Maike Hofmann

E-cadherin promotes accumulation of a unique memory CD8 T-cell population in murine salivary glands

The salivary glands are important effector sites for IgA-mediated humoral immunity to protect oral surfaces. Within murine submandibular glands (SMG), we identified a memory CD8 T-cell population that exhibited a unique cell-surface phenotype distinct from memory CD8 T cells in spleen but similar to memory T cells resident in the intraepithelial lymphocyte compartment of the intestinal mucosa. In mice immune to lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus(VSV), virus-specific memory CD8 T cells with this unusual phenotype were present in SMG at remarkably high frequencies. LCMV-specific memory CD8 T cells in SMG showed potent functional activities in vivo, including cytokine-induced bystander proliferation, antigen-triggered IFNγ production, and viral clearance. Adoptive transfer experiments further revealed that the capacity to accumulate in SMG decreased during CD8 T-cell differentiation and that SMG CD8 T cells were poorly replenished from the circulation, indicating that they were tissue-resident. Moreover, they preferentially relocalized within their tissue of origin after adoptive transfer and antigen rechallenge, thus revealing an imprinted differentiation status. Accumulation of memory CD8 T cells within SMG did not require local antigen presentation but was promoted by the epithelial differentiation molecule E-cadherin intrinsically expressed by these CD8 T cells. This finding extends the epithelial-restricted function of E-cadherin to an impact on lymphocyte accumulation within epithelial tissues.

Structure of Natural Killer Cell Receptor KLRG1 Bound to E-Cadherin Reveals Basis for MHC-Independent Missing Self Recognition

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y7F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1+ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.

Thymus‐resident memory CD8+ T cells mediate local immunity

The thymus is a primary lymphoid organ responsible for production and selection of T cells. Nonetheless, mature T cells and in particular activated T cells can reenter the thymus. Here, we identified memory CD8+ T cells specific for lymphocytic choriomeningitis virus or vaccinia virus in the thymus of mice long‐time after the infection. CD8+ T cells were mainly located in the thymic medulla, but also in the cortical areas. Interestingly, virus‐specific memory CD8+ T cells in the thymus expressed the cell surface markers CD69 and CD103 that are characteristic of tissue‐resident memory T cells in a time‐dependent manner. Kinetic analyses and selective depletion of peripheral CD8+ T cells by antibodies further revealed that thymic virus‐specific memory CD8+ T cells did not belong to the circulating pool of lymphocytes. Finally, we demonstrate that these thymus‐resident virus‐specific memory CD8+ T cells efficiently mounted a secondary proliferative response, exhibited immediate effector functions and were able to protect the thymus from lymphocytic choriomeningitis virus reinfection. In conclusion, the present study not only describes for the first time virus‐specific memory CD8+ T cells with characteristics of tissue‐resident memory T (TRM) cells in a primary lymphoid organ but also extends our knowledge about local T‐cell immunity in the thymus.

TCF1 + hepatitis C virus-specific CD8 + T cells are maintained after cessation of chronic antigen stimulation

Differentiation and fate of virus-specific CD8+ T cells after cessation of chronic antigen stimulation is unclear. Here we show that a TCF1+CD127+PD1+ hepatitis C virus (HCV)-specific CD8+ T-cell subset exists in chronically infected patients with phenotypic features of T-cell exhaustion and memory, both before and after treatment with direct acting antiviral (DAA) agents. This subset is maintained during, and for a long duration after, HCV elimination. After antigen re-challenge the less differentiated TCF1+CD127+PD1+ population expands, which is accompanied by emergence of terminally exhausted TCF1-CD127-PD1hi HCV-specific CD8+ T cells. These results suggest the TCF1+CD127+PD1+ HCV-specific CD8+ T-cell subset has memory-like characteristics, including antigen-independent survival and recall proliferation. We thus provide evidence for the establishment of memory-like virus-specific CD8+ T cells in a clinically relevant setting of chronic viral infection and we uncover their fate after cessation of chronic antigen stimulation, implicating a potential strategy for antiviral immunotherapy.

Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro

ABSTRACT CD8+ T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8+ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2hNTCP cells that endogenously processed viral antigens and presented them to HBV-specific CD8+ T cells. This induced cytolytic and noncytolytic CD8+ T-cell effector functions and reduction of viral loads.

Inhibition of protein geranylgeranylation and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells.

Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without having a negative impact on immune reconstitution.

Different inhibitory capacities of human and mouse KLRG1 are linked to distinct disulfide-mediated oligomerizations.

The killer cell lectin‐like receptor G1 (KLRG1) is a cadherin‐binding inhibitory receptor expressed by NK and T cells in humans and mice. Although structural and ligand‐binding properties of human (h) and mouse (m) KLRG1 are very similar, KLRG1‐mediated inhibition under physiological conditions is only observed with human lymphocytes. Using a well‐defined in vitro system, we demonstrate here that mKLRG1 exhibits a significantly lower inhibitory capacity compared with the human homolog. Biochemical analyses further showed that mKLRG1 formed monomers and disulfide‐linked dimers, trimers, and tetramers whereas hKLRG1 was exclusively present as disulfide‐linked dimer. Mutational analysis revealed a crucial role of Cys62 present in the stalk region of mKLRG1 but not of hKLRG1 for oligomer formation. Strikingly, mimicking hKLRG1 by replacement of Cys62 in mKLRG1 by glutamine prevented tri‐ and tetramer formation and increased the inhibitory capacity. Furthermore, mutated mKLRG1 molecules that were unable to form disulfide‐linked dimers at all or at a decreased level lacked inhibitory activity. These data indicate that only dimeric KLRG1 entities exhibit potent inhibitory capacities. The lower inhibitory capacity of mKLRG1 compared with hKLRG1 can thus be rationalized by a decreased proportion of dimeric entities, which can be pinpointed to a single amino acid.

MAIT be different–persisting dysfunction after DAA-mediated clearance of chronic hepatitis C virus infection

MAIT cells are an abundant innate‐like T‐cell subset that is defined by the invariant T‐cell receptor (iTCR) V‐alpha chain Vα7.2‐Jα33. Little is currently known about their frequency and function in chronic hepatitis C virus (HCV) infection and their fate after therapy‐mediated HCV elimination by direct acting antivirals (DAA). In this issue of the European Journal of Immunology, Hengst et al. [Eur. J. Immunol. 2016. 46: 2204‐2210] give important novel insights into the biological role of MAIT cells in a relevant human chronic viral infection by showing that first, MAIT cells are only present at low frequencies in chronic HCV infection; second, circulating MAIT cells in HCV patients also display an altered phenotype; third, they are impaired in their MR‐1‐dependent effector functions and finally, and maybe most importantly, MAIT‐cell frequency and function was not restored after HCV elimination by DAA therapy. These results suggest that MAIT cells are severely affected by a chronic human viral infection in their frequency and function and that this impairment is not reversed after HCV elimination. This is in contrast to rapid DAA‐mediated restorations of NK‐cell and CD8+ T‐cell functions, and indicates a differential impact of chronic infection and clearance on different immune cell subsets.

Overcoming CD8+ T-Cell Exhaustion in Viral Hepatitis: Lessons from the Mouse Model and Clinical Perspectives.

About 500 million people all over the world are chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV) and are thus at high risk of developing liver fibrosis, cirrhosis or hepatocellular carcinoma. While in adults about 90% of acutely HBV-infected patients clear the virus, only 30% of acute HCV infections clear spontaneously. Several mechanisms contribute to the failure in viral clearance. The main factors responsible for the chronification of HBV and HCV infection are, on the one hand, viral escape mutations leading to lack of recognition by antiviral immune cells and, on the other hand, loss of antiviral effector functions of virus-specific CD8+ T cells, called T-cell exhaustion. This review focuses on the latter highlighting current knowledge about the heterogeneity of exhausted CD8+ T cells and the potential for re-invigoration of exhausted T-cell populations during chronic viral hepatitis. Although direct-acting antivirals successfully clear chronic HCV infection, there is still the need for a prophylactic vaccine to prevent primary infection. Moreover, a therapeutic strategy eliminating HBV infection still does not exist. A better understanding of T-cell exhaustion and the potential for functional recovery will help to develop new immunotherapeutic approaches for chronic viral hepatitis.