Kalle Söderström

HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C.

The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules,. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and β2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.

A Signal Peptide Derived from hsp60 Binds HLA-E and Interferes with CD94/NKG2A Recognition

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule which presents a restricted set of nonameric peptides, derived mainly from the signal sequence of other MHC class I molecules. It interacts with CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and T cell subsets. Here we demonstrate that HLA-E also presents a peptide derived from the leader sequence of human heat shock protein 60 (hsp60). This peptide gains access to HLA-E intracellularly, resulting in up-regulated HLA-E/hsp60 signal peptide cell-surface levels on stressed cells. Notably, HLA-E molecules in complex with the hsp60 signal peptide are no longer recognized by CD94/NKG2A inhibitory receptors. Thus, during cellular stress an increased proportion of HLA-E molecules may bind the nonprotective hsp60 signal peptide, leading to a reduced capacity to inhibit a major NK cell population. Such stress induced peptide interference would gradually uncouple CD94/NKG2A inhibitory recognition and provide a mechanism for NK cells to detect stressed cells in a peptide-dependent manner.

IFN-γ protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2A-dependent mechanism

IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.

Presence of human 65 kD heat shock protein (hsp) in inflamed joints and subcutaneous nodules of RA patients.

Monoclonal antibodies to the human homologue of the bacterial 65 kD heat shock protein (hsp) were used to investigate the tissue distribution of endogenous hsp 65 in normal versus rheumatoid synovial tissue, in subcutaneous nodules of patients with rheumatoid arthritis (RA) and in several instances of non‐rheumatoid inflammation. A strong reactivity of the anti‐hsp antibody was found in the cartilage‐pannus junction in rheumatoid joints and in rheumatoid nodules, but not in normal joints or in normal or inflamed kidney or liver (irreversible graft rejection, chronic glomerulonephritis or primary biliary cirrhosis). The findings provide a new hypothetical explanation for a role of T cells reactive with the 65 kD hsp in the generation of both articular and extra‐articular lesions in chronic rheumatoid arthritis.

Role of hsp60 during Autoimmune and Bacterial Inflammation

Heat-shock proteins (hsp) are a group of proteins with a highly conserved structure in evolution, which originally attracted the interest of molecular biologists studying gene regulation, but now are challenging also for the immunologist with a number of fundamental questions in relation to infection and autoimmunity. The immunogenicity of several hsp constituents of bacteria and parasites for the host T-cell system makes them of central interest for those who are trying to manipulate the protective immune responses against microorganisms. The hsp60 protein, originally characterized in E. coli as the groEL protein, and subsequently shown to have homologues in several other species, including humans (Jindal et al. 1989), is probably among the most studied in this regard, partly because recombinant myeobacterial hsp60 was made easily available for experimental immunologists. An almost bewildering number of observations suggest that T cells specific for hsp60 are involved in phenomena ranging from organ-specific inflammatory diseases like arthritis (van Eden et al. 1988) or diabetes (Elias et al. 1990) to tolerance induction (van den Broek et al. 1989), suggesting that many of the T cells recognizing bacterial hsp60 also crossreact with endogenous hsp60. Starting with our original interests in T-cell responsiveness against mycobacteria and local immunity in arthritis, respectively, we considered that at least two basic questions needed to be addressed from the findings related above; one question being when, where and how mammalian hsp60 is expressed; particularly, when do the determinants of this protein become available for recognition by immune cells?

Natural killer cells trigger osteoclastogenesis and bone destruction in arthritis

Osteoclasts are bone-eroding cells that develop from monocytic precursor cells in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Osteoclasts are essential for physiological bone remodeling, but localized excessive osteoclast activity is responsible for the periarticular bone destruction that characteristically occurs in patients with rheumatoid arthritis (RA). The origin of osteoclasts at sites of bone erosion in RA is unknown. Natural killer (NK) cells, as well as monocytes, are abundant in the inflamed joints of patients with RA. We show here that such NK cells express both RANKL and M-CSF and are frequently associated with CD14+ monocytes in the RA synovium. Moreover, when synovial NK cells are cocultured with monocytes in vitro, they trigger their differentiation into osteoclasts, a process dependent on RANKL and M-CSF. As in RA, NK cells in the joints of mice with collagen-induced arthritis (CIA) express RANKL. Depletion of NK cells from mice before the induction of CIA reduces the severity of subsequent arthritis and almost completely prevents bone erosion. These results suggest that NK cells may play an important role in the destruction of bone associated with inflammatory arthritis.

Human Cytomegalovirus Strain-Dependent Changes in NK Cell Recognition of Infected Fibroblasts

NK cells play a key role in the control of CMV infection in mice, but the mechanism by which NK cells can recognize and kill CMV-infected cells is unclear. In this study, the modulation of NK cell susceptibility of human CMV (hCMV)-infected cells was examined. We used a human lung and a human foreskin fibroblast cell line infected with clinical isolates (4636, 13B, or 109B) or with laboratory strains (AD169, Towne). The results indicate that all three hCMV clinical isolates confer a strong NK resistance, whereas only marginal or variable effects in the NK recognition were found when the laboratory strains were used. The same results were obtained regardless of the conditions of infection, effector cell activation status, cell culture conditions, and/or donor-target cell combinations. The NK cell inhibition did not correlate with HLA class I expression levels on the surface of the target cell and was independent of the leukocyte Ig-like receptor-1, as evaluated in Ab blocking experiments. No relevant changes were detected in the adhesion molecules ICAM-I and LFA-3 expressed on the cell surface of cells infected with hCMV clinical and laboratory strains. We conclude that hCMV possesses other mechanisms, related neither to target cell expression of HLA-I or adhesion molecules nor to NK cell expression of leukocyte Ig-like receptor-1, that confer resistance to NK cell recognition. Such mechanisms may be lost during in vitro passage of the virus. These results emphasize the differences between clinical hCMV isolates compared with laboratory strains.

Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules.

The functional properties, regarding parasite growth inhibition in vitro, the cytotoxic potential and cytokine profiles of human γδ+ and αβ+ T cells, T‐cell lines and clones stimulated with Plasmodium falciparum‐antigen‐or T‐cell mitogen in vitro were investigated. Using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and specific primers, mRNA for the cytolytic molecules perforin, granzyme A and B, Fas and Fas ligand (FasL) were detected in both the γδ‐ and the αβT cells. Despite this fact, only γδT cells inhibited, both Vδ1+ and Vδ2+, the in vitro growth of the asexual blood stages in a dose dependent manner. The inhibition required cell‐to‐cell contact and was not observed until the second parasite replication implied that the likely γδT‐cell target was the extracellular merozoite or schizont. The failure of αβT cells to inhibit the growth of the parasite suggests requirement of additional cytolytic molecules/signals or different receptor specificities exhibited by the γδT cells. Both the γδ‐ and αβT cells expressed mRNA for a large number of cytokines. Interferon (IFN)‐γ, interleukin (IL) IL‐5, IL‐6, IL‐8, tumour necrosis factor alpha (TNFα), tumour necrosis factor beta (TNF‐β)/lymphotoxin (LT) and T‐cell growth factor beta‐1 (TGF‐β1) were observed in all activated clones tested. No IL‐3 was detected, while IL‐1β, IL‐2, IL‐4, IL‐10 and GM‐CSF were variably expressed. In conclusion, our data show that γδT cells in malaria nonimmune individuals inhibit the asexual blood stages of P. falciparum malaria, while similarly activated αβT cells do not. Thus, it is likely that the γδT cells could play a mandatory role in the elimination of parasites and/or the regulation of the early immune response to malaria infection.

Synergistic effect of IFN-γ and human cytomegalovirus protein UL40 in the HLA-E-dependent protection from NK cell-mediated cytotoxicity

Human CMV (HCMV) has evolved several strategies to evade the immune system of the infected host. Here, we investigated the role of the HCMV‐encoded protein UL40 in the modulation of NK cell lysis. UL40 carries in its leader sequence a nonameric peptide similar to that found in many HLA class I molecules leader sequences. This peptide up‐regulates the expression of HLA‐E, the ligand for the NK cell inhibitory receptor CD94 / NKG2A. The UL40‐encoded HLA‐E‐binding peptide was present in all HCMV clinical (4636, 13B, 109B, 3C) and laboratory (AD169) strains analyzed. However, transfection of UL40 in different cell lines (293T, 721.221, K562) did not consistently confer protection from NK lysis (as measured using NKL and the newly generated NK line Nishi), despite a moderate up‐regulation of HLA‐E. Interestingly, combined transfection and treatment with IFN‐γ increased the inhibitory effect, via an HLA‐E‐ and CD94 / NKG2A‐dependent mechanism. Although cells transfected with UL40 derived from either AD169 or 3C showed protection from NK cell lysis, infection of fibroblasts with the viruses resulted in a strong inhibition only with the clinical strain 3C. Our results suggest that UL40 and IFN‐γ‐dependent up‐regulation of HLA‐E is only one possible mechanism to avoid NK cell recognition of HCMV infected cells.

Activating and inhibitory receptors on synovial fluid natural killer cells of arthritis patients: role of CD94/NKG2A in control of cytokine secretion

Natural killer (NK) cells are activated early during inflammatory events and contribute to the shaping of the ensuing adaptive immune response. To further understand the role for NK cells in inflammation, we investigated the phenotype and function of synovial fluid (SF) NK cells from patients with chronic joint inflammation, as well as from patients with transient inflammation of the knee following trauma. We confirm that synovial NK cells are similar to the well‐characterized CD56bright peripheral blood (PB) NK‐cell subset present in healthy individuals. However, compared to this PB subset the synovial NK cells express a higher degree of activation markers including CD69 and NKp44, the latter being up‐regulated also on CD56bright NK cells in the PB of patients. Activated synovial NK cells produced interferon‐γ and tumour necrosis factor, and the production was further up‐regulated by antibody masking of CD94/NKG2A, and down‐regulated by target cells expressing human leucocyte antigen‐E in complex with peptides known to engage CD94/NKG2A. We conclude that synovial NK cells have an activated phenotype and that CD94/NKG2A is a key regulator of synovial NK‐cell cytokine synthesis.