Dedicated immunosensing of the mouse intestinal epithelium facilitated by a pair of genetically coupled lectin-like receptors.

The integrity of the intestinal epithelium is constantly surveyed by a peculiar subset of innate-like T lymphocytes embedded in the epithelial cell layer, hence called intestinal intraepithelial lymphocytes (IELs). IELs are thought to act as "first-line" sentinels sensing the state of adjacent epithelial cells via both T-cell receptors and auxiliary receptors. Auxiliary receptors modulating IEL activity include C-type lectin-like receptors encoded in the natural killer gene complex such as NKG2D. Here, we report that the CTLR Nkrp1g is expressed by a subpopulation of mouse CD103(+) IELs allowing immunosensing of the intestinal epithelium through ligation of the genetically coupled CTLR Clr-f that is almost exclusively expressed on differentiated intestinal epithelial cells (IECs). Most of these Nkrp1g-expressing IELs exhibit a γδTCR(bright)Nkg2a(-) phenotype and are intimately associated with the intestinal epithelium. As Clr-f expression strongly inhibits effector functions of Nkrp1g-expressing cells and is upregulated upon poly(I:C) challenge, Clr-f molecules may quench reactivity of these IELs towards the epithelial barrier that is constantly provoked by microbial and antigenic stimuli. Altogether, we here newly characterize a genetically linked C-type lectin-like receptor/ligand pair with a highly restricted tissue expression that apparently evolved to allow for a dedicated immunosurveillance of the mouse intestinal epithelium.

NK cells and cancer immunosurveillance.

Natural killer (NK) cells are lymphocytes of the innate immune system that monitor cell surfaces of autologous cells for an aberrant expression of MHC class I molecules and cell stress markers. Since their first description more than 30 years ago, NK cells have been implicated in the immune defence against tumours. Here, we review the broadly accumulating evidence for a crucial contribution of NK cells to the immunosurveillance of tumours and the molecular mechanisms that allow NK cells to distinguish malignant from healthy cells. Particular emphasis is placed on the activating NK receptor NKG2D, which recognizes a variety of MHC class I-related molecules believed to act as 'immuno-alerters' on malignant cells, and on tumour-mediated counterstrategies promoting escape from NKG2D-mediated recognition.

The innate immune response in the central nervous system and its role in glioma immune surveillance.

The innate immune system encompasses natural killer (NK) cells, macrophages and granulocytes, the complement system and antimicrobial peptides. Recognition pathways of the innate immune system include microbial non-self recognition, missing-self recognition and induced- self recognition. The central nervous system (CNS) participates in responses of the innate immune system. However, immune inhibitory and anti-inflammatory mechanisms physiologically outbalance and counteract immune activity and thereby limit immune-mediated tissue damage in the brain. Human gliomas appear to take advantage of this immunosuppressive milieu. Moreover, glioma cells themselves interfere with anti-tumor immune responses by expressing immune inhibitory cell surface molecules, such as HLA-G, or by releasing soluble immunosuppressants such as transforming growth factor (TGF)-beta. Yet, although glioma cells exhibit all cellular features of malignancy, these tumors very rarely metastasize outside the brain, raising the possibility of immune-mediated control of these cells outside, but not inside, the brain. Accordingly, activating the innate immune system by forcing glioma cells to express danger signals such as NKG2D ligands is a promising strategy of immunotherapy for these tumors.

Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family.

NKG2D is an activating receptor that is expressed on most natural killer (NK) cells, CD8 alphabeta T cells, and gammadelta T cells. Among its ligands is the distant major histocompatibility complex class I homolog MICA, which has no function in antigen presentation but is induced by cellular stress. To extend previous functional evidence, the NKG2D-MICA interaction was studied in isolation. NKG2D homodimers formed stable complexes with monomeric MICA in solution, demonstrating that no other components were required to facilitate this interaction. MICA glycosylation was not essential but enhanced complex formation. Soluble NKG2D also bound to cell surface MICB, which has structural and functional properties similar to those of MICA. Moreover, NKG2D stably interacted with surface molecules encoded by three newly identified cDNA sequences (N2DL-1, -2, and -3), which are identical to the human ULBP proteins and may represent homologs of the mouse retinoic acid-early inducible family of NKG2D ligands. Because of the substantial sequence divergence among these molecules, these results indicated promiscuous modes of receptor binding. Comparison of allelic variants of MICA revealed large differences in NKG2D binding that were associated with a single amino acid substitution at position 129 in the alpha2 domain. Varying affinities of MICA alleles for NKG2D may affect thresholds of NK-cell triggering and T-cell modulation.

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin-like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor-MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the alpha1 or alpha2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the alpha2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.

Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA.

Stress-inducible MICA, a distant homolog of major histocompatibility complex (MHC) class I, functions as an antigen for gammadelta T cells and is frequently expressed in epithelial tumors. A receptor for MICA was detected on most gammadelta T cells, CD8+ alphabeta T cells, and natural killer (NK) cells and was identified as NKG2D. Effector cells from all these subsets could be stimulated by ligation of NKG2D. Engagement of NKG2D activated cytolytic responses of gammadelta T cells and NK cells against transfectants and epithelial tumor cells expressing MICA. These results define an activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses.

NF-kappaB/Rel activation in cerulein pancreatitis.

BACKGROUND & AIMS: Recent evidence suggests that a number of rapid signaling cascades are initiated during secretagogue-induced pancreatitis. However, little is known about the nuclear events. The aim of this study was to explore activation of the transcription factor NF-kappaB/Rel after supramaximal stimulation with the cholecystokinin analogue cerulein in the pancreas. METHODS & RESULTS: Nuclear appearance of NF-kappaB/Rel-binding activity was detectable 15 minutes after cerulein injection. The DNA-binding activity consisted of NF-kappaB1 p50, NF-kappaB2 p52, and RelA p65 as judged by supershift assays and Western blot analysis. The onset and termination of NF-kappaB/Rel activation correlated with the degradation and reappearance of IkappaBalpha. Cerulein in supramaximal but not in physiological doses activated NF-kappaB/Rel in vitro. After blocking of NF-kappaB/Rel activation with pyrrolidine dithiocarbamate, the degree of morphological alterations was more pronounced than in controls, serum amylase and lactate dehydrogenase levels were significantly increased, and messenger RNA levels of pancreatitis-associated protein were more strongly induced, reflecting a more severe degree of pancreatitis. Similar results were obtained when N-acetyl-L-cysteine was used as an inhibitor of NF-kappaB activation. CONCLUSIONS: These data show that NF-kappaB/Rel is rapidly activated during cerulein pancreatitis. This activation may induce a self-defending genetic program before the onset of cellular injury, which might prevent higher degrees of damage of pancreatic acinar cells after secretagogue hyperstimulation.

Diversification, expression, and gamma delta T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules.

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial gamma delta T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative alpha-helical regions of the alpha1 alpha2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their alpha1 alpha2 domains, they were recognized by diverse human intestinal epithelial gamma delta T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of gamma delta T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells.

T cells with variable region Vdelta1 gammadelta T cell receptors (TCRs) are distributed throughout the human intestinal epithelium and may function as sentinels that respond to self antigens. The expression of a major histocompatibility complex (MHC) class I-related molecule, MICA, matches this localization. MICA and the closely related MICB were recognized by intestinal epithelial T cells expressing diverse Vdelta1 gammadelta TCRs. These interactions involved the alpha1alpha2 domains of MICA and MICB but were independent of antigen processing. With intestinal epithelial cell lines, the expression and recognition of MICA and MICB could be stress-induced. Thus, these molecules may broadly regulate protective responses by the Vdelta1 gammadelta T cells in the epithelium of the intestinal tract.

[Effect of cellular growth factors on hepatocytes in experimental infection--regulation of NF-kappa B and glutathione homeostasis]. / Der Einfluss zellulärer Wachstumsfaktoren auf Hepatozyten in der experimentellen Sepsis: Regulation von NF-kappa B und des Glutathionhaushaltes.

Infections, sepsis and trauma lead to cellular damage of different degrees. The formation of nitrogen and reactive oxygen intermediates (NOI and ROI) play a central role in cellular damage. In addition, it is well established that the intracellular GSH content can control both radical species whereas GSH levels are controlled by the presence of cellular growth factors. The aim of the following study was to investigate the ROI and nitric oxide formation depending on the GSH levels and the presence or absence of hepatocellular growth factors. In addition, we investigated their effects on hepatocellular injury and the status of activation of the nuclear transcriptional factor NF-kappa B which is influenced by various radical forms and the cellular GSH contents. Our data clearly demonstrate that hepatocellular growth factors such as EGF and TGF alpha can increase the GSH contents and the NOx production. In addition, we found a reduction of cellular injury and NF-kappa B expression when hepatocytes were preincubated with growth factors. Taken together, we conclude that growth factors are able to protect against hepatocellular injury in experimental sepsis by increasing the cellular GSH contents either to reduce superoxide anion formation or to induce increased NO synthesis activity with subsequent increased NO production.

Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.

The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.

Human pancreas-specific protein. A diagnostic and prognostic marker in acute pancreatitis and pancreas transplantation.

CONCLUSION: Human pancreas-specific protein (hPASP) is a very sensitive reflector of the extent of pancreatic necrosis on the cellular level, and is of both diagnostic and prognostic value in acute pancreatitis. Furthermore, it allows the estimation of the severity of graft pancreatitis soon after simultaneous renal and pancreatic transplantation. BACKGROUND: Diagnosis of acute pancreatitis (AP) has been improved in the past 15 yr as new methods for the determination of specific pancreatic enzymes have been developed. However, these enzymes have no prognostic implications. In this prospective study, we evaluated the role of human pancreas-specific protein (hPASP) in comparison with pancreatic amylase and C-reactive protein (CRP) in acute pancreatitis and pancreas transplantation. PATIENTS AND METHODS: The study included 40 patients (22 female, 18 male; mean age 51 yr, range 22-88 yr) with AP and 7 patients (2 female, 5 male; mean age 37 yr, range 25-49 yr) with type I diabetes and renal insufficiency who underwent simultaneous kidney and pancreas transplantation. By means of contrast-enhanced computed tomography (CT) and/or intraoperative findings, patients were judged to have edematous-interstitial (AIP, n = 20, mean age 55.2 yr, range 24-88 yr) or necrotizing pancreatitis (NP, n = 20, mean age 46.3 yr, range 22-81 yr). Serum hPASP concentration was measured daily by a commercial radioimmunoassay technique. In 25 healthy subjects and in several control groups (35 patients with chronic pancreatitis, 20 patients with pancreatic carcinoma and 80 patients with different gastrointestinal diseases) a single blood specimen was taken at hospital admission for the determination of the normal range of hPASP and for specificity analysis. RESULTS: The upper normal value for hPASP in healthy subjects was found to be 52 ng/mL. Serum hPASP was elevated in all patients suffering from AP, with a median of 343 ng/mL (lower-upper quartile: 192-478 ng/mL) at hospital admission. In the daily serum monitoring with respect to the onset of symptoms, significantly higher hPASP levels were found in NP compared with AIP after day 2 (p < 0.001). In patients with NP, peak values of hPASP correlated significantly with the extent of pancreatic necroses measured by contrast-enhanced CT-scanning, whereas CRP did not. Six patients of the transplantation group had the same serum hPASP course as AIP, with almost normal values on the third postoperative day. One patient had elevated levels throughout the observation period. This patient suffered from necrotizing graft pancreatitis, confirmed by relaparotomy, and died because of subsequent septic complications.

Heterogeneity of rearranged T cell receptor V alpha and V beta gene transcripts in synovial fluid T cells of HLA-B27 positive reactive arthritis patients.

To analyze the T cell receptor (TCR) V-alpha/beta gene usage by synovial fluid (SF) and peripheral blood (PB) T cells of HLA-B27+ reactive arthritis (ReA) patients. The TCR V-alpha/beta gene usage was determined by the polymerase chain reaction on freshly isolated SF and PB mononuclear cells (MNC) of five HLA-B27+ ReA patients. A total of 30 TCR V alpha and 23 V beta (sub)family specific primers in combination with a C alpha or C beta specific primer, respectively, were used. In five patients most of the TCR V alpha and V beta gene segments expressed by PB T cells were also detected in the paired SF samples. Although one patient showed an increased expression of TCR V alpha2 in SF when compared to PB, the SF samples showed a heterogeneous TCR V-gene repertoire similar to PB. Although this study was limited to a small group of patients, the apparent lack of a restricted TCR V-gene repertoire in SF does not support the involvement of a single or limited number of T cell subsets in the disease process of HLA-B27+ ReA patients.

Expression of HLA-C molecules confers target cell resistance to some non-major histocompatibility complex-restricted T cells in a manner analogous to allospecific natural killer cells.

Specific HLA molecules have recently been shown to confer target cell resistance to lysis by some CD3- natural killer (NK) cells. For certain NK clones, resistance is governed by two specificities (NK1 and NK2) that are associated with particular HLA-C alleles: in general, target cells expressing Cw1, Cw3, Cw7, or Cw8 are susceptible to NK1 but resistant to NK2 clones, whereas target cells expressing Cw2, Cw4, Cw5, or Cw6 are susceptible to NK2 and resistant to NK1 cells. These two clusters of HLA-C alleles are distinguished by a dimorphism in the alpha 1 helical region, localized at amino acid positions 77 and 80. In this report, we show that highly enriched CD3+/CD56- cytotoxic T cell sublines and CD3-/CD56+ NK sublines derived from the same donor have identical cytolytic specificities when tested against a panel of allogeneic LCL and various HLA-B and -C transfectant cell lines. The lysis pattern of the allogeneic cells appeared to be related to the NK2 specificity for both effector cells: LCL expressing HLA-Cw2, Cw4, Cw5, or Cw6 alleles were lysed, while LCL expressing HLA-Cw1, Cw3, or Cw7 molecules were resistant. Resistance to lysis could be conferred to susceptible target cells by transfection with a Cw*0702 gene, while expression of a Cw*0602 gene did not provide protection. Similar patterns of HLA-C-mediated resistance were also found with two polyclonal T cell lines generated from the peripheral blood lymphocytes of unrelated donors. Thus, major histocompatibility complex (MHC) molecules that induced resistance to particular NK cells also regulated target cell resistance to lysis by these non-MHC-restricted effector T cells. For both types of effector cells, direct binding to HLA-C molecules was necessary to achieve inhibition since preincubation with mAb specific for class I molecules destroyed the protection from lysis of HLA-Cw7 expressing target cells. mAbs specific for CD3 and CD8 molecules had no influence on lysis or inhibition of the NK-like T cells. Formation of MHC complexes with particular peptides did not appear to be essential to confer resistance, since a cell line with defective peptide transporter genes (TAP genes), when transfected with an appropriate HLA-C allele, was as resistant to lysis as HLA-C transfectant lines with normal TAP function. These results suggest that HLA-C molecules may deliver negative regulatory signals to some non-MHC-restricted T cells in a manner similar to that described previously for particular NK cells.

In vivo expansion of HLA-B35 alloreactive T cells sharing homologous T cell receptors: evidence for maintenance of an oligoclonally dominated allospecificity by persistent stimulation with an autologous MHC/peptide complex.

The nature of alloantigens seen by T lymphocytes, in particular the role of peptides in allorecognition, has been studied intensively whereas knowledge about the in vivo emergence, diversity, and the structural basis of specificity of alloreactive T cells is very limited. Here we describe human T cell clones that recognize HLA-B35 alloantigens in a peptide-dependent manner. TCR sequence analysis revealed that several of these allospecific clones utilize homologous TCR: they all express TCRAV2S3J36C1 and TCRBV4S1J2S7C2 chains with highly related CDR3 sequences. Thus peptide-specific alloreactivity is reflected in homologous CDR3 sequences in a manner similar to that described for T cells that recognize nominal peptide/self-MHC complexes. The in vivo frequency of this TCR specificity was studied in unstimulated PBL of the responding cell donor who was not sensitized against HLA-B35. The vast majority (approximately 75%) of the VA2S3J36 junctional regions obtained from two samples of PBL, isolated at a 9-yr interval, encode CDR3 identical or homologous to those of the functionally characterized HLA-B35 allospecific T cells. These data are most easily explained by a model of alloreactivity in which persistent or recurrent exposure to a foreign peptide/self-MHC complex led to the in vivo expansion and long-term maintenance of specific T cells that show fortuitous crossrecognition of an HLA-B35/peptide complex and dominate the alloresponse against HLA-B35.

HLA-DQ-restricted, islet-specific T-cell clones of a type I diabetic patient. T-cell receptor sequence similarities to insulitis-inducing T-cells of nonobese diabetic mice.

We established a T-cell line and 20 CD4+ T-cell clones from the peripheral blood lymphocytes of a type I diabetic patient using a membrane preparation of a rat insulinoma cell line (beta membrane antigen [BMA]) as a source of antigen. The T-cell line and three selected clones proliferated specifically to stimulation with BMA and to membranes prepared from human islets, rat pancreas, and NOD pancreas, but not to control antigens. Proliferation-inhibition studies using human leukocyte antigen (HLA)-specific monoclonal antibodies revealed HLA-DQw1-restricted recognition of BMA. An analysis of the T-cell receptor (TCR) repertoire of the T-cell line after 8 and 40 days of culture showed a prominent usage of the V alpha 1 and V alpha 12 gene families. Although sequencing of the TCR V alpha and V beta chains of the three clones demonstrated that each used different V alpha and V beta gene segments, structural similarities were found in complementary-determining region 3 (CDR3), the region that is postulated to interact with the peptide component of the TCR ligand. When we compared these TCR sequences with published sequences of disease-inducing T-cells of NOD mice, highly related TCR V beta families were detected. Furthermore, stretches of identical amino acids within the CDR3 region were found between two pairs of human and mouse T-cells. If one considers the species differences in TCR genes and sequence differences in the restriction elements for these cells (HLA-DQ vs. H-2 I-A nod), these sequence similarities are striking and may be useful for pinpointing T-cells of primary importance in the development of disease.