A ubiquitous bone marrow reservoir of preexisting SARS-CoV-2-reactive memory CD4+ T lymphocytes in unexposed individuals.

Circulating, blood-borne SARS-CoV-2-reactive memory T cells in persons so far unexposed to SARS-CoV-2 or the vaccines have been described in 20-100% of the adult population. They are credited with determining the efficacy of the immune response in COVID-19. Here, we demonstrate the presence of preexisting memory CD4+ T cells reacting to peptides of the spike, membrane, or nucleocapsid proteins of SARS-CoV-2 in the bone marrow of all 17 persons investigated that had previously not been exposed to SARS-CoV-2 or one of the vaccines targeting it, with only 15 of these persons also having such cells detectable circulating in the blood. The preexisting SARS-CoV-2-reactive memory CD4+ T cells of the bone marrow are abundant and polyfunctional, with the phenotype of central memory T cells. They are tissue-resident, at least in those persons who do not have such cells in the blood, and about 30% of them express CD69. Bone marrow resident SARS-CoV-2-reactive memory CD4+ memory T cells are also abundant in vaccinated persons analyzed 10-168 days after 1°-4° vaccination. Apart from securing the bone marrow, preexisting cross-reactive memory CD4+ T cells may play an important role in shaping the systemic immune response to SARS-CoV-2 and the vaccines, and contribute essentially to the rapid establishment of long-lasting immunity provided by memory plasma cells, already upon primary infection.

Rapid Isolation of Functional ex vivo Human Skin Tissue-Resident Memory T Lymphocytes.

Studies in animal models have shown that skin tissue-resident memory T (TRM) cells provide enhanced and immediate effector function at the site of infection. However, analyses of skin TRM cells in humans have been hindered by the lack of an optimized isolation protocol. Here, we present a combinatorial strategy-the 6-h collagenase IV digestion and gentle tissue dissociation - for rapid and efficient isolation of skin TRM cells with skin tissue-specific immune features. In comparison with paired blood circulating memory T cells, these ex vivo isolated skin T cells express typical TRM cell markers and display higher polyfunctional properties. Moreover, these isolated cells can also be assessed for longer periods of time in ex vivo cultures. Thus, the optimized isolation protocol provides a valuable tool for further understanding of human skin TRM cells, especially for direct comparison with peripheral blood T cells at the same sample collection time.

Inhibitory effect and mechanism of 1,25-dihydroxy vitamin D3 on RANKL expression in fibroblast-like synoviocytes and osteoclast-like cell formation induced by IL-22 in rheumatoid arthritis.

OBJECTIVES: To explore the inhibitory effect and mechanism of 1,25-dihydroxy vitamin D3 (l,25(OH)2D3) on receptor activator of nuclear factor-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLSs) and osteoclastogenesis induced by interleukin (IL)-22 in patients with rheumatoid arthritis (RA). METHODS: Fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLSs) were cultured and stimulated for RANKL expression with IL-22 in the absence or presence of various concentrations of l,25(OH)2D3, and JAK-2 inhibitor or p38 MAPK inhibitor at the optimised time point of IL-22 treatment. The level of RANKL messenger RNA (mRNA) or protein was measured using real-time polymerase chain reaction (RT-PCR) or western blot method. To assess the impact of l,25(OH)2D3 on osteoclastogenesis, isolated monocytes were activated by M-CSF and RANKL or cocultured with FLSs stimulated by IL-22 in the presence or absence of l,25(OH)2D3 and those inhibitors. TRAP-positive cells as differentiated osteoclasts were stained for alkaline phosphatase. RESULTS: FLSs stimulated with IL-22 for 72 hours were used in further experiment because of the highest expression of RANKL at this time point. The expression of RANKL mRNA and protein in IL-22-stimulated FLSs were significantly inhibited by 1 nM of 1,25(OH)2D3 (p<0.05). Interestingly, this inhibition was reversed by inhibitor of JAK-2/STAT-3 or p38 MAPK/NF-κB signalling. In monocytes cocultured with IL-22-stimulated FLSs in the presence of exogenous RANKL and M-CSF, 1,25(OH)2D3 could block the process of osteoclastogenesis by JAK-2/STAT-3 or p38 MAPK/NF-κB signalling. CONCLUSIONS: 1,25(OH)2D3 may exert inhibitory effect on osteoclastogenesis of RA-FLSs by down-regulating RANKL expression, which could be mediated by IL-22 through JAK-2/STAT-3 and p38 MAPK/NF-κB signalling.