Elektra R Amp;s

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Aron Eugine

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Aug 3, 2024, 2:42:38 PM8/3/24

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The immune system maintains a vast repertoire of B cells and T cells waiting to respond to microbial invasion. These quiescent, naive lymphocytes may be activated by antigen engagement and costimulation, triggering an exit from the G0 phase of the cell cycle, entry into active cell cycle and higher metabolism as cells both expand their populations and acquire effector functions. Published studies have demonstrated that lymphocyte quiescence, a state of reversible growth arrest in which cells remain responsive to activating stimuli and resistant to apoptosis (and are therefore not anergic), must be actively maintained by the action of molecules that include transcription factors and cell-cycle regulators1. DNA microarray experiments suggest that specific transcriptional programs are associated with the quiescent state2,3 and that cellular activation involves not only increased expression of genes that promote growth and differentiation but also suppression of a quiescent gene-expression program4,5. A growing number of known genes, including Foxo1, Foxo2, Foxo3 (ref. 6), Klf2 (ref. 7) and Tob8, have been linked to the regulation of the quiescence of cells of the immune response.

Here we describe an inherited immune deficiency characterized by susceptibility to both bacterial and viral infections, in which thymocytes developed normally but peripheral T cells died in response to activating or homeostatic expansion stimuli. Activation also triggered death of inflammatory monocytes. The majority of peripheral T cells had a semiactivated phenotype, and this population of cells specifically underwent apoptosis through the intrinsic apoptotic pathway. We positionally ascribed the cause of this immune deficiency to a mutation (elektra) in Slfn2, which demonstrates a role for Slfn2 in maintaining quiescence in cells of the immune response in vivo.

Signals from the TCR control the activity of several pathways, including those of the transcription factors NF-κB and NFAT and the mitogen-activated protein kinases Akt, Erk1/2, Jnk and p38, which together determine whether T cells will survive and proliferate or die through apoptosis12,13,14,15,16. We examined the activation status of each pathway in the first hour after TCR stimulation of wild-type or elektra-homozygous CD8+ T cells with beads coated with anti-CD3ε and anti-CD28. In elektra-homozygous cells, we found normal TCR-stimulated calcium influx (Supplementary Fig. 2a), dephosphorylation of NFAT (Supplementary Fig. 2b), nuclear translocation of NF-κB (Supplementary Fig. 2c) and phosphorylation of Erk and Akt (Supplementary Fig. 2d,e), which demonstrated intact signaling through these T cell activation and survival pathways during the initial response to TCR stimulation. T cells from elektra homozygotes also showed normal induction of the activation markers CD25 and CD69 at 24 h after TCR activation (Supplementary Fig. 2f). However, p38 and Jnk, which are activated through phosphorylation, were constitutively phosphorylated under basal conditions and were further phosphorylated after TCR stimulation in elektra-homozygous CD8+ T cells (Fig. 3e).

Collectively, the data reported above demonstrated that compared with wild-type cells, elektra-homozygous CD8+ T cells initiated responses to TCR activation normally, with a greater proportion of cells replicating DNA after 24 h of activation. By 48 h, most elektra cells had become apoptotic. These results suggest that the elektra phenotype entails both hypersensitivity to an activating stimulus and fragility after activation.

To further test the proliferative capacity of elektra-homozygous Tcells, we examined their response to homeostatic proliferation signals. We labeled wild-type and elektra-homozygous (Ly5.2+) splenocytes with the cytosolic dye CFSE and adoptively transferred them into sublethally irradiated wild-type (Ly5.1+) recipients. Wild-type T cells underwent proliferation as expected, but we detected no elektra-homozygous T cells in the spleen 7 d after infusion (Supplementary Fig. 3a). When we collected spleens 2 and 4 d after adoptive transfer, we found that an excess of the elektra-homozygous mutant cells were apoptotic, as indicated by annexin V staining (Fig. 4a). B cells homozygous for the elektra mutation were present in percentages similar to those of wild-type B cells in recipient mice (Supplementary Fig. 3b). To monitor the fate of existing peripheral T cells, we blocked their replenishment with new thymic emigrants by thymectomy of adult mice. Wild-type T cell numbers decreased by 30% after thymectomy and then remained stable for more than 60 d. In contrast, elektra-homozygous T cells decreased considerably within 12 d of thymectomy and almost completely disappeared from the blood by 60 d after thymectomy (Fig. 4b).

Homeostatic lymphoid cell death is required for the elimination of cells that are no longer needed once an infection is resolved and for the eliminatation of potentially autoreactive cells. It is mediated both by the intrinsic apoptotic signaling pathway (controlled by the balance of pro- and antiapoptotic Bcl-2 family members) and the extrinsic apoptotic signaling pathway (controlled by signals from receptors for tumor necrosis factor, the CD95 ligand and the proapoptotic molecule TRAIL). We examined the status of both pathways in elektra-homozygous mice. We excluded the Fas-mediated extrinsic pathway as the mechanism of cell death in elektra-homozygous Tcells because we observed no restoration of CD8+ or CD4+ T cells in double-mutant mice homozygous for both the elektra mutation and the lymphoproliferation mutation of Fas (Fig. 5a).

Our results have demonstrated that a mutation in Slfn2 caused immunodeficiency as a consequence of T cell and monocyte apoptosis secondary to a semiactivated phenotype. Many of our findings suggested that this semiactivated state in Slfn2eka/eka T cells stemmed from a loss of cellular quiescence. First, Slfn2eka/eka T cells had a higher percentage of cells replicating DNA both in the steady state and during the initial response to TCR stimulation, suggestive of a greater readiness to enter S phase. Second, unstimulated Slfn2eka/eka T cells had a unique phenotype in which naive (CD44lo) T cells had low expression of CD62L and IL-7Rα, and mature (CD44hi) T cells had no expression of CD62L, IL-7Rα or CD5 but had normal expression of CD69, indicative of a semiactivated phenotype.

The activation of several pathways, including the NF-κB, NFAT, Akt, Erk1/2, Jnk and p38 pathways, regulates T cell survival. In Slfn2eka/eka mice, we presume that as a result of a loss of quiescence, the balance among these pathways is altered to favor apoptosis rather than expansion. Indeed, we observed that p38 and Jnk were constitutively activated in unstimulated Slfn2eka/eka T cells. Activation of these kinases is associated with T cell apoptosis15,16 and may represent a factor contributing to the apoptosis of Slfn2eka/eka T cells. The enhancement of p38 and Jnk phosphorylation observed after TCR stimulation of Slfn2eka/eka T cells and the fact that inflammatory monocytes (of the myeloid lineage) also undergo apoptosis after activation together suggest that the proliferative signals negatively regulated by Slfn2 may be distinct from the TCR signaling pathways that lead to the development of activated T cell effector functions and full T cell activation.

We have shown that the elektra allele of Slfn2 confers susceptibility to two very different viral infections (LCMV and MCMV) in mice. The susceptibility to LCMV is probably related to impairment of CD8+ T cell function. Notably, the mutation had no effect on NK cell numbers or function, although these cells are known to be essential for defense against MCMV. We consider it possible that inflammatory monocytes fulfill an essential function as well, given that lymphocytes are not needed for survival during the first several weeks after inoculation with MCMV10. Indeed, monocyte and macrophage cells are suggested to be protective in influenza infection40, and inflammatory monocytes are essential for defense against L. monocytogenes infection28. A nonredundant role has been directly demonstrated for inflammatory monocytes in controlling viral infection41.

Several orthopoxviruses contain either full-length or fragmentary Slfn homologs. The full-length camelpox v-SLFN is most similar to mouse Slfn1 and Slfn2, with approximately 30% amino acid identity in the conserved region. A recombinant vaccinia virus strain expressing camelpox v-SLFN has been shown to be attenuated relative to the wild-type virus when mice are infected intranasally, resulting in less weight loss and more rapid recovery42. Viruses thus seem to have appropriated a Slfn sequence and may use it to manipulate the survival and proliferation of host cells of the immune response and, hence, the immunopathological consequences of infection.

Preparation of the MCMV stock (Smith strain) has been described9. Mice were infected with MCMV by intraperitoneal injection. The Armstrong strain of LCMV (provided by M.B.A. Oldstone) was injected intravenously. Viral loads were determined after organ homogenization in DMEM plus 3% (vol/vol) FCS by standard plaque assay on NIH3T3 fibroblast cells (for MCMV) and on Vero monkey kidney cells (for LCMV). For in vivo challenge with L. monocytogenes (strain 10403S; Xenogen), bioluminescent bacteria were prepared and then injected intravenously as described44.