Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Human primary T lymphocytes have a low capacity to amplify MLV-based amphotropic RCR and the virions produced are largely noninfectious.

The presence of replication-competent retrovirus (RCR) in retroviral-based gene therapy products poses a potential safety risk for patients. Therefore, RCR testing of clinical gene therapy products and monitoring of patients enrolled in gene therapy trials is required to assure viral safety. The requirement to test ex vivo-transduced cells originates from the presumed amplification of adventitious RCR during the transduction procedure. However, data on the capacity of different cell types to do so are lacking. In this study, we sought to analyze the amplification potential of primary human T lymphocytes after infection with amphotropic MLV-based RCR. The total number of viral particles produced after 1 or 2 weeks was measured by a quantitative 4070A env-specific RT-PCR assay. The fraction of infectious replication-competent viral particles was analyzed in the PG-4 S+L- assay. From this study, we conclude that the total number of viral particles RCR produced by T lymphocytes is 2-4 logs lower than the number produced by NIH-3T3 cells. Surprisingly, less than 1% of the viral particles produced by primary T lymphocytes appeared to be infectious, while nearly all virions produced by NIH-3T3 were. We conclude that primary human T lymphocytes are low producers of MLV-based amphotropic RCR.

Ganciclovir-mediated in vivo elimination of myeloid leukemic cells expressing the HSVtk gene induces HSVtk loss variants.

The in vivo elimination of suicide gene-expressing tumor cells with prodrug treatment can induce protective immunity against wild-type tumors. In this study, we determined the efficacy and safety of the in vivo elimination of HSVtk expressing cells with ganciclovir treatment of a bystander cell killing-insensitive leukemic cell line. The retroviral construct pLTk+NeoDeltaMo, containing the HSVtk gene and the NeoR gene in a bicistronic unit, was introduced into rat leukemic LT12 cells. LT12/Tk+N cells showed a 1000- to 10 000-fold increased sensitivity to ganciclovir in vitro. In vitro mixing experiments demonstrated that LT12 cells were not susceptible to bystander cell lysis by LT12/Tk+N-2 cells exposed to ganciclovir. Rats injected s.c. with cloned LT12/Tk+N-2 cells developed tumors reaching a diameter of 3-4 cm after 40 days. Rats treated with gan- ciclovir twice daily for 5 consecutive days starting at day 7 did not develop s.c. tumors. Large established s.c. LT12/Tk+N-2 tumors completely regressed after ganciclovir treatment. However, recurrences of s.c. tumors were observed that were no longer sensitive to ganciclovir treatment. In vitro analysis of aspirates from the recurrent tumors demonstrated loss of HSVtk expression. In vitro culture of LT12/Tk+N-2 cells in soft agar in the presence of ganciclovir indicated that the frequency with which HSVtk-loss variants occurred is approximately one per 104 cells. The in vivo occurrence of HSVtk-loss variants escaping ganciclovir-induced elimination may have important implications for vaccination protocols using HSVtk gene expressing tumor cells that are not susceptible to bystander cell killing.

Comparison of single and dual-platform assay formats for CD34+ haematopoietic progenitor cell enumeration.

Most techniques for CD34+ cell enumeration are dual platform assays. That is, they derive absolute numbers of CD34+ cells from either the flow cytometrically assessed per cent (%) CD34+ cells within the nucleated cells and/or the white blood cell count from a haematology cell analyser. Recently, so-called single-platform assays have been developed, in which the absolute number of CD34+ cells is directly derived from a single flow cytometric measurement. The present study aims to compare the variation between eight laboratories in CD34+ cell counts from paired assays of 15 samples using a common single (ProCOUNT) and the local dual-platform method. Six laboratories used the 'SIHON' and two the 'ISHAGE' protocol for CD34+ cell enumeration. Use of the single-platform method reduced the inter-laboratory variation in per cent and absolute numbers of CD34+ cells, as measured by interquartile ranges, by half but did not lead to an appreciable reduction of the inter-laboratory variation in white blood cell counts. Thus, part of the reduced inter-laboratory variation obtained with ProCOUNT may have been a result of the use of standardized procedures and reagents to detect CD34+ cells. In order to eliminate any variation arising from the use of different local protocols for percentage of CD34+ cell assessments, a comparison was made of the ProCOUNT-derived absolute CD34+ cell numbers (i.e. single platform) with the dual-platform absolute CD34+ cell numbers calculated by multiplying ProCOUNT-derived percentage of CD34+ cells and with the corresponding haematology analyser-derived white blood cell count. Regardless, the interquartile ranges of absolute CD34+ cell numbers remained almost a factor of two smaller with the use of the single platform method. Thus, these results suggest that single-platform methodology can reduce the variation in absolute CD34+ cell numbers between laboratories.

Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences.

In this study, we describe the efficiency of second gene translation in bicistronic constructs containing either a short (36bp) synthetic intercistron or known internal ribosomal entry sites (IRES). Experiments were performed using two different gene combinations: Herpes simplex virus-thymidine kinase (HSV-TK) and neomycine (NEO) or human glucocerebrosidase (hGC) and a methotrexate (MTX) resistant mutant dihydrofolate reductase (DHFR). We demonstrate that upon transfection, second gene translation is efficient using either an IRES or a 36-bp intercistron. Infection with retrovirus carrying the TK and NEO genes linked via a 36-bp intercistron resulted in both G418R (NEO expression) and gancyclovir (GCV) sensitivity (TK expression), indicating that both genes were expressed and thus that the genomic DNA and RNA of this bicistronic construct were intact. Likewise, retrovirus carrying the hGC and mutant DHFR gene separated by a short intercistron was harvested from MTXR murine PsiCRE cells. However, infection of PA317 cells with this virus supernatant did not result in the presence of hGC enzyme activity in these murine cells. Proviral DNA and RNA analyses indicated that the hGC coding region was lost from the original construct in the infected PA317 cells. In contrast, retrovirus carrying the hGC and DHFR cDNAs was linked via an IRES functioned as expected. Based on these results, we conclude that the efficiency of second gene translation using short synthetic intercistrons might prove useful in bicistronic constructs, depending on the gene combination used.

Influence of ultraviolet-B irradiation on engraftment, graft-versus-host disease and graft-versus-leukemia effect in a rat model for allogeneic bone marrow transplantation.

Ultraviolet-B (UVB) irradiation is known to inhibit lymphocyte activity and consequently to reduce the incidence of graft-versus-host disease (GVHD) in experimental models for allogeneic bone marrow transplantation (BMT). GVHD is frequently associated with morbidity and mortality, but also with the beneficial graft-versus-leukemia (GVL) effect, demonstrated by a reduction in the incidence of leukemia relapse. In this study, we investigated whether UVB treatment of allogeneic T cells could prevent GVHD while sparing the beneficial GVL effect following allogeneic BMT in the Brown Norway myelocytic leukemia (BNML) rat model analogous to human acute myelocytic leukemia (AML). The dose of UVB required to abolish lethal GVHD in the rat allogeneic BMT model (WAG/Rij donors into BN recipients) was 4000 J/m2. However, this UVB dose simultaneously abrogated all GVL activity mediated by the T cells in the graft, while the radio-protective capacity of rat BM cells was strongly reduced. The number of allogeneic BM cells required to protect lethally irradiated BN rats was increased 50 to 100-fold. It is concluded that UVB acts as a non-selective form of T cell inactivation, and that UVB pretreatment of an allogeneic marrow graft is unlikely to be useful clinically as a preventive measure for GVHD, since other means of reduction of the number of functional T cells are less damaging to bone marrow stem cells.

Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved.

Ultraviolet B (UVB) light is known to be immunosuppressive, but, probably because of a small UVC component in the emission spectra of some of the UVB lamps used, reports vary on effective dose levels. To prevent potentially lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, alloreactive donor T-cell activity must be suppressed. In this study, a narrow wavelength UVB lamp (TL01, 312 nm peak emission) was used to determine what doses of UVB were required to abolish rat lymphocyte proliferation while simultaneously preserving rat bone marrow progenitor cell and primitive hematopoietic stem cell viability. Lymphocyte proliferation, as measured by 3H-Thymidine incorporation, in response to lectin stimulation was abolished below detection at doses greater than 3,500 J/m2. When T-cell clonogenicity was measured in a limiting dilution assay, a small fraction (0.6%) was maintained at doses up to 4,000 J/m2. Cytotoxic T-lymphocyte (CTL) activity was reduced after treatment with 4,000 J/m2, but a significant level of cytotoxicity was still maintained. Natural killer cell cytolytic activity was not affected by doses up to 4,000 J/m2. At 4,000 J+m2 there was a 10% survival of colony-forming units-granulocyte-macrophage; a 1% and 4% survival of day-8 and day-12 colony-forming units-spleen, respectively; and 11% survival of marrow repopulating ability cells. Up to 25% of late cobblestone area forming cells (4 to 5 weeks), reflecting the more immature hematopoietic stem cells, were preserved in bone marrow treated with 4,000 J/m2, indicating that early stem cells are less sensitive to UVB damage than are more committed progenitor cells. Thus, a potential therapeutic window was established at approximately 4,000 J/m2 using this light source, whereby the potentially GVHD-inducing T cells were suppressed, but a sufficient proportion of the cells responsible for engraftment was maintained.

Clustered CD3/TCR complexes do not transduce activation signals after bispecific monoclonal antibody-triggered lysis by cytotoxic T lymphocytes via CD3.

Bispecfic mAb (bsmAb) directed against the CD3/TCR complex and a tumor-associated Ag (TAA) induces CTL-mediated lysis of TAA+ target cells. We have investigated whether bsmAb-pretargeted CTL can enter multiple lytic cycles. BsmAb-pretargeted CTL retained bsmAb-targeted lytic capacity for at least 24 h when exposed to medium without TAA+ target cells. Exposure of bsmAb-pretargeted CTL to TAA+ target cells resulted in a rapid loss of bsmAb-targeted cytotoxicity of TAA+ or Fc gamma R+ target cells, although the CTL retained surface bsmAb. Moreover, addition of rabbit anti-mouse lg to these CTL did not induce calcium mobilization. These CTL still showed Ag-specific cytotoxicity and cytolysis of anti-CD3 mAb-expressing hybridoma cells. Readdition of bsmAb to CTL that had lost bsmAb-targeted cytotoxicity instantly restored the bsmAb-targeted lytic activity of the CTL. Hence, as in Ag-specific cytolysis, bsmAb-pretargeted CTL can enter multiple bsmAb-targeted cytolytic cycles. Surprisingly, exposure of bsmAb-pretargeted CTL to Fc gamma R+ cells did not result in loss of bsmAb-targeted cytolysis of TAA+ cells. Fluorescence microscopic analysis revealed that bsmAb-mediated interaction with TAA+ cells, but not with Fc gamma R+ cells, resulted in clustering of bsmAb-pretargeted CD3/TCR complexes on the CTL surface. On the basis of the observed correlation between clustered bsmAb-pretargeted CD3/TCR complexes and loss of bsmAb-targeted cytotoxicity, we hypothesize that clustered CD3/TCR complexes can no longer transduce signals.

Retrovirus-mediated gene transfer into rhesus monkey hematopoietic stem cells: the effect of viral titers on transduction efficiency.

We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until > 1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for > 2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.

Activation of the immune system of cancer patients by continuous i.v. recombinant IL-2 (rIL-2) therapy is dependent on dose and schedule of rIL-2.

The effect of dose and schedule of continuous i.v. rIL-2 infusions on leucocyte subset counts, activation status of CD56+CD3- natural killer (NK) and CD3+ T lymphocytes, and cytolytic activities of peripheral blood mononuclear cells (PBMC) was studied. A single 4-day course of rIL-2 in escalating doses (0.9-11.5 x 10(6) U/m2 per day) was given to 18 patients with various types of metastatic cancer. The serum IL-2 concentration during rIL-2 therapy ranged between 23 and 64 U/ml and was proportional to the administered rIL-2 dose, as was the rebound lymphocytosis following therapy. Before therapy, the CD56+CD3- NK cells expressed low levels of the p75 chain of the IL-2 receptor (IL-2R) and virtually no IL-2R(p55). Most CD3+ T cells were IL-2R(p55-,p75-). Between 2 and 4 days following therapy, i.e. at the time of lymphocytosis, the percentage of CD56+,CD3- NK cells among the lymphocytes had increased proportional to the administered rIL-2 dose. The levels of IL-2R(p75) expression by the CD56+,CD3- NK cells had increased. The percentages of CD3+ T cells expressing IL-2R(p55), HLA-DR and CD45RO had increased proportional to the administered rIL-2 dose. The level of lymphokine- activated killer (LAK) activity against Daudi cells was also positively correlated with rIL-2 dose. Subsequently, seven patients received 4-weekly cycles of rIL-2 (2.9-4.4 x 10(6) U/m2 per day) during 4 consecutive weeks. This schedule led to marked increments in lymphocyte and eosinophil counts, and to increased cytolytic activities compared with pretreatment. We conclude that CD56+,CD3- NK and CD3+ T cells are activated differentially by continuous i.v. rIL-2 proportional to dose and duration of treatment.

Bone marrow gene therapy for adenosine deaminase deficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency disease (SCID). The cause for this is believed to be the accumulation of one of the substrates for ADA, 2'-deoxyadenosine to which especially T cells are hypersensitive. This disease can be treated successfully with bone marrow transplantation if a suitable donor is available. Alternatively, the human ADA gene could be introduced into the autologous bone marrow. We have generated a retroviral vector containing the human ADA gene. With this vector we were able to restore human ADA-activity in ADA-SCID T cells to normal levels resulting in a sensitivity to 2'-deoxyadenosine that is also found for T cells from a healthy donor. In murine studies we have shown that our retrovirus can infect pluripotent hemopoietic stem cells resulting in long-term (> 6 months) expression of human ADA in the hemopoietic system of transplanted animals. These results were confirmed in rhesus monkeys where we were able to detect the provirus in both peripheral blood mononuclear cells and granulocytes for as long as the animals were analyzed, i.e. up to more than 1 year post bone marrow transplantation. On the basis of these results we have proposed a clinical protocol for the treatment of ADA-SCID patients with bone marrow gene therapy.

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.

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.

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.

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.

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.

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.

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.