Developing effective strategies to prevent or treat COVID-19 requires understanding the natural immune response to SARS-CoV-2. We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8 + T cells of COVID-19 patients. In total, we identified 3–8 epitopes for each of the six most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8 + T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8 + T cell immunity to SARS-CoV-2.

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Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3β, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.

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Protein phosphatase 2A (PP2A) composed of a scaffold subunit, a catalytic subunit, and multiple regulatory subunits is a ubiquitously expressed serine/threonine phosphatase. We have previously shown that the PP2A catalytic subunit is increased in T cells from patients with systemic lupus erythematosus and promotes IL-17 production by enhancing the activity of Rho-associated kinase (ROCK) in T cells. However, the molecular mechanism whereby PP2A regulates ROCK activity is unknown. In this study, we show that the PP2A regulatory subunit PPP2R2A is increased in T cells from people with systemic lupus erythematosus and binds to, dephosphorylates, and activates the guanine nucleotide exchange factor GEF-H1 at Ser885, which in turn increases the levels of RhoA-GTP and the activity of ROCK in T cells. Genetic PPP2R2A deficiency in murine T cells reduced Th1 and Th17, but not regulatory T cell differentiation and mice with T cell–specific PPP2R2A deficiency displayed less autoimmunity when immunized with myelin oligodendrocyte glycoprotein peptide. Our studies indicate that PPP2R2A is the regulatory subunit that dictates the PP2A-directed enhanced Th1 and Th17 differentiation, and therefore, it represents a therapeutic target for pathologies linked to Th1 and Th17 cell expansion.

Calcium calmodulin kinase IV (CaMK4) regulates multiple processes that significantly contribute to the lupus-related pathology by controlling the production of IL-2 and IL-17 by T cells, the proliferation of mesangial cells, and the function and structure of podocytes. CaMK4 is also upregulated in podocytes from patients with focal segmental glomerulosclerosis (FSGS). In both immune and non-immune podocytopathies, CaMK4 disrupts the structure and function of podocytes. In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to CD4+ T cells suppresses both autoimmunity and the development of nephritis. Targeted delivery though to podocytes averts the deposition of immune complexes without affecting autoimmunity in lupus-prone mice and averts pathology induced by adriamycin in normal mice. Therefore, targeted delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex pathogenesis. The expression of autoimmunity and organ inflammation involves altered production of cytokines by T cells (1-3). Specifically, decreased IL-2 production may account for the reported decreased cytotoxic T cell function and the decreased function and numbers of Tregs (2), whereas increased production of IFN-γ and IL-17 may contribute directly to organ inflammation (4, 5). Treatment with low doses of IL-2 diminishes renal inflammation and the rate of kidney-infiltrating CD4 + T cells in murine lupus nephritis (6). Low-dose IL-2 in the treatment of autoimmune diseases including SLE, rheumatoid arthritis, and multiple sclerosis is in clinical trials (7). IL-2 is a pleiotropic cytokine produced by effector T (T eff) cells after antigen activation and is at the crossroads of effector responses, tolerance, and immunotherapy (8). IL-2 transcription is mediated by multiple transcription factors (TFs), including activator protein-1 (AP-1, FOS-JUN family dimers), cAMP responsive element binding protein (CREB), nuclear factor of activated T cells (NFAT) family proteins, NF-κB, ETS-related TF (Elf1), and GA binding protein α chain (GABPA) (9). Tregs have abundant expression of the IL-2 receptor α chain (CD25) but are unable to produce IL-2. IL-2 binds with low affinity to CD25 or to heterodimers of the common γ chain (CD132) and IL-2Rβ (CD122), but receptor affinity increases about 1000-fold when these subunits collectively interact with IL-2 (10). Capture of IL-2 by Tregs is critical for their suppressor function and for limiting the activation of T cells (11). Protein phosphatase 2A (PP2A) is a ubiquitously expressed and highly conserved serine/threonine phosphatase that is important in multiple cellular processes, including cell division, cytoskeletal dynamics, and various signaling pathways (12). The PP2A core enzyme consists of the scaffold subunit A (PP2A A) and the catalytic subunit C (PP2A C). To form a functional holoenzyme, the core enzyme interacts with one of the many regulatory subunits (PP2A B), which define substrate and tissue specificity (13). The regulatory subunits are classified into 4 major families, identified as B (e.g.

To address the problem of poor asthma control due to drug resistance, an antisense oligonucleotide complementary to mmu-miR-145a-5p (antimiR-145) was tested in a house dust mite mouse model of mild/moderate asthma. miR-145 was targeted to reduce inflammation, regulate epithelial-mesenchymal transitions, and promote differentiation of structural cells. In addition, several chemical variations of a nontargeting oligonucleotide were tested to define sequence-dependent effects of the miRNA antagonist. After intravenous administration, oligonucleotides complexed with a pegylated cationic lipid nanoparticle distributed to most cells in the lung parenchyma but were not present in smooth muscle or the mucosal epithelium of the upper airways. Treatment with antimiR-145 and a nontargeting oligonucleotide both reduced eosinophilia, reduced obstructive airway remodeling, reduced mucosal metaplasia, and reduced CD68 immunoreactivity. Poly(A) RNA-seq verified that antimiR-145 increased levels of many miR-145 target transcripts. Genes upregulated in human asthma and the mouse model of asthma were downregulated by oligonucleotide treatments. However, both oligonucleotides significantly upregulated many genes of interferon signaling pathways. These results establish effective lung delivery and efficacy of locked nucleic acid/DNA oligonucleotides administered intravenously, and suggest that some of the beneficial effects of oligonucleotide therapy of lung inflammation may be due to normalization of interferon response pathways.