Knocking 'em Dead: Pore-Forming Proteins in Immune Defense.

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain-like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

Cryo-EM structures of perforin-2 in isolation and assembled on a membrane suggest a mechanism for pore formation.

Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 ß-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

Radiotherapy transiently reduces the sensitivity of cancer cells to lymphocyte cytotoxicity.

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.

Extracellular CIRP induces CD4CD8αα intraepithelial lymphocyte cytotoxicity in sepsis.

BACKGROUND: In sepsis, intestinal barrier dysfunction is often caused by the uncontrolled death of intestinal epithelial cells (IECs). CD4CD8αα intraepithelial lymphocytes (IELs), a subtype of CD4+ T cells residing within the intestinal epithelium, exert cytotoxicity by producing granzyme B (GrB) and perforin (Prf). Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently identified alarmin which stimulates TLR4 on immune cells to induce proinflammatory responses. Here, we hypothesized that eCIRP enhances CD4CD8αα IEL cytotoxicity and induces IEC death in sepsis. METHODS: We subjected wild-type (WT) and CIRP-/- mice to sepsis by cecal ligation and puncture (CLP) and collected the small intestines to isolate IELs. The expression of GrB and Prf in CD4CD8αα IELs was assessed by flow cytometry. IELs isolated from WT and TLR4-/- mice were challenged with recombinant mouse CIRP (eCIRP) and assessed the expression of GrB and Prf in CD4CD8αα by flow cytometry. Organoid-derived IECs were co-cultured with eCIRP-treated CD4CD8αα cells in the presence/absence of GrB and Prf inhibitors and assessed IEC death by flow cytometry. RESULTS: We found a significant increase in the expression of GrB and Prf in CD4CD8αα IELs of septic mice compared to sham mice. We found that GrB and Prf levels in CD4CD8αα IELs were increased in the small intestines of WT septic mice, while CD4CD8αα IELs of CIRP-/- mice did not show an increase in those cytotoxic granules after sepsis. We found that eCIRP upregulated GrB and Prf in CD4CD8αα IELs isolated from WT mice but not from TLR4-/- mice. Furthermore, we also revealed that eCIRP-treated CD4CD8αα cells induced organoid-derived IEC death, which was mitigated by GrB and Prf inhibitors. Finally, histological analysis of septic mice revealed that CIRP-/- mice were protected from tissue injury and cell death in the small intestines compared to WT mice. CONCLUSION: In sepsis, the cytotoxicity initiated by the eCIRP/TLR4 axis in CD4CD8αα IELs is associated with intestinal epithelial cell (IEC) death, which could lead to gut injury.

Pore-forming aegerolysin and MACPF proteins in extremotolerant or extremophilic fungi.

Aegerolysin proteins are involved in various interactions by recognising a molecular receptor in the target organism. The formation of pores in combination with larger, non-aegerolysin-like protein partners (such as membrane attack complex/perforin proteins [MACPFs]) is one of the possible responses in the presumed competitive exclusion of other organisms from the ecological niche. Bicomponent pairs are already observed at the gene level. Fungi growing under extreme conditions can be divided into ubiquitous and extremotolerant generalists which can compete with mesophilic species and rare, isolated extremophilic and extremotolerant specialists with narrow ecological amplitude that cannot compete. Under extreme conditions, there are fewer competitors, so fungal specialists generally produce less diverse and complicated profiles of specialised molecules. Since extremotolerant and extremophilic fungi have evolved in numerous branches of the fungal tree of life and aegerolysins are unevenly distributed across fungal genomes, we investigated whether aegerolysins, together with their partner proteins, contribute to the extreme survival ecology of generalists and specialists. We compiled a list of 109 thermo-, psihro-, acido-, alkali-, halo-, metallo- and polyextremo-tolerant/-philic fungal species. Several challenges were identified that affected the outcome: renaming fungal species, defining extremotolerant/extremophilic traits, identifying extremotolerant/extremophilic traits as metadata in databases and linking fungal isolates to fungal genomes. The yield of genomes coding aegerolysins or MACPFs appears to be lower in extremotolerant/extremophilic fungi compared to all fungal genomes. No candidates for pore-forming gene pairs were identified in the genomes of extremophilic fungi. Aegerolysin and MACPFs partner pairs were identified in only two of 69 species with sequenced genomes, namely in the ubiquitous metallotolerant generalists Aspergillus niger and A. foetidus. These results support the hypothesised role of these pore-forming proteins in competitive exclusion.

Concomitant assessment of PD-1 and CD56 expression identifies subsets of resting cord blood Vδ2 T cells with disparate cytotoxic potential.

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.

Immunohistochemical expression of perforin in adult systemic lupus erythematosus associated macrophage activation syndrome: Clinicohematological correlation and literature review.

OBJECTIVE: To study the bone marrow (BM) immunohistomorphological characteristics in adult systemic lupus erythematosus (SLE) associated macrophage activation syndrome (SLE-MAS). MATERIALS AND METHODS: Immunohistochemical (IHC) expression of CD3, CD8, perforin (PFN), and CD163 was studied on BM trephine biopsies from 30 cytopenic adult SLE cases (male: female = 1:5, age; 24 years, range; 19-32) and compared them with ten age matched controls. Clinicopathological parameters were compared among the cases likely (L) or unlikely (U) to have MAS using probability scoring criteria. The best cut off laboratory parameters to discriminate between the two were obtained through receiver operator curve (ROC) analysis. RESULTS: MAS occurred in 12/30 (40%) cases and was more commonly associated with prior immunosuppressive therapy (p = .07), ≥ 3 system involvement (p = .09), lower fibrinogen (p < .01), increased triglyceride (p = .002), increased BM hemophagocytosis (p = .002), and higher MAS score [185 (176-203) vs. 105 (77-119), p < .01] than MAS-U subgroup. Although PFN+CD8+ T lymphocytes significantly decreased among cases than controls (p < .05), it was comparable between MAS-L and MAS-U subgroups. Fibrinogen (< 2.4 g/L, AUC; 0.93, p < .01), hemophagocytosis score (> 1.5, AUC; 0.71, p = .03), and an MAS probability score of ≥ 164 (AUC; 1, p < .01) discriminated MAS from those without MAS. CONCLUSION: We noted a decrease in perforin mediated CD8 + T cell cytotoxicity in SLE. Immunohistochemical demonstration of the same along with histiocytic hemophagocytosis on BM biopsy may be useful adjunct in early diagnosis and management of MAS in SLE.

CD8+ T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease.

BACKGROUND: CD4+ (cluster of differentation) and CD8+ T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown. METHODS: We describe how CD8+ T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors. RESULTS: In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4+ and CD8+ T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy. Interestingly, the depletion of CD8+ T cells but not CD4+ T cells reduced retinal neovascularization and vascular leakage. Using reporter mice expressing gfp (green fluorescence protein) in CD8+ T cells, these cells were localized near neovascular tufts in the retina, confirming that CD8+ T cells contribute to the disease. Furthermore, the adoptive transfer of CD8+ T cells deficient in TNF (tumor necrosis factor), IFNγ (interferon gamma), Prf (perforin), or GzmA/B (granzymes A/B) into immunocompetent Rag1-/- mice revealed that CD8+ T cells mediate retinal vascular disease via these factors, with TNF influencing all aspects of vascular pathology. The pathway by which CD8+ T cells migrate into the retina was identified as CXCR3 (C-X-C motif chemokine receptor 3) with the CXCR3 blockade reducing the number of CD8+ T cells within the retina and retinal vascular disease. CONCLUSIONS: We discovered that CXCR3 is central to the migration of CD8+ T cells into the retina as the CXCR3 blockade reduced the number of CD8+ T cells within the retina and vasculopathy. This research identified an unappreciated role for CD8+ T cells in retinal inflammation and vascular disease. Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies.

Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.