A large-scale type I CBASS antiphage screen identifies the phage prohead protease as a key determinant of immune activation and evasion.

Cyclic oligonucleotide-based signaling system (CBASS) is an antiviral system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by the recognition of viral DNA, but the molecular cues activating CBASS are incompletely understood. Using a screen of 975 type I CBASS operon-phage challenges, we show that operons with distinct cGAS/DncV-like nucleotidyltransferases (CD-NTases) and CD-NTase-associated protein (Cap) effectors exhibit marked patterns of phage restriction. We find that some type I CD-NTase enzymes require a C-terminal AGS-C immunoglobulin (Ig)-like fold domain for defense against select phages. Escaper phages evade CBASS via protein-coding mutations in virion assembly proteins, and acquired resistance is largely operon specific. We demonstrate that the phage Bas13 prohead protease interacts with the CD-NTase EcCdnD12 and can induce CBASS-dependent growth arrest in cells. Our results define phage virion assembly as a determinant of type I CBASS immune evasion and support viral protein recognition as a putative mechanism of cGAS-like enzyme activation.

Phages overcome bacterial immunity via diverse anti-defence proteins.

It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems1-4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.

Bacterial SEAL domains undergo autoproteolysis and function in regulated intramembrane proteolysis.

Gram-positive bacteria use SigI/RsgI-family sigma factor/anti-sigma factor pairs to sense and respond to cell wall defects and plant polysaccharides. In Bacillus subtilis, this signal transduction pathway involves regulated intramembrane proteolysis (RIP) of the membrane-anchored anti-sigma factor RsgI. However, unlike most RIP signaling pathways, site-1 cleavage of RsgI on the extracytoplasmic side of the membrane is constitutive and the cleavage products remain stably associated, preventing intramembrane proteolysis. The regulated step in this pathway is their dissociation, which is hypothesized to involve mechanical force. Release of the ectodomain enables intramembrane cleavage by the RasP site-2 protease and activation of SigI. The constitutive site-1 protease has not been identified for any RsgI homolog. Here, we report that RsgI's extracytoplasmic domain has structural and functional similarities to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. We show that site-1 proteolysis in B. subtilis and Clostridial RsgI family members is mediated by enzyme-independent autoproteolysis of these SEA-like domains. Importantly, the site of proteolysis enables retention of the ectodomain through an undisrupted ß-sheet that spans the two cleavage products. Autoproteolysis can be abrogated by relief of conformational strain in the scissile loop, in a mechanism analogous to eukaryotic SEA domains. Collectively, our data support the model that RsgI-SigI signaling is mediated by mechanotransduction in a manner that has striking parallels with eukaryotic mechanotransducive signaling pathways.

Bacterial SEAL domains undergo autoproteolysis and function in regulated intramembrane proteolysis.

Gram-positive bacteria use SigI/RsgI-family sigma factor/anti-sigma factor pairs to sense and respond to cell wall defects and plant polysaccharides. In Bacillus subtilis this signal transduction pathway involves regulated intramembrane proteolysis (RIP) of the membrane-anchored anti-sigma factor RsgI. However, unlike most RIP signaling pathways, site-1 cleavage of RsgI on the extracytoplasmic side of the membrane is constitutive and the cleavage products remain stably associated, preventing intramembrane proteolysis. The regulated step in this pathway is their dissociation, which is hypothesized to involve mechanical force. Release of the ectodomain enables intramembrane cleavage by the RasP site-2 protease and activation of SigI. The constitutive site-1 protease has not been identified for any RsgI homolog. Here, we report that RsgI's extracytoplasmic domain has structural and functional similarities to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. We show that site-1 proteolysis in B. subtilis and Clostridial RsgI family members is mediated by enzyme-independent autoproteolysis of these SEA-like (SEAL) domains. Importantly, the site of proteolysis enables retention of the ectodomain through an undisrupted ß-sheet that spans the two cleavage products. Autoproteolysis can be abrogated by relief of conformational strain in the scissile loop, in a mechanism analogous to eukaryotic SEA domains. Collectively, our data support the model that RsgI-SigI signaling is mediated by mechanotransduction in a manner that has striking parallels with eukaryotic mechanotransducive signaling pathways. SIGNIFICANCE: SEA domains are broadly conserved among eukaryotes but absent in bacteria. They are present on diverse membrane-anchored proteins some of which have been implicated in mechanotransducive signaling pathways. Many of these domains have been found to undergo autoproteolysis and remain noncovalently associated following cleavage. Their dissociation requires mechanical force. Here, we identify a family of bacterial SEA-like (SEAL) domains that arose independently from their eukaryotic counterparts but have structural and functional similarities. We show these SEAL domains autocleave and the cleavage products remain stably associated. Importantly, these domains are present on membrane-anchored anti-sigma factors that have been implicated in mechanotransduction pathways analogous to those in eukaryotes. Our findings suggest that bacterial and eukaryotic signaling systems have evolved a similar mechanism to transduce mechanical stimuli across the lipid bilayer.

T cell receptor signaling strength establishes the chemotactic properties of effector CD8+ T cells that control tissue-residency.

Tissue-resident memory (TRM) CD8+ T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation within tissue microenvironments remain unresolved. Here, using an IFNγ-YFP reporter system to identify CD8+ T cells executing antigen-dependent effector functions, we define the transcriptional consequences and functional mechanisms controlled by TCR-signaling strength that occur within the skin during viral infection to promote TRM differentiation. TCR-signaling both enhances CXCR6-mediated migration and suppresses migration toward sphingosine-1-phosphate, indicating the programming of a 'chemotactic switch' following secondary antigen encounter within non-lymphoid tissues. Blimp1 was identified as the critical target of TCR re-stimulation that is necessary to establish this chemotactic switch and for TRM differentiation to efficiently occur. Collectively, our findings show that access to antigen presentation and strength of TCR-signaling required for Blimp1 expression establishes the chemotactic properties of effector CD8+ T cells to promote residency within non-lymphoid tissues.

Convergent mutations in phage virion assembly proteins enable evasion of Type I CBASS immunity.

CBASS is an anti-phage defense system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by viral DNA but the stage of phage replication which activates bacterial CBASS remains unclear. Here we define the specificity of Type I CBASS immunity using a comprehensive analysis of 975 operon-phage pairings and show that Type I CBASS operons composed of distinct CD-NTases, and Cap effectors exhibit striking patterns of defense against dsDNA phages across five diverse viral families. We demonstrate that escaper phages evade CBASS immunity by acquiring mutations in structural genes encoding the prohead protease, capsid, and tail fiber proteins. Acquired CBASS resistance is highly operon-specific and typically does not affect overall fitness. However, we observe that some resistance mutations drastically alter phage infection kinetics. Our results define late-stage virus assembly as a critical determinant of CBASS immune activation and evasion by phages.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.

Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity.

The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3-10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.

Endophthalmitis: a bibliographic review.

PURPOSE: To analyze the 100 most cited articles pertaining to endophthalmitis using bibliometric analysis. METHODS: An all-time Web of Science literature search and refined to peer-reviewed articles in the field of ophthalmology with the keyword "endophthalmitis" was completed. Total citation count of articles published pertaining to endophthalmitis, topic, incidence rate, publishing journals, year published, language, country of origin, number of authors, names of the first and last authors, study type, and number of patients/eyes studied. RESULTS: The top 100 most cited articles pertaining to endophthalmitis had a mean citation count of 362.92, with a range of 175 to 3583. They were published in 20 peer-reviewed journals, with Ophthalmology publishing the most (n = 42). Thirteen different countries were represented, with the majority (n = 77) originating from the USA. The most common study type was clinical experiences (n = 52), though eight of the top ten were clinical trials. The number of patients varied widely, represented by a mean of 9680, but with a median of only 229. The majority (n = 67) examined the incidence of endophthalmitis which included 24 articles after anti-vascular endothelial growth factor injections, 18 after ocular surgeries/procedures, 15 after intraocular steroid injections, eight after chemotoxic drug use, and three after ocular injuries. CONCLUSION: This bibliographic study serves as a unique historical analysis of the top 100 cited scholarly articles pertaining to endophthalmitis with many of the articles related to post-procedural endophthalmitis.

POSTERIOR SEGMENT INJURIES IN OPERATION IRAQI FREEDOM AND OPERATION ENDURING FREEDOM: 2001 to 2011.

BACKGROUND/PURPOSE: To characterize the nature of posterior segment ocular injuries in combat trauma. METHODS: Eyes in the Walter Reed Ocular Trauma Database were evaluated for the presence of posterior segment injury. Final visual outcomes in open-globe versus closed-globe injuries and by zone of injury and the types of posterior segment injuries in open-globe versus closed-globe injuries were assessed. RESULTS: Four hundred fifty-two of 890 eyes (50.8%) had at least one posterior segment injury. The mechanism of injury was most commonly an improvised explosive device in 280 (62.0%) eyes. Sixty-one patients (13.5%) had a Zone I injury, 50 (11.1%) a Zone II injury, and 341 (75.4%) a Zone III injury. Patients with Zone I injuries were more likely to have a final visual acuity of 20/200 or better compared with patients with either a Zone II (P < 0.001) or Zone III injury (P = 0.007). Eyes with a closed-globe injury were more likely to have a final visual acuity of 20/200 or better compared with those with an open-globe injury (P < 0.001). Furthermore, closed-globe injury compared with open-globe injury had a lower risk of vitreous hemorrhage (odds ratio 0.32, P < 0.001), proliferative vitreoretinopathy (odds ratio 0.14, P < 0.001), and retinal detachment (odds ratio 0.18, P < 0.001) but a higher risk of chorioretinal rupture (odds ratio 2.82, P < 0.001) and macular hole (odds ratio 3.46, P = 0.004). CONCLUSION: Patients with combat ophthalmic trauma had similar posterior segment injury patterns to civilian trauma in open-globe versus closed-globe injuries. Zone II and III injuries were associated with a worse visual prognosis.

Transcriptional regulator-induced phenotype screen reveals drug potentiators in Mycobacterium tuberculosis.

Transposon-based strategies provide a powerful and unbiased way to study the bacterial stress response1-8, but these approaches cannot fully capture the complexities of network-based behaviour. Here, we present a network-based genetic screening approach: the transcriptional regulator-induced phenotype (TRIP) screen, which we used to identify previously uncharacterized network adaptations of Mycobacterium tuberculosis to the first-line anti-tuberculosis drug isoniazid (INH). We found regulators that alter INH susceptibility when induced, several of which could not be identified by standard gene disruption approaches. We then focused on a specific regulator, mce3R, which potentiated INH activity when induced. We compared mce3R-regulated genes with baseline INH transcriptional responses and implicated the gene ctpD (Rv1469) as a putative INH effector. Evaluating a ctpD disruption mutant demonstrated a previously unknown role for this gene in INH susceptibility. Integrating TRIP screening with network information can uncover sophisticated molecular response programs.

The Association Between Obstructive Sleep Apnea and Cotton-Wool Spots in Nonproliferative Diabetic Retinopathy.

Purpose: This work reports the association of obstructive sleep apnea (OSA) and cotton-wool spots (CWS) seen in patients with nonproliferative diabetic retinopathy (DR). Methods: A random sample of patients diagnosed with DR between January 1, 2015 and December 31, 2018, were selected from medical-billing codes. Dilated funduscopic examination findings and medical history were analyzed by reviewing medical records. Results: CWS were present in 12 of 118 patients without OSA, compared with 11 of 32 patients with OSA (10.2% vs 34.4%, respectively; P = .002). OSA was more common in men (68.8%, P = .03) and associated with a higher body mass index (30.0 ± 5.0 without OSA vs 33.6 ± 5.5 with OSA, P < .001). When comparing those with and without OSA, there was no association with age; glycated hemoglobin A1c; stage of DR; insulin dependence; presence of diabetic macular edema; smoking status; or a history of hypertension, hyperlipidemia, cardiovascular disease, or other breathing disorder. Conclusions: The presence of OSA is associated with CWS in patients with DR, as well as male sex and a higher body mass index. Further research is needed to determine the ophthalmologist's role in the timely referral of patients with CWS for OSA evaluation.

A Comparison of Radial and Horizontal Macular Optical Coherence Tomography on Detection of Macular Pathology: A Prospective Study at an Academic Institution.

Purpose: Optical coherence tomography (OCT) is useful in diagnosing and monitoring retinal pathology such as age-related macular degeneration, diabetic macular edema (DME), central serous chorioretinopathy, and epiretinal membrane, among others. This study compared the ability of horizontal (H) 25-, 13-, and 7-cut macular OCT vs 24-, 12-, and 6-cut radial (R) macular OCT in identifying various macular pathology. Methods: This was a prospective study of 161 established patients evaluated at Wilford Hall Eye Center Retina Clinic between September and October of 2019. Pathology included age-related macular degeneration, central serous chorioretinopathy, DME, and epiretinal membrane, among others. Patients obtained 25-, 13-, and 7-cut H raster OCT as well as 24-, 12-, and 6-cut R OCT. Primary outcomes were sensitivity in detecting macular fluid and each macular abnormality. Results: The 24-cut radial (R24) OCT equally or out-performed the H25 (horizontal 25-cut OCT) in detecting macular fluid across all pathological groups. Generally, a higher number of cuts correlated with better detection of fluid. In detecting any macular abnormalities, H25, R24, and R12 had 100% sensitivity. R6 OCT had near 100% sensitivity across all groups, except for DME (95%). Overall, R OCT had better sensitivity (0.960) than H OCT (0.907) in detecting macular pathology. Conclusions: R outperformed H macular OCT in detecting fluid and other abnormalities. Clinically, both scanning patterns can be used by ophthalmologists in diagnosis and management of commonly encountered macular diseases. Technicians may be able to use a variety of these scans to screen for pathology prior to physician evaluation.

A History of Anti-VEGF Inhibitors in the Ophthalmic Literature: A Bibliographic Review.

Purpose: This work aimed to analyze the 100 most-cited articles on antivascular endothelial growth factor (anti-VEGF) inhibitors. Methods: A literature search for anti-VEGF inhibitors using the Web of Science was completed using the bibliographic databases for peer-reviewed literature published in Ophthalmology, the New England Journal of Medicine, Journal of the American Medical Association, and Lancet. Primary outcomes were the most frequently cited articles and journals with the most citations as well as the specific drug and disease process studied. Results: There were 42 696 cumulative citations among the top 100 articles. The articles were published between 2004 to 2016, with most articles published in 2006. Ophthalmology published the greatest number of articles among the top 100 at 48, whereas the New England Journal of Medicine has the most citations per publication at 1714. Ranibizumab was the medication researched in most articles at 56, followed by bevacizumab at 48, aflibercept at 10, and pegaptanib at 9. Forty-two articles investigated treatment of age-related macular degeneration, followed by 24 investigating diabetic macular edema, 10 for retinal vein occlusion, 8 for proliferative diabetic retinopathy, 2 for retinopathy of prematurity and polypoidal choroidal vasculopathy, and 1 for corneal neovascularization. Conclusions: As evidenced by the considerable number of citations accumulated over the past 20 years, anti-VEGF inhibitors have led to significant research in ophthalmology.

Long-Term Outcomes After Central Retinal Artery Occlusion Treated Acutely With Hyperbaric Oxygen Therapy: A Case Series.

Purpose: This study assesses the long-term outcomes, including neovascular complications, of central retinal artery occlusion (CRAO) treated acutely with hyperbaric oxygen therapy (HBOT). Methods: Four cases of CRAO treated acutely with HBOT were reviewed. Visual and structural outcomes were reviewed. Ocular complications including neovascularization were assessed and risk factors determined. Results: Two patients with a history of non-insulin dependent diabetes mellitus (NIDDM) developed early-onset ocular neovascularization within 1 month following treatment, with final vision of light perception over 1 year after injury. One patient with NIDDM and 1 patient without NIDDM did not develop ocular neovascularization; both had improvement in final visual acuity to 20/400 and 20/250, respectively. Conclusions: Patients treated acutely with HBOT for CRAO may require more frequent and earlier monitoring for complications, especially in patients with diabetes. Further research is needed to determine the long-term safety and efficacy of HBOT for CRAO, especially in the setting of systemic disease such as diabetes.

Vaccinia Virus Vectors Targeting Peptides for MHC Class II Presentation to CD4+ T Cells.

CD4+ helper T cells play important roles in providing help to B cells, macrophages, and cytotoxic CD8+ T cells, but also exhibit direct effector functions against a variety of different pathogens. In contrast to CD8+ T cells, CD4+ T cells typically exhibit broader specificities and undergo less clonal expansion during many types of viral infections, which often makes the identification of virus-specific CD4+ T cells technically challenging. In this study, we have generated recombinant vaccinia virus (VacV) vectors that target I-Ab-restricted peptides for MHC class II (MHC-II) presentation to activate CD4+ T cells in mice. Conjugating the lymphocytic choriomeningitis virus immunodominant epitope GP61-80 to either LAMP1 to facilitate lysosomal targeting or to the MHC-II invariant chain (Ii) significantly increased the activation of Ag-specific CD4+ T cells in vivo. Immunization with VacV-Ii-GP61-80 activated endogenous Ag-specific CD4+ T cells that formed memory and rapidly re-expanded following heterologous challenge. Notably, immunization of mice with VacV expressing an MHC-II-restricted peptide from Leishmania species (PEPCK335-351) conjugated to either LAMP1 or Ii also generated Ag-specific memory CD4+ T cells that underwent robust secondary expansion following a visceral leishmaniasis infection, suggesting this approach could be used to generate Ag-specific memory CD4+ T cells against a variety of different pathogens. Overall, our data show that VacV vectors targeting peptides for MHC-II presentation is an effective strategy to activate Ag-specific CD4+ T cells in vivo and could be used to study Ag-specific effector and memory CD4+ T cell responses against a variety of viral, bacterial, or parasitic infections.

Posterior Segment Findings in Patients on Extracorporeal Membrane Oxygenation.

Purpose: Extracorporeal membrane oxygenation (ECMO) is an established treatment modality for critically ill patients with cardiopulmonary failure, yet little is known of the ocular pathology in this population. The aim of this study is to characterize the posterior segment findings of ECMO patients. Methods: This study is a retrospective analysis of 20 ECMO patients evaluated by ophthalmology from September 2012 to May 2019 at a level 1 trauma center. Comprehensive examinations assessed for intraocular pathology. Demographic data, exam findings, and mortality were analyzed. Results: The sample size consisted of 20 patients; a majority were male (75%), and mean age was 37.4 years (interquartile range, 26.75-50 years). All patients received ECMO for care of acute respiratory distress syndrome (ARDS). Average duration of ECMO therapy was 9.6 ± 6.5 days. Eleven (55%) patients had acute retinal pathology, including Purtscher-like retinopathy (20%), intraocular hemorrhage (50%), and septic chorioretinitis (bacterial or fungal, 10%). Location of hemorrhage included the retina (40%), vitreous (30%), and optic disc (15%). Sixty percent (n = 12) of patients were unable to provide a subjective history on initial assessment. Ultimately, 5 out of 20 patients (25%) died of systemic illness during their hospital stay. Conclusion: This study demonstrates high rates of retinal pathology, most commonly vitreous and/or retinal hemorrhage alongside a Purtscher-like retinopathy. This is likely secondary to complications of anticoagulation, microthrombi, septicemia, and hemodynamic instability. We found a mortality rate slightly lower than that of prior ECMO studies. Prospective studies with pre-ECMO and post-ECMO fundus photography is warranted for better understanding of these medically complex patients.

Targeted Expansion of Tissue-Resident CD8+ T Cells to Boost Cellular Immunity in the Skin.

Tissue-resident memory (TRM) CD8+ T cells are positioned within environmental barrier tissues to provide a first line of defense against pathogen entry, but whether these specialized T cell populations can be readily boosted to increase protective immunity is ill defined. Here, we demonstrate that repeated activation of rare, endogenous TRM CD8+ T cells, using only topical application of antigenic peptide causes delayed-type hypersensitivity and increases the number of antigen-specific TRM CD8+ T cells, specifically in the challenged skin by ∼15-fold. Expanded TRM CD8+ T cells in the skin are derived from memory T cells recruited out of the circulation that became CD69+ tissue residents following a local antigen encounter. Notably, recruited circulating memory CD8+ T cells of a different antigen specificity could be coerced to become tissue resident using a dual-peptide challenge strategy. Expanded TRM CD8+ T cells significantly increase anti-viral protection, suggesting that this approach could be used to rapidly boost tissue-specific cellular immunity.

Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance.

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.