Mesenchymal stem cells (MSCs) are recognized as potential treatments for multiple degenerative and inflammatory disorders as a number of animal and human studies have indicated their therapeutic effects. There are also several clinically approved medicinal products that are manufactured using these cells. For such large-scale manufacturing requirements, the in vitro expansion of harvested MSCs is essential. Multiple subculturing of MSCs, however, provokes cellular senescence processes which is known to deteriorate the therapeutic efficacy of the cells. Strategies to rejuvenate or selectively remove senescent MSCs are therefore highly desirable for fostering future clinical applications of these cells. In this present study, we investigated gene expression changes related to cellular senescence of MSCs derived from umbilical cord blood and found that CD26, also known as DPP4, is significantly upregulated upon cellular aging. We further observed that the inhibition of CD26 by genetic or pharmacologic means delayed the cellular aging of MSCs with their multiple passaging in culture. Moreover, the sorting and exclusion of CD26-positive MSCs from heterogenous cell population enhanced in vitro cell attachment and reduced senescence-associated cytokine secretion. CD26-negative MSCs also showed superior therapeutic efficacy in mouse lung emphysema model. Our present results collectively suggest CD26 is a potential novel target for the rejuvenation of senescent MSCs for their use in manufacturing MSC-based applications.
Heterologous ChAdOx1-BNT162b2 vaccination induces a stronger immune response than two doses of BNT162b2 or ChAdOx1. Yet, the molecular transcriptome, the germline allelic variants of immunoglobulin loci and anti-Omicron antibody levels induced by the heterologous vaccination have not been formally investigated. Moreover, there is a paucity of COVID vaccine studies including diverse genetic populations. Here, we show a robust molecular immune transcriptome and antibody repertoire in 51 office workers from the Republic of Korea after a heterologous ChAdOx1-BNT162b2 vaccination or a homologous ChAdOx1-ChAdOx1 vaccination. Anti-spike-specific IgG antibody levels in the heterologous group increased from 14,000 U/ml to 142,000 AU/ml within eight days after the BNT162b2 vaccination. In contrast, antibody levels in the homologous group increased two-fold after the second ChAdOx1 dose. Antibody titers against the Omicron spike protein as compared to the ancestral strain were reduced to a lesser extent in the heterologous group. RNA-seq conducted on immune cells demonstrated a stronger activation of interferon-induced genetic programs in the heterologous cohort. An increase of specific IGHV clonal transcripts encoding neutralizing antibodies was preferentially detected in the heterologous cohort. Enrichment of B cell and CD4+ T cell responses were observed following both heterologous and homologous vaccination using scRNA-seq, but clonally expanded memory B cells were relatively stronger in the ChAdOx1-BNT162b2 cohort. In summary, a heterologous vaccination with ChAdOx1 followed by BNT162b2 provides an innate and adaptive immune response exceeding that seen in homologous ChAdOx1 vaccinations but equivalent to that seen in homologous BNT162b2 vaccination.
Background MSCs have long been thought to be immune-privileged with low levels of major histocompatibility complex (MHC) class I and rare expression of MHC class II. However, growing evidence indicates that these cells may not actually be hypoimmunogenic, particularly when exposed to cytokines such as IFN-γ. IFN-γ primed increase of MHC class I expression can promote the rejection of allogenic MSCs in the host recipient. A strategy to overcome this drawback is urgently required. Methods We knocked out β2-microglobulin (B2M) in MSCs, which is a component of MHC class I, using the ribonucleoprotein (RNP)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) system. The expression of MSC surface markers, MHC class I, and B2M was assayed by flow cytometry and western blotting. Upon co-culture of MSCs with CD4+ and CD8+ T cells, the survival and proliferation of both cell types were examined by cell counting kit (CCK-8) and carboxy fluorescein succinimidyl ester (CFSE), respectively. The levels of immunomodulatory molecules in MSCs were evaluated by both enzyme-linked immunosorbent assay (ELISA) and western blotting. Results B2M-knockout MSCs expressed low levels of MHC class I even upon IFN-γ priming, but maintained their native properties as evidenced by the expression of specific surface markers. CD8+ T cell proliferation was also far less stimulated by B2M-knockout MSCs than by control cells. Under these conditions, B2M-knockout MSCs had a significantly longer survival duration (> 2.4 fold) than did control cells. B2M-knockout MSCs showed significantly elevated levels of immune-modulatory molecules including indoleamine 2, 3-dioxygenase 1 (IDO-1), prostaglandin E2 (PGE2), C-C motif chemokine ligand 2 (CCL-2), and interleukin-6 (IL-6); conversely, B2M-knockout MSCs produced significantly lower levels of proinflammatory molecules (e.g., IL-1b, CXCL10) compared with control cells. Conclusion The loss of B2M in MSCs potentiated the immunomodulatory effects of IFN-γ priming while mitigating its potential inflammatory effects. B2M-knockout MSCs are a potentially promising treatment for immune-related inflammatory diseases.
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