In-depth analysis of human virus-specific CD8+ T cells delineates unique phenotypic signatures for T cell specificity prediction.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes.

Patients with altered skin immunity, such as individuals with atopic dermatitis (AD), can have a life-threatening disruption of the epidermis known as eczema vaccinatum after vaccinia virus (VV) infection of the skin. Here, we sought to better understand the mechanism(s) by which VV associates with keratinocytes. The class A scavenger receptor known as MARCO (macrophage receptor with collagenous structure) is expressed on human and mouse keratinocytes and found to be abundantly expressed in the skin of patients with AD. VV bound directly to MARCO, and overexpression of MARCO increased susceptibility to VV infection. Furthermore, ligands with affinity for MARCO, or excess soluble MARCO, competitively inhibited VV infection. These findings indicate that MARCO promotes VV infection and highlights potential new therapeutic strategies for prevention of VV infection in the skin.

HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection.

Herpes simplex virus type 1 is an important epithelial pathogen and has the potential for significant morbidity in humans. Here we demonstrate that a cell surface scavenger receptor, macrophage receptor with collagenous structure (MARCO), previously thought to enhance antiviral defense by enabling nucleic acid recognition, is usurped by herpes simplex virus type 1 and functions together with heparan sulphate proteoglycans to mediate adsorption to epithelial cells. Ligands of MARCO dramatically inhibit herpes simplex virus type 1 adsorption and infection of human keratinocytes and protect mice against infection. Herpes simplex virus type 1 glycoprotein C closely co-localizes with MARCO at the cell surface, and glycoprotein C binds directly to purified MARCO with high affinity. Increasing MARCO expression enhances herpes simplex virus type 1 infection while MARCO(-/-) mice have reduced susceptibility to infection by herpes simplex virus type 1. These findings demonstrate that herpes simplex virus type 1 binds to MARCO to enhance its capacity for disease, and suggests a new therapeutic target to alter pathogenicity of herpes simplex virus type 1 in skin infection.

Commensal bacteria lipoteichoic acid increases skin mast cell antimicrobial activity against vaccinia viruses.

Mast cells (MCs) are considered sentinels in the skin and mucosa. Their ability to release antimicrobial peptides, such as cathelicidin, protects against bacterial infections when the epithelial barrier is breached. We recently described that MCs defend against bacterial and viral infections through the release of cathelicidin during degranulation. In this study, we hypothesize that cathelicidin expression is induced in MCs by the activation of TLR2 from bacterial products (lipoteichoic acid) produced by commensal bacteria at the epithelial surface. Our research shows that signaling through TLR2 increases the production and expression of cathelicidin in mast cells, thereby enhancing their capacity to fight vaccinia virus. MCs deficient in cathelicidin were less efficient in killing vaccinia virus after lipoteichoic acid stimulation than wild-type cells. Moreover, the activation of TLR2 increases the MC recruitment at the skin barrier interface. Taken together, our findings reveal that the expression and control of antimicrobial peptides and TLR signaling on MCs are key in fighting viral infection. Our findings also provide new insights into the pathogenesis of skin infections and suggest potential roles for MCs and TLR2 ligands in antiviral therapy.

Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1PR2 and releasing antimicrobial peptides.

Mast cells (MCs) are well-known effectors of allergic reactions and are considered sentinels in the skin and mucosa. In addition, through their production of cathelicidin, MCs have the capacity to oppose invading pathogens. We therefore hypothesized that MCs could act as sentinels in the skin against viral infections using antimicrobial peptides. In this study, we demonstrate that MCs react to vaccinia virus (VV) and degranulate using a membrane-activated pathway that leads to antimicrobial peptide discharge and virus inactivation. This finding was supported using a mouse model of viral infection. MC-deficient (Kit(wsh-/-)) mice were more susceptible to skin VV infection than the wild type animals, whereas Kit(wsh-/-) mice reconstituted with MCs in the skin showed a normal response to VV. Using MCs derived from mice deficient in cathelicidin antimicrobial peptide, we showed that antimicrobial peptides are one important antiviral granule component in in vivo skin infections. In conclusion, we demonstrate that MC presence protects mice from VV skin infection, MC degranulation is required for protecting mice from VV, neutralizing Ab to the L1 fusion entry protein of VV inhibits degranulation apparently by preventing S1PR2 activation by viral membrane lipids, and antimicrobial peptide release from MC granules is necessary to inactivate VV infectivity.

TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea.

Activation of TLR2 by a small molecule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections.

Production of antimicrobial peptides by epithelia is an essential defense against infectious pathogens. In this study we evaluated whether the commensal microorganism Staphylococcus epidermidis may enhance production of antimicrobial peptides by keratinocytes and thus augment skin defense against infection. Exposure of cultured undifferentiated human keratinocytes to a sterile nontoxic small molecule of <10 kDa from S. epidermidis conditioned culture medium (SECM), but not similar preparations from other bacteria, enhanced human beta-defensin 2 (hBD2) and hBD3 mRNA expression and increased the capacity of cell lysates to inhibit the growth of group A Streptococcus (GAS) and S. aureus. Partial gene silencing of hBD3 inhibited this antimicrobial action. This effect was relevant in vivo as administration of SECM to mice decreased susceptibility to infection by GAS. Toll-like receptor 2 (TLR2) was important to this process as a TLR2-neutralizing antibody blocked induction of hBDs 2 and 3, and Tlr2-deficient mice did not show induction of mBD4. Taken together, these findings reveal a potential use for normal commensal bacterium S. epidermidis to activate TLR2 signaling and induce antimicrobial peptide expression, thus enabling the skin to mount an enhanced response to pathogens.

Selective antimicrobial action is provided by phenol-soluble modulins derived from Staphylococcus epidermidis, a normal resident of the skin.

Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) gamma and delta produced by S. epidermidis have an alpha-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

Bcl-3 acts as an innate immune modulator by controlling antimicrobial responses in keratinocytes.

Innate immune responses involve the production of antimicrobial peptides (AMPs), chemokines, and cytokines. We report here the identification of B-cell leukemia (Bcl)-3 as a modulator of innate immune signaling in keratinocytes. In this study, it is shown that Bcl-3 is inducible by the Th2 cytokines IL-4 and IL-13 and is overexpressed in lesional skin of atopic dermatitis (AD) patients. Bcl-3 was shown to be important to cutaneous innate immune responses as silencing of Bcl-3 by small-interfering RNA (siRNA) reversed the downregulatory effect of IL-4 on the HBD3 expression. Bcl-3 silencing enhanced vitamin D3 (1,25D3)-induced gene expression of cathelicidin AMP in keratinocytes, suggesting a negative regulatory function on cathelicidin transcription. Furthermore, 1,25D3 suppressed Bcl-3 expression in vitro and in vivo. This study identified Bcl-3 as an important modulator of cutaneous innate immune responses and its possible therapeutic role in AD.