonella is in large part achieved by the activity of gene products encoded on Salmonella Pathogenicity Islands -1 and - 2. The understanding of some of the remarkable molecular virulence mechanisms displayed by Salmonella has contributed to the design of new vaccines capable of inducing protective immunity against this pathogen in mouse models. Here we describe recent data underscoring the virulence mechanisms used by Salmonella to exploit DC function and discuss strategies based on this new knowledge aimed at the design of new efficient and safe vaccines against this pathogen.

A de novo unequal cross-over mutation between CYP11B1 and CYP11B2 genes causes familial hyperaldosteronism type I.

UNLABELLED: Familial hyperaldosteronism type I (FH-I) is an autosomal dominant disorder caused by an unequal cross-over of the gene encoding steroid 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), giving rise to a chimeric CYP11B1/CYP11B2 gene that displays aldosterone synthase activity regulated by ACTH instead of angiotensin II. AIM: To report an unprecedented case of a de novo unequal crossover mutation between CYP11B1 and CYP11B2 genes causing FH-I. PATIENTS AND METHODS: The index case is a 45-yr-old Chilean male diagnosed with primary aldosteronism (PA). All family members were also studied: his biological parents, 1 brother, 6 sisters, 2 daughters, and 1 son. Plasma renin activity, serum aldosterone, and its ratio were measured in all patients. Genetic analyses were performed using long-extension PCR (XL-PCR), DNA sequencing and Southern blot methods. RESULTS: PA was diagnosed for the index case, 1 of his daughters, his son but not for his parents or siblings. XLPCR and Southern blotting demonstrated the presence of the chimeric CYP11B1/CYP11B2 gene solely in PA-affected subjects, suggesting a case of a de novo mutation. Sequence analysis showed the unequal cross-over CYP11B1/CYP11B2 at intron 2 (c.2600-273 CYP11B2). We also identified a polymorphism at the same intron (c.2600-145C>A CYP11B2) in the genome of the index case's father. CONCLUSION: We describe an unprecedented case of unequal cross-over mutation for the chimeric CYP11B1/CYP11B2 gene causing FH-I, which may be linked to a polymorphism in the index case's father germ line.

Virulence mechanisms displayed by Salmonella to impair dendritic cell function.

Dendritic cells (DCs) link innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) on bacteria. DCs can capture and degrade bacteria and present their antigens on MHC molecules to T cells. PAMP recognition promotes DC maturation, a phenotypic change that empowers them to prime naïve T cells. As a result, an adaptive immune response that specifically targets bacteria-derived antigens is initiated. Consequently, any impairment of DC function might contribute to bacterial survival and dissemination in the host. Therefore, the characterization of DC-bacteria interactions is required to understand the mechanisms used by virulent bacteria to avoid adaptive immunity. An example of a bacterial pathogen capable of interfering with DC function is Salmonella enterica serovar Typhimurium (S. Typhimurium), which causes a typhoid-like disease in mice. Virulent strains of S. Typhimurium are able to differentially modulate the entrance to DCs and avoid lysosomal degradation, to prevent antigen presentation on MHC molecules. These features of virulent S. Typhimurium are controlled by virulence factors encoded by Salmonella Pathogenicity Islands 1 and 2. Modulation of DC functions by the activity of these gene products is supported by several recent studies, which have shown that pathogenesis might depend on this attribute of virulent S. Typhimurium. Here we discuss recent data showing that several virulence factors from Salmonella are required to differentially modulate DC function and adaptive immunity in the host.

Primary aldosteronism can alter peripheral levels of transforming growth factor beta and tumor necrosis factor alpha.

UNLABELLED: Primary aldosteronism (PA) is the most common secondary cause of hypertension that has recently been implicated in alterations of the immune system and progression of cardiovascular disease. OBJECTIVE: To study the cytokines transforming growth factor beta1 (TGF-beta1), tumor necrosis factor alpha (TNF-alpha), and interleukin 10 (IL-10) in patients with PA and essential hypertensives (EH) and evaluate its association with the renin-angiotensin-aldosterone system. PATIENTS AND METHODS: We studied 26 PA and 52 EH patients as controls, adjusted by their blood pressure, body mass index, age, and gender. In both groups, PA and EH, we measured serum aldosterone (SA), plasma renin activity (PRA), and cytokines TGF- beta1, TNF-alpha, and IL-10. In addition, 17 PA patients were treated for 6 months with spironolactone, a mineralocorticoid receptor (MR) antagonist. RESULTS: PA patients had lower levels of TGF-beta1 (17.6+/-4.1 vs 34.5+/-20.5 pg/ml, p<0.001) and TNF-alpha (17.0+/-4.4 vs 35.6+/-21.7 pg/ml, p<0.001) and similar IL-10 levels (99.7+/-18.7 vs 89.4+/-49.5 pg/ml, p: ns), as compared with EH controls. TGF-beta1 and TNF-alpha levels showed a remarkable correlation with SA/PRA ratio in the total group (PA+EH). The treatment of PA patients with spironolactone increased the TGF-beta1 levels (18.3+/-5.9 to 28.4+/-6.3 pg/ml, p<0.001), while TNF-alpha, and IL-10 remained unchanged. CONCLUSION: Our results showed that PA patients have lower TGF-beta1 and TNF-alpha cytokine serum levels than EH. TGF-beta1 levels were restored with spironolactone, showing a MR-dependent regulation. In this way, the chronic aldosterone excess modifies the TGF-beta1 levels, which could produce an imbalance in the immune system homeostasis that may promote an early proinflammatory cardiovascular phenotype.

Maternal hypothyroxinemia impairs spatial learning and synaptic nature and function in the offspring.

Neurological deficits in the offspring caused by human maternal hypothyroxinemia are thought to be irreversible. To understand the mechanism responsible for these neurological alterations, we induced maternal hypothyroxinemia in pregnant rats. Behavior and synapse function were evaluated in the offspring of thyroid hormone-deficient rats. Our data indicate that, when compared with controls, hypothyroxinemic mothers bear litters that, in adulthood, show prolonged latencies during the learning process in the water maze test. Impaired learning capacity caused by hypothyroxinemia was consistent with cellular and molecular alterations, including: 1) lack of increase of phosphorylated c-fos on the second day of the water maze test; 2) impaired induction of long-term potentiation in response to theta-burst stimulation to the Schaffer collateral pathway in the area 1 of the hippocampus Ammon's horn stratum radiatum, despite normal responses for input/output experiments; 3) increase of postsynaptic density protein 95 (PSD-95), N-methyl-D-aspartic acid receptor subunit 1, and tyrosine receptor kinase B levels in brain extracts; and 4) significant increase of PSD-95 at the PSDs and failure of this molecule to colocalize with N-methyl-D-aspartic acid receptor subunit 1, as it was shown by control rats. Our findings suggest that maternal hypothyroxinemia is a harmful condition for the offspring that can affect key molecular components for synaptic function and spatial learning.

Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8(+) T cells.

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR alpha- or beta-chain of a VSV8 (unmodified)/H-2K(b)-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3alpha and beta loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3beta residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jbeta region, while the Jalpha region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3alpha and beta loops to allow interaction with a haptenated peptide.

Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3alpha and an antigenic peptide bound to MHC class I.

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR beta-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2K(b). We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3alpha sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3alpha and preferred Jalpha usage, indicating that multiple residues of CDR3alpha are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jalpha usage, suggesting a potential interaction between CDR3alpha and position 4. Cross-reactivity data revealed the foremost importance of the Jalpha region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3alpha to a positively charged residue, suggesting that CDR3alpha may interact with position 6 in certain circumstances. Analogous interactions between the TCR alpha-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.

The effect of CD28/B7 blockade on alloreactive T and B cells after liver cell transplantation.

BACKGROUND: Hepatocyte cell lines are beginning to be developed as universal donors for isolated liver cell transplantation, which is a less invasive method than orthotopic liver transplantation for treatment of metabolic liver disease. The immune response to isolated liver cell transplantation and its modification by costimulatory blockade are as yet not well delineated. METHODS: Adenovirus expressing CTLA4Ig was used to study blockade of the costimulatory CD28/B7 pathway in murine models of hepatocyte transplantation, and the effects on alloreactive T and B cells were studied. RESULTS: CTLA4Ig delayed rejection of subcutaneously administered C57L-derived murine hepatoma cells in CBA/J recipients for >50 days. Activation and cytokine secretion by allospecific CD4+ and CD8+ T cells were initially blocked by CTLA4Ig; delayed rejection was associated with tumor infiltration by CD8+ T cells that did not secrete interferon-gamma. CTLA4Ig failed to block transplant rejection in primed mice, indicating that memory effector T cells were resistant to its action. In contrast, CTLA4Ig suppressed both naive and memory alloreactive B cells. High levels of CTLA4Ig mediated acceptance of hepatoma cells delivered directly into the spleen. However, isolated primary C57BL/6 mouse hepatocytes delivered into the spleen were rejected with only moderately delayed kinetics. CONCLUSIONS: Transplant antigenicity, transplant site, and CTLA4Ig dose all affected the survival of transplanted liver cells. CD8+ T cells are significant mediators of hepatocyte transplant rejection and are relatively resistant to costimulatory blockade with CTLA4Ig. Strategies to specifically antagonize CD8+ T cells or to modulate MHC class I expression in association with costimulatory blockade by CTLA4Ig may enhance the clinical feasibility of transplanting allogeneic hepatocytes.

A structural difference limited to one residue of the antigenic peptide can profoundly alter the biological outcome of the TCR-peptide/MHC class I interaction.

The vesicular stomatitis virus (VSV) octapeptide RGYVYQGL binds to H-2K(b) and triggers a cytotoxic T cell response in mice. A variant peptide, RGYVYEGL (E6) with a glutamic acid for glutamine replacement at position 6 of the VSV peptide, elicits a T cell response with features that are quite different from those elicited by the wild-type VSV peptide. The differences found in the nature of the T cells responding to the E6 peptide include changes in both the V beta elements and the sequences of the complementarity-determining region 3 loops of their TCRs. Further experiments found that the E6 peptide can act as an antagonist for VSV-specific T cell hybridomas. To determine whether these differences in V beta usage, complementarity-determining region 3 sequences, and the switch from agonism to antagonism are caused by a conformational change on the MHC, the peptide, or both, we determined the crystal structure of the variant E6 peptide bound to H-2K(b). This structure shows that the only significant structural difference between H-2K(b)/E6 and the previously determined H-2K(b)/VSV is limited to the side chain of position 6 of the peptide, with no differences in the MHC molecule. Thus, a minor conformational change in the peptide can profoundly alter the biological outcome of the TCR-peptide/MHC interaction.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex.

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 beta of an MHC class I-restricted TCR.

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.