Altered Basal Lipid Metabolism Underlies the Functional Impairment of Naive CD8+ T Cells in Elderly Humans.

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8+ T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

TCR-induced alteration of primary MHC peptide anchor residue.

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.

Subtle changes at the variable domain interface of the T-cell receptor can strongly increase affinity.

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vß) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vß interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vß association or direct contact with the ligand. More surprisingly, many Vα/Vß interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45ßY) was most prevalent. Further analysis of F45ßY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vß interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.

Endpoint-restricted adiabatic free energy dynamics approach for the exploration of biomolecular conformational equilibria.

A method for calculating the free energy difference between two structurally defined conformational states of a chemical system is developed. A path is defined using a previously reported collective variable that interpolates between two or more conformations, and a restraint is introduced in order to keep the system close to the path. The evolution of the system along the path, which typically presents a high free energy barrier, is generated using enhanced sampling schemes. Although the formulation of the method in terms of a path is quite general, an important advance in this work is the demonstration that prior knowledge of the path is, in fact, not needed and that the free energy difference can be obtained using a simplified definition of the path collective variable that only involves the endpoints. We first validate this method on cyclohexane isomerization. The method is then tested for an extensive conformational change in a realistic molecular system by calculating the free energy difference between the α-helix and ß-hairpin conformations of deca-alanine in solution. Finally, the method is applied to a biologically relevant system to calculate the free energy difference of an observed and a hypothetical conformation of an antigenic peptide bound to a major histocompatibility complex.

Artificial human antigen-presenting cells are superior to dendritic cells at inducing cytotoxic T-cell responses.

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T-cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I-peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T-cell response, we generated artificial antigen-presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen-specific CTLs were then induced using either peptide-loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen-specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA-A2-positive tumour antigen-bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA-NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.

Specific roles of each TCR hemichain in generating functional chain-centric TCR.

TCRα- and ß-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3ß sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRß genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance.

T-cell receptors: tugging on the anchor for a tighter hold on the tumor-associated peptide.

Although it has been shown that human tumor-associated, HLA anchor residue modified "heteroclitic" peptides may induce stronger immune responses than wild-type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor-expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584-591] highlight a novel twist on T-cell receptor (TCR) recognition of HLA-peptide complexes. Tumor-associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T-cell cross-reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan-A/MART-126-35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8(+) T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA-A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide "lifts" its amino-terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue "optimized" heteroclitic peptide, which cannot be "pulled" from the HLA groove.

Structural basis for ineffective T-cell responses to MHC anchor residue-improved "heteroclitic" peptides.

MHC anchor residue-modified "heteroclitic" peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26-35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to "pull" the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.

Specific increase in potency via structure-based design of a TCR.

Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive "self" proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in ∼30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag-MHC (peptide-MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.

Computational design of the affinity and specificity of a therapeutic T cell receptor.

T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. Here we explored the efficacy of computational design with the clinically relevant TCR DMF5, which recognizes nonameric and decameric epitopes from the melanoma-associated Melan-A/MART-1 protein presented by the class I MHC HLA-A2. We tested multiple mutations selected by flexible and rigid modeling protocols, assessed impacts on affinity and specificity, and utilized the data to examine and improve algorithmic performance. We identified multiple mutations that improved binding affinity, and characterized the structure, affinity, and binding kinetics of a previously reported double mutant that exhibits an impressive 400-fold affinity improvement for the decameric pMHC ligand without detectable binding to non-cognate ligands. The structure of this high affinity mutant indicated very little conformational consequences and emphasized the high fidelity of our modeling procedure. Overall, our work showcases the capability of computational design to generate TCRs with improved pMHC affinities while explicitly accounting for peptide specificity, as well as its potential for generating TCRs with customized antigen targeting capabilities.

T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting.

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8(+) T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8(+) T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201(+) individuals that were capable of efficient HLA A*0201(+) melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.

Process development and production of cGMP grade Melan-A for cancer vaccine clinical trials.

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.

TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms.

T cells engineered to express TCRs specific for tumor Ags can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Although DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity toward both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same Ags and the finding that TCR-binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.

A full GMP process to select and amplify epitope-specific T lymphocytes for adoptive immunotherapy of metastatic melanoma.

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108 pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

Targeted silencing of MART-1 gene expression by RNA interference enhances the migration ability of uveal melanoma cells.

Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy.

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

Synthesis and Biological Evaluation of Hapten-Clicked Analogues of The Antigenic Peptide Melan-A/MART-126(27L)-35.

A click-chemistry-based approach was implemented to prepare peptidomimetics designed in silico and made from aromatic azides and a propargylated GIGI-mimicking platform derived from the altered Melan-A/MART-126(27L)-35 antigenic peptide ELAGIGILTV. The CuI -catalyzed Huisgen cycloaddition was carried out on solid support to generate rapidly a first series of peptidomimetics, which were evaluated for their capacity to dock at the interface between the major histocompatibility complex class-I (MHC-I) human leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite being a weak HLA-A2 ligand, one of these 11 first synthetic compounds bearing a p-nitrobenzyl-triazole side chain was recognized by the receptor proteins of Melan-A/MART-1-specific T-cells. After modification of the N and C termini of this agonist, which was intended to enhance HLA-A2 binding, one of the resulting seven additional compounds triggered significant T-cell responses. Thus, these results highlight the capacity of naturally circulating human TCRs that are specific for the native Melan-A/MART-126-35 peptide to cross-react with peptidomimetics bearing organic motifs structurally different from the native central amino acids.

In vivo targeting of DNA vaccines to dendritic cells using functionalized gold nanoparticles.

The clinical success of dendritic cell (DC)-based genetic immunization remains critically dependent on the availability of effective and safe nano-carriers for targeting antigen-encoded DNA vaccines to DCs, the most potent antigen-presenting cells in the human body in vivo. Recent studies revealed the efficacies of mannose receptor-mediated in vivo DC-targeted genetic immunization by liposomal DNA vaccine carriers containing both mannose-mimicking shikimoyl and transfection enhancing guanidinyl functionalities. However, to date, the efficacies of this approach have not been examined for metal-based nanoparticle DNA vaccine carriers. Herein, we report for the first time, the design, synthesis, physico-chemical characterization and bioactivities of gold nanoparticles covalently functionalized with a thiol ligand containing both shikimoyl and guanidinyl functionalities (Au-SGSH). We show that Au-SGSH nanoparticles can deliver DNA vaccines to mouse DCs under in vivo conditions. Subcutaneous administration of near infrared (NIR) dye-labeled Au-SGSH showed significant accumulation of the NIR dye in the DCs of the nearby lymph nodes compared to that for the non-targeting NIR-labeled Au-GSH nanoconjugate containing only a covalently tethered guanidinyl group, not the shikimoyl-functionality. Under prophylactic settings, in vivo immunization (s.c.) with the Au-SGSH-pCMV-MART1 nanoplex induced a long-lasting (180 days) immune response against murine melanoma. Notably, mannose receptor-mediated in vivo DC-targeted immunization (s.c.) with the Au-SGSH-MART1 nanoplex significantly inhibited established melanoma growth and increased the overall survivability of melanoma-bearing mice under therapeutic settings. The Au-SGSH nanoparticles reported herein have potential use for in vivo DC-targeted genetic immunization against cancer and infectious diseases.

Generation and molecular recognition of melanoma-associated antigen-specific human γδ T cells.

Antigen recognition by T cells bearing αß T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αß TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.

Heat-induced Epitope Retrieval for Immunohistochemistry at High Altitude: A Quality Assurance Study.

INTRODUCTION: Heat-induced epitope retrieval (HIER) of formalin-fixed paraffin-embedded tissues is now a standard practice in immunohistochemistry (IHC). In this study, we aimed to test the effect of altering HIER temperature on IHC staining quality at high altitude, the hypothesis being that lower HIER temperatures would result in improved staining patterns. MATERIALS AND METHODS: In a laboratory at high altitude (Aurora, CO), we used a platform with automated onboard epitope retrieval, and systematically tested 3 different HIER temperatures (100°C, 95°C, 90°C) with 4 IHC stains that are commonly used in routine practice: CD3, Ki67, CK20, and Melan A (n=10 for each antibody/epitope retrieval temperature combination). A scoring system was devised, the slides were scored in a blinded manner, and statistical analysis was performed. For comparison, the same study was performed in a laboratory near sea level (Atlanta, GA). RESULTS: At high altitude, lower HIER temperatures resulted in improved staining patterns, as quantified by stronger staining intensity and greater area of the slides stained. The scores obtained with HIER temperatures of 95°C and 90°C were higher than those obtained with HIER of 100°C, and the difference was found to be statistically significantly for some antibody/epitope retrieval temperature combinations (P<0.05). This effect was not seen in the laboratory near sea level. CONCLUSIONS: We show that alternate epitope retrieval recommendations are warranted for laboratories at high altitude. Furthermore, we suggest that manufactures should consider how their instruments will perform at high altitude as they further automate the process of IHC.