Helmut J. Kolb

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm restricted expression in peripheral blood mononuclear cells (PBMCs) from healthy donors but also observe overexpression in different leukemias and aberrant expression in transformed cell lines derived from solid tumors. We isolated allorestricted T-cell clones expressing a single defined TCR recognizing a particular HLA-A2-presented peptide derived from FMNL1. This T-cell clone showed potent antitumor activity against lymphoma and renal cell carcinoma cell lines, Epstein-Barr virus (EBV)-transformed B cells, and primary tumor samples derived from patients with chronic lymphocytic leukemia (CLL), whereas nontransformed cells with the exception of activated B cells were only marginally recognized. Allorestricted TCRs with specificity for naturally presented FMNL1-derived epitopes may represent promising reagents for the development of adoptive therapies in lymphoma and other malignant diseases.

Modulation of autocrine TNF-α-stimulated matrix metalloproteinase 9 (MMP-9) expression by mitogen- activated protein kinases in THP-1 monocytic cells

Abstract Matrix metalloproteinase 9 (MMP-9) is implicated in various physiological processes by its ability to degrade the extracellular matrix (ECM) and process multiple regulatory proteins. Normally, MMP-9 expression is tightly controlled in cells. Sustained or enhanced MMP-9 secretion, however, has been demonstrated to contribute to the pathophysiology of numerous diseases, including arthritis and tumor progression, rendering this enzyme a major target for clinical interventions. Here we show that constitutive MMP-9 secretion was abrogated in THP-1 monocytic leukemia cells by addition of neutralizing antibodies against tumor necrosis factor α (TNF-α) or TNF receptor type 1 (TNF-R1), as well as by inhibition of TNF-α converting enzyme (TACE). This indicates that MMP-9 production in these cells is maintained by autocrine stimulation, with TNF-α acting via TNF-R1. To investigate the intracellular signaling routes involved in MMP-9 gene transcription, cells were treated with different inhibitors of major mitogen-activated protein kinase (MAPK) pathways. Interruption of the extracellular signal-regulated kinase pathway 1/2 (ERK1/2) using PD98059 significantly downregulated constitutive MMP-9 release. In contrast, blockage of p38 kinase activity by addition of SB203580 or SB202190, as well as inhibition of c-Jun N-terminal kinase (JNK) using L-JNK-I1, clearly augmented MMP-9 expression and secretion by an upregulation of ERK1/2 phosphorylation. Moreover, exogenously added TNF-α augmented MMP-9 synthesis and secretion in THP-1 cells via enhancement of ERK1/2 activity. Taken together, our results indicate that ERK1/2 activity plays a pivotal role in TNF-α-induced MMP-9 production and demonstrate its negative modulation by p38 and JNK activity. These findings suggest ERK1/2 rather than p38 and JNK as a reasonable target to specifically block MMP-9 expression using MAPK inhibitors in therapeutic applications.

Proteolytic activity in the yolk sac membrane of quail eggs

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.

Low-Pigment Skin Type and Predisposition for Development of Type I Diabetes

To ascertain whether skin pigmentation type and sensitivity to ultraviolet (UV) light are associated with susceptibility to type I (insulin-dependent) diabetes, 55 type I diabetic patients were examined, 38 new-onset and 17 long-term cases. They were compared to 72 control subjects of the same geographic region and nationality. To evaluate the individual skin pigmentation type, a standardized questionnaire was developed. Reactivity to UV light was determined by a stepwisegraded UV irradiation. Significantly more diabetic patients in southern Germany had blue eyes than nondiabetic control subjects (55 vs. 26%, P < 0.01), and significantly more diabetic patients had a low-pigment eye color (blue or green) than control subjects (66 vs. 38%, P < 0.01). In addition, more fair skin color was noted among diabetic versus control subjects (84 vs. 60%, P < 0.01). In response to UV irradiation, diabetic patients more often snowed an increased UV-light sensitivity than control subjects (83 vs. 23%, P < 0.001). The relative risk for susceptibility to type I diabetes in subjects with low-pigment eye color was 3.1, in subjects with fair skin type 3.4, and in subjects with increased UV-light sensitivity 5.8. The highest risk for the development of diabetes was seen in subjects who had low-pigment eye color and/or increased UV-light sensitivity (95 vs. 51%, P = 0.00002, odds ratio 17.4). We conclude that a low-pigment skin type may predispose for the development of type I diabetes.

Proteolytic enzymes in yolk-sac membrane of quail egg. Purification and enzymatic characterisation.

Degradation of yolk protein is essential for the early development of the avian embryo. In Japanese quail (Coturnix coturnix japonica), proteolysis in the surrounding tissue of the yolk, the yolk-sac membrane, can be inhibited by class-specific inhibitors of cysteine proteinases as well as of aspartic proteinases. Purification of the enzymes leads to one cysteine proteinase and one aspartic proteinase with an apparent molecular mass of 29 kD and 44 kD, respectively. Both enzymes were purified in a two-chain form, although a single-chain form is also present in the homogenate of yolk-sac membrane. The cysteine proteinase was identified by NH2-terminal sequence analysis as well as by kinetic studies as a new cathepsin B from quail. Like mammalian cathepsin B, this avian cathepsin B exhibits two different kinds of proteolytic activity, an endopeptidase activity and a dipeptidyl carboxypeptidase activity. Chicken egg white cystatin, a protein-aceous cysteine proteinase inhibitor, inhibits quail cathepsin B with an equilibrium dissociation constant (Ki) of 3.3 nM. Likewise the aspartic proteinase was identified as a new cathepsin D from quail. This avian cathepsin D has a different processing site to all known mammalian cathepsins D. In quail cathepsin D one NH2-termini is homologous to amino acids 211-230 in mammalian cathepsin D. This is more than 100 amino acids downstream of the mammalian processing site. Comparison of the enzymatic properties of quail and bovine cathepsin D indicate that the different processing site has no influence on the enzymatic properties.

Quail cystatin: Isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B

Quail cystatin, a new cysteine proteinase inhibitor protein of the cystatin superfamily, was purified from egg albumen of Japanese quail Coturnix coturnix japonica. Amino acid sequencing and mass spectrometry revealed the complete 116 amino acid residue primary structure of a phosphorylated form (13u2008173 Da). The inhibitor has a 90% sequence identity with chicken cystatin. Its interaction with papain is rapid and tight (K i=4.4 pM; k on=1.8×107 M−1 s−1; k off=0.8×10−4 s−1) and very similar to that of chicken cystatin. Surprisingly, however, cathepsin B was inhibited 15‐fold more strongly by quail cystatin (K i=47 pM; k on=19×107 M−1 s−1; k off=9×10−4 s−1) than by chicken cystatin (K i=784 pM; k on=2.9×107 M−1 s−1; k off=24×10−4 s−1). Intuitive comparative conformational inspection of related inhibitors and of cognate enzymes suggest that: (i) the 3D structure of quail cystatin is nearly identical to that of chicken cystatin, (ii) quail cystatin can interact with cathepsin B analogous to the stefin B–papain interaction, if the `occluding loop of cathepsin B possesses an `open conformation, (iii) the greater inhibition of cathepsin B by quail cystatin compared to chicken cystatins probably arises from two additional ionic interactions between residues Arg15 and Lys112 of the inhibitor and Glu194 and Asp124 of the enzyme, respectively. The two potential salt bridges are located outside of the known contact regions between cystatins and peptidases of the papain family.