Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.     

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs

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Comparison of whole-cell protein electrophoretic profiles of Haemophilus influenzae: implementation of a microcomputer mainframe linked system and description of a new similarity coefficient

A microcomputer mainframe linked system is described which allows video camera data capture and storage of one-dimensional whole-cell protein electrophoresis gel images, processing of normalized traces to produce a similarity matrix, and analysis of the matrix using the commercial cluster analysis program CLUSTAN. A new similarity coefficient is introduced which takes into account both band position and intensity. Forty-five strains of Haemophilus influenzae, including the eight biotypes and six serotypes, were analyzed using this system. Results demonstrated groupings which are consistent with known genetic relationships.     

Structure and expression of mouse alpha 1-acid glycoprotein gene-3 (AGP-3).

The genome of Mus domesticus has multiple genes of the alpha 1-acid glycoprotein (AGP). Two cDNA clones were identified corresponding to AGP-1 and AGP-2. Moreover, two alleles of AGP-1 exist in inbred mice. The genomic DNA of the AGP-2 gene has been cloned and studied. Here we report the genomic organization of three M. domesticus AGP genes, the sequence analysis of the AGP-3 genomic DNA, and the expression of the AGP-3 gene. The major structural differences between AGP-2 and AGP-3 genes are located in introns 1 and 5. The low level of AGP-3 mRNA can be detected by the polymerase chain reaction (PCR). The molecular basis of the low level expression of AGP-3 and the possible classification of AGP-3 as a pseudogene are discussed.     

Intra- and inter-specific effects of mast seeding on seed fates of two sympatric <Emphasis Type="Italic">Corylus</Emphasis> species

We released seeds of two sympatric tree species, Corylus mandshurica (seed with thinner seed hull, higher nutrition) and C. heterophylla (seeds with thicker seed hull, lower nutrition) in the masting year of C. mandshurica in 2008, and C. heterophylla in 2009, respectively, to investigate how seed masting of the two sympatric Corylus species affects seed removal and dispersal fitness of the two species differently at both intra- and inter-specific levels. At intra-specific level, the authors found mast seeding of both C. mandshurica and C. heterophylla significantly reduced seed removal, seed consumption, but increased seed dispersal distance and seed dispersal fitness of the released seeds. Mast seeding of C. mandshurica increased seed caching of C. mandshurica. At inter-specific level, the authors found mast seeding of C. mandshurica reduced seed removal of C. heterophylla, but mast seeding of C. heterophylla did not significantly reduce seed removal of C. mandshurica. Mast seeding of C. mandshurica reduced seed consumption of C. heterophylla, while mast seeding of C. heterophylla reduced seed consumption of C. mandshurica. We found mast seeding of C. mandshurica significantly reduced seed dispersal distance of C. heterophylla, while mast seeding of C. heterophylla significantly increased seed dispersal distance of C. mandshurica. We found that mast seeding of C. mandshurica significantly increased seed dispersal fitness of C. heterophylla, while mast seeding of C. heterophylla did not significantly increase seed dispersal fitness of C. mandshurica. More studies are needed to reveal the ecological consequences of mast seeding at inter-specific or community-level. Seed traits may attribute the differences of mast seeding at inter-specific level. Because seeds with thinner seed hull and higher nutrition were more harvested and eaten by rodents, mast seeding of C. mandshurica might have reduced seed removal and seed consumption, but increased dispersal fitness of C. heterophylla (seeds with thicker seed hull, lower nutrition). Therefore, synchrony among species is, or is not, selectively beneficial to the focus species depends on seed traits which determine gains from mast seeding at inter-specific level.     

Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFα is an apoptotic inducer in some cancer cells, it activates NFκB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFκB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFα, thus converting TNFα from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTα, FasL, and TRAIL. Mechanistically, CCNs function through integrin α₆β₁ and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding α₆β₁-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an α₆β₁-HSPG-binding defective CCN1 are blunted in TNFα- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFα and related cytokines.     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.