Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen: induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo.

Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.

Continuous ambulatory peritoneal dialysis impairs T lymphocyte selection in the peritoneum.

Peritoneal lymphocytes (PCL) of 45 healthy individuals, four uremic patients with end-stage renal disease (ESRD) and 25 long-term continuous ambulatory peritoneal dialysis (CAPD) patients were characterized by flow cytometry to investigate whether CAPD alters the phenotype of PCL. B lineage cells constitute a minority of PCL (2.5% of cells). Although the majority of peritoneal T cells expressed alpha beta T cell receptor (TcR), 7% expressed gamma delta TcR, a proportion which was significantly higher than that in peripheral blood (PBMC) (approximately 4%). The majority of PCL T cells exhibited markers of the thymus-dependent lineage (CD2, CD3, TcR alpha beta, CD8 alpha beta or CD4) and surface antigens associated with memory and activation (CD45RO, CD11a, CD18, CD49d, HLA-DR). An average of 75% of both CD4+ and CD8+ PCL T cells of healthy subjects and CAPD patients were CDw60+, thus characterizing the T cell subset containing the helper activity for the mitogen-driven B cell differentiation. CD44s was abundantly expressed on PCL T cells. In contrast to PCL T cells of healthy subjects peritoneal T lymphocytes of CAPD patients exhibited CD44 splice variants containing products of exon-v9 and the proportion of CD44v9+ cells correlated with the frequency of peritonitis episodes the patients had gone through. The majority of PCL T cells of both healthy subjects and CAPD patients were CD8+. A large proportion of CD8+ PCL T cells from healthy subjects expressed the homodimeric CD8 alpha alpha isoform; however, such cells were not found in CAPD patients. In healthy subjects mRNA for the recombination activating gene 1 (RAG-1) was detectable in a PCL population containing CD7-CD34+ and CD7+CD34+ cells. In contrast, neither mRNA transcripts of the RAG-1 gene nor CD34+ cells were detectable in PCL of CAPD patients.

T lymphocytes from normal human peritoneum are phenotypically different from their counterparts in peripheral blood and CD3- lymphocyte subsets contain mRNA for the recombination activating gene RAG-1.

The surface antigens of peritoneal lymphocytes of healthy human individuals were studied. B lineage cells comprised 2.3% of the total peritoneal lymphocyte population. Although the majority of peritoneal cavity lymphocyte (PCL) T cells expressed alpha beta T cell receptor (TcR), up to 17% expressed gamma delta TcR. The majority of PCL exhibited markers of the thymus-dependent lineage (CD2+ CD3+ TcR alpha beta + CD4+ or CD8 alpha + beta +) and surface antigens associated with memory and activation (CD45RO+ CD11a+ CD18+ CD49d+ HLA-DR). Up to 92% of both CD4+ and CD8+ T cells bore CDw60, thus characterizing the T cell subset containing helper activity for mitogen-driven B cell differentiation. The majority of PCL T cells were CD8+ and, in addition, up to 60% of this population expressed the homodimeric CD8 alpha + beta -. Messenger RNA for the recombination activating gene RAG-1 was examined in CD3- PCL depleted of CD19+ lineage cells. The PCL population which comprised cells containing RAG-1 mRNA transcripts was CD19-, surface IgM-, cytoplasmic IgM- and CD2- CD3- CD4- CD8- CD56-. However, this population was CD7+ (approx. 75%), and contained both CD7- CD34+ (up to 3%) and CD7- CD34+ (up to 3%) cells. These findings are compatible with the hypothesis that the adult human peritoneum provides a microenvironment capable of supporting a thymus-indenpendent differentiation of T lymphocytes.

[Sensible diagnosis in suspected immune deficiency]. / Rationelle Diagnostik bei Verdacht auf Immundefekt.

Patients reporting a marked proclivity for developing infections often present the care-providing physician with a diagnostic problem. If the available diagnostic possibilities are to be used rationally and with proper understanding, a thorough knowledge of the nature of an immune reaction is essential. The present paper contains a step-by-step scheme that may serve as an aid for the diagnostic approach. In addition to the clinical examination and routine laboratory investigations, simple tests such as Mérieux multitest and serum antibody titer serve to orientate the doctor on the patient's immune status. More sophisticated time-consuming and expensive tests, such as flow cytometric analysis of immune cells or functional in vitro investigations permit the identification of immune deficiency syndromes. These tests should, however, be undertaken only when severe immunodeficiency is suspected clinically or the orienting studies make it appear likely.

Prevention of graft-versus-host disease in DLA-haplotype mismatched dogs and hemopoietic engraftment of CD6-depleted marrow with and without cG-CSF treatment after transplantation.

Prevention of graft-versus-host disease by depletion of CD6-positive T cells was studied in the dog. Donors were DLA-homozygous, recipients DLA-heterozygous with one DLA haplotype identical to the donor. Seven control dogs received untreated marrow and died of GvHD after full hemopoietic recovery within 28 days of transplantation. For prevention of GvHD, immunomagnetic separation of T cells with a monoclonal antibody against human CD6 that crossreacted with canine T cells was evaluated. Depletion of CD6-positive cells depleted CD4-positive cells completely, but only part of CD8-positive cells and DR-positive cells. CD6-depleted marrow exhibited strong nonspecific "natural" suppression of the generation of cytotoxic T cells in vitro. Eleven dogs received CD6-depleted marrow. Only 1 dog developed GvHD and died. Sustained engraftment was seen in 8 dogs. Hemopoietic recovery was delayed and slower after transplantation of CD6-depleted marrow than after transplantation of untreated marrow. Four of these dogs were treated with G-CSF, and this accelerated the recovery of leukocytes, but did not prevent rejection. Chimerism was mixed in 7 of 10 evaluable dogs and 1 dog recovered its own hemopoiesis 2 years after transplantation. CD6 depletion prevents GvHD across a DLA-haplotype difference, but rejection and mixed chimerism may occur. Treatment with G-CSF accelerates leukocyte recovery, but cannot prevent rejection.

In vivo treatment with a monoclonal chimeric anti-CD4 antibody results in prolonged depletion of circulating CD4+ cells in chimpanzees.

Chimeric M-T412 (cM-T412), an anti-CD4 antibody, was tolerated in chimpanzees at a dosage of 5 mg/kg per day for up to 7 consecutive days, or 5 mg/kg per dose, twice weekly for 4 weeks. All cM-T412-treated chimpanzees showed a prolonged CD4-cell depression. Weak chimpanzee antibody responses to chimeric M-T412 were observed. One of the chimpanzees on the biweekly dosage regimen exhibited a hypersensitivity reaction immediately after receiving its seventh dose. Following supportive treatment, the animal recovered and remained asymptomatic during the non-treatment observation period. The hypersensitivity reaction was not an unexpected response considering the animal received repeated intermittent i.v. administration of a foreign protein. This animal also showed a chimpanzee antibody response to chimeric M-T412 after the seventh dose. Chimeric M-T412 also induced an anti-cM-T412 response in some of the other animals. The level of this response was lower than the anti-mouse responses observed in animals treated with murine anti-CD4. Moreover, the anti-cM-T412 response was mainly directed to idiotypic determinants. The decrease in CD4+ cells observed for all chimeric M-T412-treated chimpanzees is an expected effect of the anti-CD4 antibody. The duration of this CD4+ cell decrease is, however, much longer than observed for other CD4-specific MoAbs described. No selective loss of either memory or naive CD4+ cells was observed after either the single, 7-day or twice-weekly treatments. The CD4+ cell depression was reversible, although individual variation in time to recovery was observed. Therefore, cM-T412 could be a good candidate for clinical use in autoimmune conditions.

Immunological characterization of canine hematopoietic progenitor cells.

Canine hematopoietic progenitor cells were characterized by separation with monoclonal antibodies. Depleted and enriched fractions were studied for growth of CFU-GM in semisolid agar and for repopulating capacity of lethally irradiated dogs. CFU growth was not reduced by depletion of marrow using monoclonal antibodies F 3-20-7 (anti-dog Thy-1), MT606 (anti-human CD 6), and IOT2a (anti-human DR). CFU growth was variable following treatment with the anti-canine T-cell antibody MdT-P 1 and immunomagnetic bead separation. It was regularly enriched when MdT-P 1 treatment was followed by immunorosetting with staphylococcal protein A-loaded sheep red blood cells and density gradient separation. Lethally irradiated dogs were reconstituted by autologous marrow depleted of MdT-P 1-positive cells using immunorosetting and density gradient centrifugation, whereas immunomagnetic bead-depleted marrow was ineffective. Fluorescence-activated cell sorting showed enrichment of hematopoietic progenitor cells in the weakly MdT-P 1-positive fraction.

Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1.

Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B.

Functional characterization of canine lymphocyte subsets.

Functional characterization of subsets of T lymphocytes is essential for transplantation studies in dogs, as it is in other species. We studied the function of T cells separated by two mouse monoclonal antibodies recognizing complementary subsets--an antibody directed to canine T cells (MdT-P1) with an up-regulating function, and an antibody directed to human CD 8 (MT811) that cross-reacts with down-regulating canine T cells. Immunorosetting with sheep red blood cells and Percoll gradient allowed us to study depleted and enriched fractions. Their function was tested in mixed lymphocyte culture (MLC), cell-mediated cytotoxicity (CML), and coculture with B cells in a hemolytic plaque assay (PFC). In MLC, MdT-P1-positive cells showed a high proliferative response, and MT811-positive cells responded poorly to allogeneic cells. Vice versa, MT811- negative cells responded strongly, and MdT-P1-negative cells were poor responders but strong stimulators. Effector cells of CML were separated following 8 days of culture and prior to mixing with target cells. Enriched and depleted fractions with either antibody showed low cytotoxic activity as compared with unseparated cells. When added to unseparated effector cells MT 811-positive cells suppressed cytotoxicity. B cells were obtained by rosetting with staphylococcal protein A (SPA). Their immunoglobulin production was studied following 6 days of culture stimulated by pokeweed mitogen in a reverse hemolytic plaque assay. Again, MT 811-positive cells added to the culture suppressed, and MT 811-negative cells enhanced immunoglobulin production. In conclusion, immunorosetting with two monoclonal antibodies allowed us to distinguish subpopulations of canine T cells with up-regulating (helper/inducer) from those with down-regulating (suppressor) activity.

Anti-idiotypic antibodies against anti-CD4 antibodies MT151 and OKT4A.

Anti-idiotypic antibodies (Ab2) binding to the antigen-combining site of other antibodies may functionally and even structurally mimic antigen. Ab2 to antibodies directed against the lymphocyte CD4 receptor for human immunodeficiency virus type 1 (HIV-1) may mimic the receptor and therefore inhibit viral infectivity. We have produced Ab2 against monoclonal anti-CD4 receptor antibodies (Ab1). The Ab1 strongly inhibit HIV-1 binding to the receptor. Six monoclonal rat Ab2 and two polyclonal rabbit Ab2 were produced against the Ab1 MT151 and nine monoclonal Ab2 against the Ab1 OKT4A. These Ab2 bound only to Ab1 and not to a panel of nine unrelated murine monoclonal antibodies (MAbs). The Ab2 completely inhibited the binding of the homologous Ab1 to CD4-positive target cells, and recombinant soluble CD4 inhibited binding of Ab2 to Ab1. Thus, the Ab2 seemed to mimic the Ab1-binding site of the CD4 receptor, although the results of inhibition assays did not exclude steric hindrance of antibody-combining sites. However, none of the 17 Ab2 bound to gp120 of HIV-1 envelope or inhibited syncytia formation between cells infected and uninfected with HIV-1. These results suggest that the Ab2 do not mimic the HIV-1 binding site of the CD4 receptor. They further suggest that the Ab1 may not bind within the virus-binding site of the CD4 receptor.

Reconstitution of transferrin receptor in mixed lipid vesicles. An example of the role of elastic and electrostatic forces for protein/lipid assembly.

We studied the interaction of transferrin receptors (of cell line Molt-4) with mixed model membranes as a function of lipid chain length (phospholipids with C14:0 and C18:1 hydrocarbon chains) and of the surface charge of the membrane using mixtures of C14:0 lecithin (DMPC) with C14:0 phosphatidylglycerol (DMPG) and C14:0 phosphatidylserine (DMPS). Spontaneous self-assembly of receptors and lipids was achieved by freeze-thaw cycles of a codispersion of mixed vesicles and receptors in buffer and subsequent separation of receptor-loaded and receptor-free vesicles by density gradient centrifugation. Information on specific lipid/protein interaction mechanisms was obtained by evaluation of protein-induced shifts of phase boundaries of lipid mixtures by calorimetry and by FTIR spectroscopy of partially deuterated lipid mixtures. The important role (1) of minimizing the elastic forces caused by the mismatch of the lengths of hydrophobic cores of the protein (lp) and the bilayer (lL) and (2) of the electrostatic coupling of protein head groups with the charged membrane/water interface for the lipid/protein self-assembly is established. The electrostatic interaction energy per receptor is about 10(3) kBT (by coupling to about 1000 charged lipids) which is sufficient to overcompensate the elastic energy associated with a mismatch of lp - lL approximately 1.0 nm. The maximum receptor concentration incorporated was measured as a function of membrane surface charge and lipid chain length. The maximum receptor molar fraction varied from xpmax = 5 x 10(-5) for DMPC to xpmax = 4 x 10(-4) for 1:1 DMPC/DMPG; moreover xpmax is higher for DMPS than for DMPG as charged component. For the long-chain lipids, xpmax is higher for a 9:1 DEPE/DEPC mixture [(4.2-9) x 10(-4)] than for pure DEPC (ca. 3.5 x 10(-4)). By decomposition of reconstituted receptors with proteases, we demonstrated the homogeneous orientation of the receptor with its extracellular head group pointing to the convex side of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-CD4 antibody treatment of patients with rheumatoid arthritis: I. Effect on clinical course and circulating T cells.

Eight patients with arthritis (seven with rheumatoid, one with psoriatic arthritis) were treated for 7 d with a daily injection of 10 mg of mouse monoclonal anti-CD4 antibodies (three with VIT4, five with MT151). With the exception of a short-lasting low-grade fever in one patient, no side effects were observed. Clinical symptoms (morning stiffness, number of swollen joints, pain assessment and Ritchie articular index) improved in all patients within 7 d of treatment. Improvement lasted from 3 weeks to greater than or equal to 5 months (mean approximately 11 weeks). Rheumatoid factors, immune complexes and other laboratory parameters did not change during or after treatment. Skin reactivity to recall antigens was suppressed in four out of six patients during treatment but returned to pretreatment levels within 6 weeks. Immunofluorescent analysis revealed a short-lasting drop of T cells, mainly of the CD4+ CDw29+ subset, but monocytes were also affected. The injected antibody was detectable on circulating cells for about 10 h. Within 20-24 h, the cell distribution returned to pretreatment levels. In six out of eight patients an anti-mouse-Ig response was seen. We conclude that mouse anti-CD4 monoclonal antibody (MoAb) treatment is well tolerated and that the cellular immunological changes observed are short-lasting. The low incidence of side effects may justify further clinical studies to evaluate the clinical efficacy of such treatment.

Anti-CD4 antibody treatment of patients with rheumatoid arthritis: II. Effect of in vivo treatment on in vitro proliferative response of CD4 cells.

In treating rheumatoid arthritis (RA) patients with anti-CD4 antibodies (MT151 10 mg/day, 7 d) we observed diminished skin-test response to recall antigens and reduced proliferative response to antigens and mitogens (in vitro) 2-4 h following the injection. To investigate whether this diminished response is due to functional impairment of CD4+ cells, we analyzed the proliferative response of CD4+ cells to various stimuli, in conditions where the CD4 structure was either cross-linked or not. We found that in vivo anti-CD4-antibody-coated cells could be induced to proliferate if the anti-CD4 antibody was cross-linked with an anti-CD3 antibody [BMA030-F(ab')2] added in vitro and per se non-mitogenic. Also, without cross-linking the cell-bound anti-CD4 antibodies, no impairment of the proliferative capacity of CD4-antibody-coated cells could be detected; double fluorescence analyses of phytohaemagglutinin (PHA)- or CD3-stimulated cells showed that a similar proportion of CD4+ cells proliferated before and after anti-CD4-antibody treatment. The diminished proliferation observed following anti-CD4 treatment correlated to the reduced number of CD3+ and CD4+ cells in culture, which suggests that altered cell distribution might be one factor contributing to the impaired immune response.

Towards a better definition of human leucocyte surface molecules.

At the 4th International Workshop on Human Leucocyte Differentiation Antigens workers from 525 laboratories jointly evaluated and/or submitted about 1100 different monoclonal antibodies to human leucocyte surface molecules. At the final conference of the workshop agreement was reached on 35 new cluster determinants (CDs) and subclusters. In addition, seven previously established clusters were redefined. This report provides basic information concerning the molecules defined by these antibodies and some highlights of the conference.