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R L Bolhuis

Publications and source records attributed to R L Bolhuis.

At least 19 recordsLinked to original sources
PubMed1992

Heterodimeric complex formation with CD8 and TCR by bispecific antibody sustains paracrine IL-2-dependent growth of CD3+ CD8+ T cells.

During physiologic activation of mature CD8+ T cells, TCR and CD8 bind to the same Ag-complexed MHC class I molecule. Thereby, close proximity is induced between CD8 and the TCR/CD3 complex. During this engagement, CD8 may deliver TCR-independent signals via its associated protein tyrosine kinase, p56lck. We studied the potential biologic effects of close association between CD8 and TCR/CD3 complexes by using a bispecific antibody (bsAb) directed against both TCR and CD8 molecules. This hybrid hybridoma (quadroma)-produced bsAb binds as a monomeric molecule to CD3+ CD8+ but not CD3+ CD4+ T cells. The bsAb proved capable of inducing the cytotoxic effector function of cloned CD3+ CD8+ T cells but not of CD3+ CD4+ T cells. When the bsAb was presented to resting T cells by monocytes, proliferation of the CD3+ CD4+ but not the CD3+ CD8+ subset of T lymphocytes was induced. Parental anti-TCR antibody induced vigorous growth of cells of both subsets. Essentially identical results were obtained when bsAb was presented in an immobilized fashion. The unresponsiveness of the CD3+ CD8+ T cells with respect to mitogenesis could be restored by exogenous rIL-2. The data suggest that bsAb-induced activation differs from activation by monospecific anti-TCR antibody. The former appears to more closely mimic physiologic Ag-induced signaling, because it leads to a similar paracrine IL-2-dependent growth pattern. The bsAb may, therefore, be instrumental in studying T cell signaling pathways, in particular the role of CD8-associated p56lck therein.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Optimization of culture conditions for activation and large-scale expansion of human T lymphocytes for bispecific antibody-directed cellular immunotherapy.

We investigated the optimal culture conditions (i.e., activation procedure, medium composition and type of culture vessel) for rapid in vitro expansion of large numbers (greater than 5 x 10(9) of blood T lymphocytes. These expanded lymphocytes can be targeted to be cytotoxic to ovarian carcinoma cells with a bispecific monoclonal antibody (BsAb) specific for CD3 and for the ovarian carcinoma-associated antigen MOv18. Both phytohemagglutinin (PHA) and monoclonal antibody (MAb) CD3 induced rapid T-cell proliferation, although the growth kinetics after PHA activation were slightly faster. A 50-fold increase in cell number was obtained after 14 and 16 days for PHA and CD3 MAb, respectively. The induction of BsAb-directed cytolysis was faster after CD3 MAb than after PHA activation of lymphocytes, but became similar around day 20. A mixture of media consisting of 78% RPMI 1640, 20% AIM-V and 2% human plasma (Mix-med) yielded better results than 100% AIM-V medium. Culture of lymphocytes in polyolefin bags, compared with tissue culture flasks, or cryopreservation did not affect lymphocyte yield and function. In most cultures the proportion of CD8+ lymphocytes increased, suggesting a growth advantage of CD8+ over CD4+ lymphocytes in this culture system. A protocol employing PHA activation, Mix-med and polyolefin bags has been used successfully to activate and expand blood lymphocytes for the first 5 patients entered into a phase-I/II clinical trial for the intraperitoneal treatment of ovarian carcinoma using CD3 x anti-MOv18 BsAb-directed T lymphocytes.

Antibodies, Monoclonal↗
PubMed1992

Genetic correction of cultured T cells from an adenosine deaminase-deficient patient: characteristics of non-transduced and transduced T cells.

T lymphocytes derived from peripheral blood of a patient with adenosine deaminase (ADA) deficiency were expanded in vitro. The human ADA (hADA) gene was introduced into these replicating ADA- T cells with the use of an amphotropic recombinant retrovirus carrying the hADA gene. Subsequently, infected T cells were selected on the basis of their ADA expression, by exposure to a combination of the toxic agent xylofuranosyl-adenine and the specific ADA inhibitor 2'-deoxycoformycin. CD4+ and CD8+ T cells could be infected and selected with equal efficiencies. The genetically modified T cells were shown to contain intact copies of the provirus and to express normal levels of hADA. As expected, uninfected T cells from the ADA-deficient patient displayed an increased sensitivity to 2'-deoxyadenosine. Following genetic modification, however, this sensitivity was restored to normal levels in both CD4+ and CD8+ T cells. The introduction of the hADA gene into the genome of the in vitro expanded T cells did not alter their phenotype, proliferative capacity or cytotoxic potential. These characteristics were identical to those of T cells derived from healthy individuals. These findings are of critical importance for the clinical application of hADA gene-transducted T cells.

Adenosine Deaminase↗
PubMed1992

T-cell receptor gamma/delta: comparison of gene configurations and function between humans and chimpanzees.

The human and chimpanzee T-cell receptor gamma-delta (TCR gamma delta) bearing cells represent a minor subset (3-8%) of T lymphocytes. In the periphery, the TCR gamma delta population has a restricted combinatorial repertoire. The TCRD-V1 and -V2 gene products are expressed in a mutually exclusive fashion, whereas, the TCRD-V2 and the TCRG-V9 encoded proteins show, in general, a coordinated expression. Restriction fragment length polymorphism analysis showed conservation of the restriction sites that identify the TCRG-V9 and TCRD-V2 rearrangements. The human TCRG-V9 locus has two alleles, TCRG-V9A1 and TCRG-V9A2 differing at codon position 31. The chimpanzee TCRG-V9 gene product differs from the products of the human TCRG-V9A1 and TCRG-V9A2 allele by two and three amino acid replacements, respectively. The human and the chimpanzee TCRG-V9-TCRD-V2 lymphocytes show a similar specific proliferative and cytolytic response to human Daudi Burkitt's lymphoma cells. Therefore, the amino acid replacements found in the chimpanzee TCRG-V9 gene product do not change the superantigen specificity across this species barrier.

Alleles↗
PubMed1992

Epstein-Barr virus infection in allogeneic marrow grafting: lessons for transplant physicians and virologists.

The relationship between Epstein-Barr virus (EBV) and the host is profoundly disturbed by allogeneic bone marrow transplantation (BMT) because EBV resides in the recipient's hematopoietic system, which has to be destroyed in the majority of cases, and in the donor's hematopoietic system, i.e., the marrow graft. We have shown that EBV may be eradicated from some BMT recipients and that the virus may be transferred with the marrow graft. During the immediate post-transplant period oropharyngeal EBV excretion may occur which, by infecting passing B lymphocytes, may act as co-factor for acute graft-versus-host disease and help the virus to survive, despite the temporary depletion of its reservoir. The coexistence of totally different EBV strains in BMT recipients but not in healthy, untransfused controls, suggests that superinfection may by possible in case of immunodeficiency; alternatively, transfer of the virus by the reservoir itself (the B lymphocytes) might be the only effective route for superinfection. The generation of 'variant' strains during viral replication may form the basis of the vast polymorphism between wild-type EBV isolates in the population.

Adolescent↗
PubMed1992

CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes.

We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3- CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.

Animals↗
PubMed1992

Interleukin-2 and interferon-alpha in the treatment of patients with advanced non-small-cell lung cancer.

The role of combination chemotherapy in the treatment of advanced non-small-cell lung cancer is controversial. At best, a small survival benefit can be achieved. Therefore, other treatment modalities are needed. On the basis of the promising treatment results with interleukin-2 (IL-2) -containing immunotherapy in renal cell cancer and melanoma, we performed a phase I-II study with IL-2 and interferon alpha (IFN-alpha). Eligible patients were treated with IL-2 18 x 10(6) IU/m2/day by continuous intravenous infusion (c.i.v.) for 3 days. On the same days, 5 x 10(6) U/m2/day IFN-alpha was given intramuscularly. After a rest period of 4 days, patients at the first dose level received IL-2 2.4 x 10(6) IU/m2/day c.i.v. for a period of 28 days, followed by 14 days' rest, 14 days' treatment, 7 days' rest, and a final treatment for 14 days. Patients at the second dose level were treated according to the same schedule, in which the dose of IL-2 was increased to 3.6 x 10(6) IU/m2/day. During low-dose IL-2 treatment, patients received IFN-alpha 5 x 10(6) U/m2/day on days 1 and 4 of each week. Eleven patients were admitted to the study, six at the first and five at the second dose level. Median age was 54 years; all patients had a performance status of 0 or 1. The most important adverse effects included anorexia, fatigue, nausea, and headache, which were not dose limiting. In the 11 patients treated, no responses were seen. Nine patients developed progressive disease during the first 5 weeks of treatment. We concluded that this regimen of IL-2 and IFN-alpha is ineffective.

Adult↗
PubMed1992

Repetitive weekly cycles of 4-day continuous infusion of recombinant interleukin-2: a phase I study.

A phase I trial was performed with a new interleukin-2 (IL-2) given as a continuous intravenous infusion in patients with solid tumors. The objectives of the study were to examine the feasibility of administering IL-2 in 4-day cycles for 4 consecutive weeks, and to investigate the response pattern of peripheral blood lymphocytes. Tumor necrosis factor (TNF) and IL-2 serum concentrations were also measured. Prior to this study, IL-2 had been tested at increasing dosages during one 4-day cycle, and it appeared that a dose of 1300 mcg/m2/day was tolerated. However, when this treatment schedule was maintained for 4 consecutive weeks, the maximum tolerated dose was 430 mcg/m2/day. In this schedule, a dose-dependent progressive increase in rebound lymphocyte count occurred after each weekly cycle, resulting in a 5-70-fold increase after the 4th cycle. Serum TNF peak concentrations also showed a tendency to increase during each subsequent cycle, while serum IL-2 peak concentrations showed a paradoxical decrease. Clinical toxicity comprised several events, which, possibly, could be ascribed to autoimmune phenomena. Myocardial infarction as a late toxicity of IL-2 is suggested. One complete response (renal carcinoma) and two partial responses (renal and breast carcinoma) were documented, one of these occurring in a patient who previously had shown a transient response on interferon therapy.

Adult↗
PubMed1992

Adoptive immunotherapy of ovarian carcinoma with bs-MAb-targeted lymphocytes: a multicenter study.

A bispecific monoclonal antibody (bs-MAb) (OC/TR) was produced in large quantities for the intraperitoneal (i.p.) treatment of ovarian cancer. The bs-MAb recognizes the folate-binding protein on ovarian cancer cells on the one hand and the CD3 activation site on T lymphocytes on the other. T lymphocytes were expanded ex vivo, targeted with OC/TR in vitro and administered to the i.p. cavity in the presence of soluble OC/TR. All patients developed human anti-mouse-antibodies (HAMA). In the Dutch study, 2 complete remissions (CR) were seen, 2 partial regressions (PR), I stable disease (SD) and I progressive disease (PD). In the Italian study 3 CR, I PR, I SD and 2 PD were seen.

Antibodies, Monoclonal↗
PubMed1991

Simultaneous flow cytometric detection of antibodies against platelets, granulocytes and lymphocytes.

We present a time-saving and objective flow cytometric immunofluorescence assay for the simultaneous detection of antibodies against platelets, granulocytes or lymphocytes using a reconstituted mixture of these cell populations. Platelets, granulocytes and lymphocytes could be distinguished on the basis of their forward (FSC) and sideways (SSC) light scattering properties plotted on scales of 4 log orders. After setting FSC/SSC gates around the platelets, granulocytes and lymphocytes, the reactivity of the sera with the cell populations was determined by histogram analyses of immunofluorescence for each gate. The flow cytometric assay of reconstituted cell mixtures showed a strong, positive correlation with a reference microscopic immunofluorescence assay of separate cell suspensions. The reproducible procedures for the isolation and staining of the cells and the electronic stability of the flow cytometer permitted the use of the same gate and marker settings throughout the experiments. Consequently, the entire analysis of data stored in list mode could be performed using a keystroke, so that time consuming and subjective manual analyses were avoided.

Antibodies↗
PubMed1991

Exogenous interleukin 2 recruits in vitro lymphokine-activated killer activity by in vivo activated lymphocytes.

Cryopreserved and thawed lymphocytes can be used instead of fresh lymphocytes to avoid test-to-test variability in studies of fluctuations of natural killer (NK) and lymphokine-activated killer (LAK) activities as a function of time. We investigated the effects of 1-h versus 18-h resting of lymphocytes on their lytic activities, because the process of cryopreservation and thawing decreases NK and LAK activities. Lymphocytes from renal cell cancer patients receiving adoptive immunotherapy were studied. An 18-h versus 1-h resting period led to a significant increase in NK activity but had no significant effect on LAK activity. The presence of 1200 IU/ml interleukin 2 (IL-2) in the medium 1 h prior to and during the cytotoxicity (CTX) assay increased in vivo and in vitro IL-2-induced LAK activities. This phenomenon has been interpreted as IL-2 dependency of effector lymphocytes (J.A. Hank, P.C. Kohler, G. Weil-Hillman, N. Rosenthal, K. H. Moore, B. Storer, D. Minkoff, J. Bradshaw, R. Bechhofer, and P. M. Sondel. Cancer Res., 48: 1965-1971, 1988). We performed kinetic studies to assess the role of effector lymphocyte recruitment in these experiments. LAK activity was tested in the presence or absence of IL-2 during preincubations and CTX assays varying between 0 and 120 min. These kinetic studies showed that effector lymphocyte recruitment indeed contributed to the increased level of LAK activity when IL-2 was added to the CTX assay. A minimal incubation period of 30 min was required to detect recruitment of lymphocytes. Effector lymphocytes could be recruited for periods varying between 90 and greater than 240 min, depending on the lymphocyte donor. We conclude that: (a) in vitro, IL-2-mediated recruitment of lymphocytes due to presence of IL-2 in the CTX assay may lead to an overestimate of the actual LAK activity; and (b) in vivo, prolonged IL-2 infusion after the administration of activated lymphocytes seems warranted in order to recruit maximal levels of effector lymphocytes with LAK activity.

Carcinoma, Renal Cell↗
PubMed1991

Engineering T lymphocyte antigen specificity.

Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review.

Animals↗
PubMed1991

T cell targeting in cancer therapy.

Targeting of immune cells by bispecific antibodies has proven a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction are the major topics of this review.

Antibodies, Monoclonal↗
PubMed1991

Expression of CD45 isoforms by fresh and activated human gamma delta T lymphocytes and natural killer cells.

Naive and primed alpha beta T cells can be distinguished on the basis of their differential expression of CD45RA and CD45RO, respectively. The present study indicates that these CD45-isoforms also identify naive and primed maturational stages of gamma delta T cells and natural killer (NK) cells. In peripheral blood, all V gamma 9-V delta 2 gamma delta T cells reportedly express CD45RO whereas all V delta gamma delta T cells lack CD45RO. Here, we show that these CD45RO- V delta gamma delta T cells all express CD45RA and the CD45RO+ V.9-V delta 2 gamma delta cells lack expression of CD45RA. The V delta T cells acquired CD45RO expression and lost part of their surface CD45RA, following in vitro activation with phytohaemagglutinin or IL-2. Also the CD3-CD16+ NK cells in peripheral blood that are uniformly CD45RA+ CD45RO- completely converted to the CD45RA-CD45RO+ phenotype upon in vitro activation. Moreover, all cloned V.9-V delta 2 and V delta 1 T cells and NK cells express CD45RO and lack expression of CD45RA. Our results strongly suggest that CD45RA and CD45RO are genuine markers for naive and primed lymphocytes that represent distinct differentiation lineages.

Antigens, CD↗
PubMed1991

Clinical experience with the combined use of recombinant interleukin-2 (IL2) and interferon alfa-2a (IFN alpha) in metastatic melanoma.

A multicentre study of IL2 and IFN alpha has been performed in 58 patients with metastatic melanoma. The scheme consisted of IL2 3.0 BRMP MU/m2/d as a continuous infusion for 4 d combined with subcutaneous administration of IFN alpha 6 MU/m2/d, day 1 + 4. The cycle was repeated every 2 weeks for a maximum duration of 26 weeks. 54 patients were evaluable for response. One (2%) achieved a complete and 10 (19%) a partial response. 19 (35%) patients were stable and 24 (44%) showed progressive disease. Common side-effects included fever, chills, fatigue, skin rash, anorexia, nausea and diarrhoea. Hypothyroidism was noted in 10% of the patients. These results show that this regimen of IL2 and IFN alpha is active but, in contrast to what could be expected, not superior to IL2 alone possibly due to suboptimal dosing. In an ongoing study in Rotterdam and Nijmegen, a more intense schedule was chosen, consisting of three daily i.v. doses of IL2 4.5 BRMP MU/m2 and IFN alpha 3.0 MU/m2 for 5 d. This regimen is repeated at intervals of 3 weeks for a total of three cycles. Presently, nine patients have been entered. One patient achieved a complete response, four a partial response (overall 56%), three had stable disease and one progressed. Toxicity was severe and treatment was prematurely stopped in five patients: myocardial infarction (one patient), atrial fibrillation (one patient), negative T waves and myocardial hypokinesia (one patient) and psychosis (two patients). This regimen can only be justified if the therapeutic results are superb, which has yet to be awaited.

Adolescent↗