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[This corrects the article DOI: 10.3389/fimmu.2021.621138.].[This corrects the article DOI: 10.3389/fimmu.2021.621138.].

In pre-sensitizing events, immunological memory is mainly created via indirect allorecognition where CD4 + T cells recognize foreign peptides in the context of self-HLA class II (pHLA) presented on antigen-presenting cells. This recognition makes it possible for naive CD4 + T-helper cells to differentiate into memory cells, resulting in the creation of further antibody memory. These responses contribute to effective secretion of donor-specific anti-HLA antibodies (DSA) after second encounters with the same peptide. Preformed donor-reactive CD4 + memory T cells may induce early immune responses after transplantation; however, the tools to evaluate them are limited. This study evaluated shared T cell epitopes (TEs) between the pre-sensitizing and donor HLA using an in silico assay, an alternative to estimate donor-reactive CD4 + memory T cells before transplantation. In 578 living donor kidney transplants without preformed DSA, 69 patients had anti-HLA antibodies before transplantation. Of them, 40 had shared TEs and were estimated to have donor-reactive CD4 + memory T cells. De novo DSA formation in the early phase was significantly higher in the shared TE-positive group than in the anti-HLA antibody- and shared TE-negative groups (p=0.001 and p=0.02, respectively). In conclusion, evaluation of shared TEs for estimating preformed donor-reactive CD4 + memory T cells may help predict the risk of early de novo DSA formation after kidney transplantation.

CD8+ T cell immune monitoring aims at measuring the size and functions of antigen-specific CD8+ T cell populations, thereby providing insights into cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical because within a complex antigen exists a multitude of potential epitopes that can be presented by HLA class I molecules. Further complicating this task, there is HLA class I polygenism and polymorphism which predisposes CD8+ T cell responses towards individualized epitope recognition profiles. In this study, we compare the actual CD8+ T cell recognition of a well-characterized model antigen, human cytomegalovirus (HCMV) pp65 protein, with its anticipated epitope coverage. Due to the abundance of experimentally defined HLA-A * 02:01-restricted pp65 epitopes, and because in silico epitope predictions are most advanced for HLA-A * 02:01, we elected to focus on subjects expressing this allele. In each test subject, every possible CD8+ T cell epitope was systematically covered testing 553 individual peptides that walk the sequence of pp65 in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. No correlation was found between epitopes’ ranking on the prediction scale and their actual immune dominance. Collectively, these data suggest that accurate CD8+ T cell immune monitoring may necessitate reliance on agnostic mega peptide pools, or brute force mapping, rather than electing individual peptides as representative epitopes for tetramer and other multimer labeling of surface antigen receptors.

The native-like, soluble SOSIP.664 trimer based on the BG505 clade A env gene of HIV-1 is immunogenic in various animal species, of which the most studied are rabbits and rhesus macaques. The trimer induces autologous neutralizing antibodies (NAbs) consistently but at a wide range of titers and with incompletely determined specificities. A precise delineation of immunogenic neutralization epitopes on native-like trimers could help strategies to extend the NAb response to heterologous HIV-1 strains. One autologous NAb epitope on the BG505 Env trimer is known to involve residues lining a hole in the glycan shield that is blocked by adding a glycan at either residue 241 or 289. This glycan-hole epitope accounts for the NAb response of most trimer-immunized rabbits but not for that of a substantial subset. Here, we have used a large panel of mutant BG505 Env-pseudotyped viruses to define additional sites. A frequently immunogenic epitope in rabbits is blocked by adding a glycan at residue 465 near the junction of the gp120 V5 loop and β24 strand and is influenced by amino-acid changes in the structurally nearby C3 region. We name this new site the \"C3/465 epitope\". Of note is that the C3 region was under selection pressure in the infected infant from whom the BG505 virus was isolated. A third NAb epitope is located in the V1 region of gp120, although it is rarely immunogenic. In macaques, NAb responses induced by BG505 SOSIP trimers are more often directed at the C3/465 epitope than the 241/289-glycan hole.

Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most--if not the most--frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy.

Crystal structures of HIV Env trimer with native glycosylation in complex with neutralizing antibodies reveal a glycan shield of high-mannose and complex-type N-glycan and indicate a path for germline-targeting vaccine design. HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46–derived CD4bs bNAbs, which have normal-length CDRL3s. The existence of bNAbs that combine features of VRC01-class and 8ANC131-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs.

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the 3D appreciation of cell and protein localization in their native organ environment. However, the sample preparations for such imaging are often onerous, and their capability for antigen detection is limited. Here, we describe FLASH (fast light-microscopic analysis of antibody-stained whole organs), a simple, rapid, fully customizable technique for molecular phenotyping of intact tissue volumes. FLASH utilizes non-degradative epitope recovery and membrane solubilization to enable the detection of a multitude of membranous, cytoplasmic and nuclear antigens in whole mouse organs and embryos, human biopsies, organoids and Drosophila . Retrieval and immunolabeling of epithelial markers, an obstacle for previous clearing techniques, can be achieved with FLASH. Upon volumetric imaging, FLASH-processed samples preserve their architecture and integrity and can be paraffin-embedded for subsequent histopathological analysis. The technique can be performed by scientists trained in light microscopy and yields results in <1 week. This protocol describes how to perform antigen retrieval and tissue clearing for volumetric imaging of whole organs, organoids and small organisms by using fast light-microscopic analysis of antibody-stained whole organs.

Endothelial dysfunction is a critical event in vascular inflammation characterized, in part, by elevated surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is heavily N-glycosylated, and like other surface proteins, it is largely presumed that fully processed, complex N-glycoforms are dominant. However, our recent studies suggest that hypoglycosylated or high mannose (HM)-ICAM-1 N-glycoforms are also expressed on the cell surface during endothelial dysfunction, and have higher affinity for monocyte adhesion and regulate outside-in endothelial signaling by different mechanisms. Whether different ICAM-1 N-glycoforms are expressed in vivo during disease is unknown. In this study, using the proximity ligation assay, we assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. Our data demonstrates that ICAM-1 harboring HM or hybrid epitopes as well as ICAM-1 bearing α-2,6-sialylated epitopes are present in human and mouse atherosclerotic lesions. Further, HM-ICAM-1 positively associated with increased macrophage burden in lesions as assessed by CD68 staining, whereas α-2,6-sialylated ICAM-1 did not. Finally, both HM and α-2,6-sialylated ICAM-1 N-glycoforms were present in hemodialysis patients who had AVF maturation failure compared to successful AVF maturation. Collectively, these data provide evidence that HM- ICAM-1 N-glycoforms are present in vivo, and at levels similar to complex α-2,6-sialylated ICAM-1 underscoring the need to better understand their roles in modulating vascular inflammation.

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.