Characterization of putative regulatory isoforms of porcine tumor necrosis factor receptor 2 in endothelial cells.

Tumor necrosis factor α (TNFα) and its receptors contribute to rejection of transplanted cells and organs. To elucidate how TNFα affects xenograft rejection, we previously cloned the cDNA of pig TNF-receptor 2 (pTNFR2) and found four isoforms: one comprising the full receptor with four cysteine-rich domains (CRD), a shorter variant (pTNFR2ΔE7-10) encoding for a soluble isoform, another lacking exon 4 (pTNFR2ΔE4) displaying only 3 CRD and poor ligand binding, and the smallest one generated by the two alternative splicings. All isoforms contained the pre-ligand assembly domain (PLAD) responsible for receptor trimerization. We now investigated their roles by structural, expression, and subcellular localization studies. Structural in silico analyses identified four amino acids potentially involved in TNFα binding and lacking in pTNFR2ΔE4. Quantitative RT-PCR determined regulated expression affecting the two pTNFR2 alternative splicings in cytokine-stimulated porcine aortic endothelial cells (PAEC). Particularly, human IL-1α and TNFα produced a strong mRNA upregulation of all isoforms, being the full receptor the predominant one. However, expression of pTNFR2 on PAEC did not correlate with mRNA and decreased after 24-hour exposure to IL-1α or TNFα. Notably, confocal microscopy confirmed the presence of pTNFR2 inside and on the plasma membrane, whereas pTNFR2ΔE4 located only intracellularly. Most interestingly, FRET analyses showed that membrane-bound isoforms pTNFR2 and pTNFR2ΔE4 colocalized intracellularly and associated through the PLAD. Our data show that pTNFR2ΔE4 bind and may retain the full receptor intracellularly. This mechanism has not been described in other species and represents a particularity that may affect the pathophysiology of pig xenografts.

Limited induction of polyfunctional lung-resident memory T cells against SARS-CoV-2 by mRNA vaccination compared to infection.

Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Here we show that the frequency of CD4+ T cells secreting IFNγ in response to S-peptides is variable but overall similar in the lung of mRNA-vaccinated patients compared to convalescent-infected patients. However, in vaccinated patients, lung responses present less frequently a TRM phenotype compared to convalescent infected individuals and polyfunctional CD107a+ IFNγ+ TRM are virtually absent in vaccinated patients. These data indicate that mRNA vaccination induces specific T cell responses to SARS-CoV-2 in the lung parenchyma, although to a limited extend. It remains to be determined whether these vaccine-induced responses contribute to overall COVID-19 control.

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model.

Structural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult α1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose α1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor.

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration.

Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.

Tools for Molecular Studies in Xenotransplantation.

Understanding the molecular bases of xenograft rejection is one of the highest priorities in the xenotransplantation field. This information is needed for the successful development of genetic modifications of the animal source of xenogeneic cells and organs that prevent rejection. Furthermore, the identification of physiological incompatibilities in the xenogeneic setting is also necessary for developing the appropriate strategies that allow long-term xenograft function. As the pig is the species of choice for the development of the majority of xenogeneic applications, the cloning of pig genes or cDNA is a key step to elucidate the interactions of pig and human molecules. In addition, there are currently multiple bioinformatic tools which facilitate the study in silico of protein structures, molecular interactions, and docking sites. Thus, we describe a basic cloning method that comprises total RNA extraction, reverse transcription (RT), and polymerase chain reaction (PCR) for cDNA amplification and include some links for databases and bioinformatics tools available on the Internet for the subsequent analyses and predictions. Finally, some procedures of protein expression and analysis of protein interactions by surface plasmon resonance (SPR) are described for elucidating the molecular mechanisms of xenograft function and rejection.