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PubMed2000

Anaphylactoid reaction to muromonab-CD3 in a pediatric renal transplant recipient.

Muromonab-CD3 (OKT3), a murine IgG2a antibody directed against the T3 (CD3) complex on mature lymphocytes, triggers adverse immune reactions. Anaphylactic reactions have occurred in patients exposed to OKT3 and are mediated by anti-OKT3 IgE antibodies. The reactions are not antibody mediated and can occur within seconds of administration of a mast cell secretogogue. A renal transplant recipient became hypotensive and hypoxic immediately after receiving her first dose of OKT3 and required advanced life support. Serum antibody tests were negative for anti-OKT3 IgG, IgE, and antimouse protein antibodies. To our knowledge, this is the first published report of a patient with an anaphylactoid reaction to the initial infusion of OKT3.

Adolescent↗

PubMed1990

Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C.

We recently reported that the myristoylated peptide N-myristoyl-Lys-Arg-Thr-Leu-Arg (N-m-KRTLR) is a novel protein kinase C inhibitor. In this study, we investigated the biological effects of N-m-KRTLR using as an in vitro model the induction of the IL-2 receptor and IL-2 secretion by Jurkat cells in response to stimulation with 12-O tetradecanoylphorbol-13-acetate (TPA) plus phytohemagglutinin (PHA) and TPA plus OKT3 mAb. N-m-KRTLR significantly suppressed induction of the IL-2 receptor on the surface of the Jurkat cells by TPA plus either PHA or OKT3 mAb. Furthermore, N-m-KRTLR inhibited the production and release of IL-2 from cultured Jurkat cells stimulated with TPA plus either PHA or OKT3 mAb. Similarly, this peptide significantly inhibited the IL-2 production in normal human peripheral blood mononuclear cells in response to stimulation by TPA and PHA. In contrast, this peptide did not affect expression of the CD3 complex on the surface of the Jurkat cells either alone or in the presence of TPA or PHA. Furthermore, N-m-KRTLR did not interfere with the spontaneous proliferation of the Jurkat cells, and its effects on IL-2 secretion and IL-2 receptor expression in the Jurkat cells were evident without loss of cell viability. These results suggest that the novel protein kinase C inhibitor N-m-KRTLR may selectively inhibit certain activation pathways of Jurkat cells and indicate the usefulness of N-m-KRTLR in the analysis of discrete events in T cell activation.

Amino Acid Sequence↗

PubMed1996

The two membrane proximal domains of CD4 interact with the T cell receptor.

During T cell activation, CD4 is intimately involved in colocalizing the T cell receptor (TCR) with its specific peptide ligand bound to class II molecules of the major histocompatibility complex (MHC). Previously, the COOH-terminal residues, Trp62/63, which flank the immunodominant epitope of hen egg lysozyme (HEL 52-61), were shown to have a profound effect on TCR recognition. CD4 maintains the fidelity of this interaction when short peptides are used. To determine which portion of CD4 was responsible for this effect, a series of CD4 mutants were made and transfected into CD4 loss variants of two HEL 52-61-specific T cell hybridomas. Surprisingly, some CD4 mutants that failed to interact with MHC class II molecules (D2 domain mutant) or with p56kk (cytoplasmic-tailless mutant) restored responsiveness. Nevertheless, a significant reduction in association between cytoplasmic-tailless CD4 and the TCR, as determined by fluorescence resonance energy transfer, was observed. Thus, neither colocalization of CD4 and the TCR nor signal transduction via CD4 was solely responsible for the functional restoration of these T cell hybridomas by wild-type CD4. However, substitution of the two membrane proximal domains of murine CD4 (D3 and D4) with domains from human CD4 or intercellular adhesion molecule 1 not only abrogated its ability to restore function, but also substantially reduced its ability to associate with the TCR. Furthermore, the mouse/human CD4 chimera had a potent dominant negative effect on T cell function in the presence of equimolar concentrations of wild-type CD4. These data suggest that the D3/D4 domains of CD4 may interact directly or indirectly with the TCR-CD3 complex and influence the signal transduction processes. Given the striking structural differences between CD4 and CD8 in this region, these data define a novel and unique function for CD4.

Amino Acid Sequence↗

PubMed2000

Cytokine profiles in early rejection following OKT3 treatment in liver transplant patients.

OKT3 , a murine monoclonal antibody specific to the human CD3 complex, induces immunosuppression by depletion of T cells. Administration of OKT3 results in significant release of proinflammatory cytokines, such as TNFalpha and IL1beta. Liver recipients who experience rejection within 3 weeks after transplantation with OKT3 prophylaxis recover their T cells by postoperative day 10 despite complete initial clearance. We sought to analyze the role of proinflammatory and Th-1 cytokines in T cell recovery and rejection after liver transplantation with OKT3 prophylaxis. In plasma samples from 32 patients, we measured TNFalpha, IL1beta and IL6 (before transplant and on postoperative days 1, 2 and 3) and IL2, IFNgamma, sIL2R and slCAM (postoperative days 5, 7 and 10) and examined possible correlations with T-cell recovery and occurrence of rejection within 3 weeks. TNFalpha, IL1beta, and IL6 did not correlate with T-cell recovery. In patients who rejected, IL2 and IFNgamma on postoperative days 5 and 7 correlated with degree of T-cell recovery by day 10; a significant rise in sIL2R over time also correlated with T-cell recovery in this group. Our results emphasize the role of Th-1 cytokines in rejection following OKT3 induction and suggest that markers of T cell activation may predict risk.

Animals↗

PubMed1999

Bispecific antibody-mediated lysis of primary cultures of ovarian carcinoma cells using multiple target antigens.

We have shown previously that a bispecific antibody (BsAb) directed against both germ-cell alkaline phosphatase (GCAP) and the CD3 complex on mouse T cells could effectively eliminate GCAP-positive tumor cells in vivo using an immunocompetent mouse model. However, some GCAP-negative tumor cells were still able to grow, suggesting that BsAb therapy, when used in a clinical setting, could benefit from targeting several tumor markers to prevent outgrowth of tumor cells lacking a targeted marker. To test this hypothesis, we developed an in vitro model based on primary human ovarian carcinoma (OC) cultures and BsAbs directed against human T cells and several tumor markers [placental alkaline phosphatase (PLAP), GCAP, folate-binding protein (FBP) and CA19.9]. OC cells, isolated from primary tumors, were co-cultured with human peripheral blood mononuclear cells in the presence or absence of various concentrations of BsAbs against PLAP/GCAP, FBP and CA19.9 administered separately or in combination. Results derived from 18 primary OC samples showed that the combination treatment was better than or equally effective as the best single BsAB treatment in 60% of cases. Sometimes targeting FBP, PLAP/GCAP or CA19.9 alone was superior to targeting all simultaneously. Combining each BsAb with a low dose of IL-2 was always beneficial. These results indicate that before using a specific BsAb in the clinic, it is important to determine the optimal BsAb for each patient using this in vitro assay on cells from the removed tumor mass.

Animals↗

PubMed1991

Tumor-infiltrating lymphocytes isolated from a Ki-1-positive large cell lymphoma of the skin. Phenotypic characterization and analysis of cytokine secretion.

Tumor-infiltrating lymphocytes (TIL) were obtained from a biopsy of a patient with a Ki-1-positive large cell lymphoma of the skin. Immunohistologic studies of the large anaplastic tumor cells showed an "aberrant" T "helper/inducer" phenotype (CD30 + CD3-CD4+ CD8-IL-2R + HLA-DR+). Using cDNA probe for the constant region of the T-cell receptor (TCR) beta gene, the cells were identified by their distinct monoclonal rearrangement of T-cell receptor (TCR)-beta DNA. Tumor cells isolated from biopsies were cultured in the presence of interleukin-2 (IL-2). Outgrowing lymphocytes were cloned, expanded in vitro, and 11 clones were subjected to phenotypic analysis: ten clones showed a predominantly CD4-positive T "helper/inducer" phenotype whereas one clone expressed CD8 T "cytotoxic/suppressor" antigens. In contrast to the tumor cells, cells of all clones grown in vitro expressed the TCR-associated CD3 complex. Furthermore, cells from all clones analyzed expressed CD5, CD7, CD45RO (UCHL1), CD11a (LFA-1), CD25, and HLA-DR antigens. Cells of two of ten CD4-positive clones expressed CD45RA (2H4) in addition to UCHL1. T-cell clones isolated from the tumor and grown in vitro exhibited individual DNA restriction band patterns different from that of a DNA tumor biopsy specimen. Therefore, the authors conclude that these T-cell clones represent presumably nonmalignant TIL. All clones tested secreted interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in vitro. Nine of 11 clones were found to secrete additionally IL-2 and IL-4 upon stimulation with phytohemagglutinin (PHA) whereas two clones did not secrete detectable amounts of IL-4. Selective growth of TIL in the presence of IL-2 opens the possibility to use these cells in adoptive immunotherapy of cutaneous T-cell lymphoma (CTCL). Cytokines secreted by TIL cells in vitro (IL-2, IL-4, IFN-gamma, TNF-alpha) may be involved in their antitumorigenic activity. Moreover, these data implicate that CD4-positive TIL derived from CTCL cannot be grouped into different subsets based on the production of IL-2, IL-4, IFN-gamma, and TNF-alpha.

Aged↗

PubMed2000

Human T cells elicit IFN-alpha secretion from dendritic cells following cell to cell interactions.

Major insights into events that control Th1/Th2 differentiation have been acquired recently, and highlight the role of Type I IFN in Th1 generation, by inducing up-regulation of the IL-12 receptor beta(2) subunit. IFN-alpha induces responsiveness to IL-12, and here we have investigated the source and the circumstances under which IFN-alpha is produced, in the absence of viral infections. Human dendritic cells (DC) were co-cultured with autologous T cells activated by cross-linking the CD3 complex. DC were also cultured with L cells expressing human CD40 ligand (CD40L). Our results show that large amounts (>200 IU IFN-alpha from 2.5x10(4) cells) of IFN-alpha are produced by DC following interaction with stimulated T cells. Similar effects were observed when DC were cultured with CD40L-expressing transfectants, although the amount of IFN-alpha produced was reduced, suggesting that the CD40-CD40L interaction may be important. These results show that stimulated T cells can solicit the signals from DC that allow their polarization towards a Th1 phenotype. Type I DC produce Type I IFN not only following viral infection but also during an immunological interaction and this may be the basic mechanism that assists in the development of a Th1 response.

Animals↗

PubMed2002

Microchip-based amperometric immunoassays using redox tracers.

A new chip-based electrochemical immunoassay protocol, based on the use of a ferrocene redox label, is described. Two reaction formats, based on direct (noncompetitive) and competitive modes of operation, were employed for illustrating the use of redox tracers in chip-based electrochemical immunoassays. The direct assay consisted of mixing the ferrocene-tagged antibody and the antigen analyte, a rapid electrophoretic separation of labeled free antibody and the labeled antigen/antibody complex, and a downstream anodic detection of the ferrocene tracer at gold-plated carbon screen-printed electrode detector. The competitive assay integrates precolumn reactions of the labeled antigen and the target antigen with the antibody with electrophoretic separation of the free and bound labeled antigens, along with amperometric detection of the redox tag. An internal standard has been used to normalize the peak area for the construction of calibration plots. Fundamental operating variables are examined and optimized. The use of a redox tracer offers the advantages of simplified protocol, wider linear range, higher stability, and higher separation efficiency compared to an analogous use of enzyme tags. The direct mouse-immunoglobulin G (IgG) assay and the competitive 3,3',5-triiodo-L-thyronine (T(3)) one were accomplished within less than 150 and 130 s (with field strengths of 256 and 192 V/cm), and offer minimum detectable concentrations of 2.5 x 10(-12) and 1 x1 0(-6) g/mL, respectively. Such use of redox labels for chip-based amperometric immunoassay protocols offers considerable promise for decentralized clinical or environmental testing.

Antigen-Antibody Complex↗

PubMed2004

Oligodeoxynucleotides containing CpG motifs can induce T cell-dependent arthritis in rats.

OBJECTIVE: To investigate whether CpG oligodeoxynucleotides (ODNs) can induce or accelerate arthritis in rats. METHODS: The CpG-induced response was studied by recording joint inflammation, cell activation in draining lymph nodes, and levels of the acute-phase reactant alpha(1)-acid glycoprotein (AGP) in sera. The role of T cells was investigated by in vivo administration of monoclonal antibodies specific for the T cell receptor alpha/beta (TCRalpha/beta), followed by analysis of cell phenotypes by flow cytometry. RESULTS: One intradermal injection of CpG ODN emulsified with Freund's incomplete adjuvant (IFA) induced arthritis in LEW and LEW.1AV1 rats, while the control ODN sequence without CpG motifs or IFA alone did not trigger disease. The CpG/IFA and control-ODN/IFA injections induced lymphoplasia as well as elevated levels of interleukin-1beta and interferon-gamma messenger RNA in lymph nodes. The arthritis was preceded by elevated levels of AGP in serum. In vivo administration of anti-TCRalpha/beta antibodies after disease induction caused decreased expression of the TCR-CD3 complex on circulating T cells and ameliorated the arthritis. CONCLUSION: We demonstrated that injection with immunostimulatory CpG, both in phosphorothioate-modified and native forms, can induce a T cell-dependent joint-specific inflammation in LEW and LEW.1AV1 rat strains. This arthritis is preceded by signs of activation of the innate immune system. Since unmethylated CG dinucleotides are common in bacterial DNA but rare in mammalian DNA, our results indicate that exposure to bacterial DNA during infection may contribute to arthritis induction by amplifying the innate immune response.

Animals↗

PubMed1992

A versatile method to produce antibodies to human T cell receptor V beta segments: frequency determination of human V beta 2+ T cells that react with toxic-shock syndrome toxin-1.

Human V beta (hV beta) regions have been expressed in the context of mouse T cell receptor (TcR)-CD3 complexes, and subsequently used to raise hV beta-specific monoclonal antibodies (mAb). The method of expression of hV beta outlined in this report contrasts in its versatility with the one reported by Choi et al. (Proc. Natl. Acad. Sci. USA 1991. 88: 8357). For instance, we have applied it successfully to the construction of mouse T cell transfectants expressing hV beta 1, hV beta 2, hV beta 3, hV beta 8, hV beta 9, hV beta 13.5, hV beta 19, hV beta 21, and hV beta 22 gene segments. mAb against the hV beta 2 and hV beta 19 regions have been raised by using these transfectants as immunogens in mice. Here, we illustrate the application of the anti-hV beta 2 mAb to the measurement of human T cells that react with the staphylococcal toxic-shock syndrome toxin-1.

Animals↗

PubMed1993

Expression of the CD28 costimulatory molecule on subsets of murine intestinal intraepithelial lymphocytes correlates with lineage and responsiveness.

The CD28 antigen has been recently demonstrated to be a costimulatory molecule and is expressed by almost all thymic and peripheral T cell receptor (TcR) alpha beta+ and gamma delta+ cells in the mouse system. We show here that expression of CD28 is heterogeneous among murine intestinal intraepithelial lymphocytes (IEL). Whereas some TcR alpha beta-expressing IEL subsets such as CD4+8- and CD4-8 alpha+ beta+ cells express CD28 at the same levels as their phenotypic counterparts in lymph node, other subsets of TcR alpha beta cells (including CD4-8 alpha+ beta- and CD4+8 alpha+ beta- cells) as well as TcR gamma delta+ IEL fail to express CD28. Parallel experiments using aged BALB/c-nu/nu mice indicated that CD28 expression patterns among IEL are quite similar to those of normal BALB/c mice. Furthermore, forward light scatter analysis showed that CD28- cells are considerably larger than CD28+ cells in the gut, although cycling cells were rare in both subsets. Finally CD28- cells in the gut did not proliferate or produce IL-2 upon stimulation by anti-CD3 monoclonal antibodies (mAb) and phorbol 12-myristate 13-acetate, whereas CD28+ cells in the gut and lymph nodes responded to these stimuli. The response of the CD28+ cells was enhanced by anti-CD28 mAb. These results suggest that CD28- IEL (CD4- 8 alpha+ beta- cells, and some CD4+ 8 alpha+ beta- cells) may follow a different developmental pathway from that of CD28+ IEL in a thymus-independent environment, and that expression of CD28 correlates with responsiveness of the cells to triggering via the TcR-CD3 complex.

Animals↗

PubMed1994

Failure of correlation between B7 expression and activation of interleukin-2-secreting T cells.

It is well established that triggering interleukin-2 (IL-2) secretion by helper T cells requires the T cell to receive at least two discrete signals. One signal is transduced by the CD3 complex, usually as the result of T cell receptor (TcR) occupancy, the second, or co-stimulatory, signal involves a non-cognate interaction between cell surface accessory molecules on the antigen-presenting cell (APC) and the T cell. A molecular interaction that has been implicated in the provision of co-stimulatory signals is that between B7/BB1 on the APC and its ligands, CD28 and CTL-A4 on the T cell. We have studied the ability of HLA-class II antigen-positive human T cells and a population of DR1-expressing transfected human fibroblasts to stimulate a proliferative response by human T cell clones, and by freshly isolated peripheral blood T cells. Despite their high levels of B7 expression, the T cell clones, were unable to induce proliferation or IL-2 secretion by DR-restricted, antigen-specific T cells. In contrast, the DR1-expressing transfectants, that were B7 negative, induced a strong proliferative response. When these two populations of DR-expressing cells were used to stimulate a primary alloresponse the results were reversed, in that the T cell clones induced a strong alloresponse but the transfected fibroblasts induced no proliferation. These results suggest that the expression of B7 may be necessary for costimulation of unprimed T cells, but not of established T cell clones. Furthermore the data show that the expression of B7 by an APC does not necessarily lead to IL-2 production or protection from the induction of tolerance. The mechanisms responsible for the inability of these T cells to provide full activation signals when used as APC is currently under investigation.

Antigen-Presenting Cells↗

PubMed1992

The T-cell receptor zeta chain contains a GTP/GDP binding site.

In a search for nucleotide binding proteins associated with the T-cell receptor (TCR)-CD3 complex, a novel labeling technique involving introduction of [alpha-32P]GTP or [alpha-32P]ATP into permeabilized cells followed by in situ periodate oxidation was developed. To test the method we first demonstrated that p21ras and other classical GTP binding proteins could be labeled in a GTP-specific manner. In human T lymphocytes the TCR zeta chain was found to be specifically labeled by GTPoxi but not by ATPoxi or CTPoxi. Labeling kinetics and competition experiments demonstrated that zeta had a capacity to bind GTP and GDP but not GMP or ATP. Proteolytic cleavage experiments identified lysine 128 as the GTP crosslinking site. This result was confirmed by studies using oligonucleotide-directed mutagenesis. Lysine residues 128, 135 and 149 were each replaced by arginine and glycine 134 by valine and mutated proteins were expressed in CHO cells. Labeling of mutants K128R and G134V was abrogated whereas mutant proteins K135R and K148R could still be specifically crosslinked to GTP. We conclude that Lys128 and Gly134 are part of a GTP/GDP binding site suggesting that zeta is a unique GTP/GDP binding structure.

3T3 Cells↗

PubMed1996

Cloning, expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.

Amino Acid Sequence↗

PubMed2004

Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy.

BACKGROUND: Designer T cells are T lymphocytes engineered toward specific antibody-type membrane antigens through chimeric immunoglobulin-T-cell receptor (IgTCR) genes that have been used for adoptive cellular immunotherapy. We have extended this approach to prostate specific membrane antigen (PSMA) as a means to attack prostate cancer. METHODS: A chimeric anti-PSMA IgTCR gene was constructed based on an anti-PSMA monoclonal antibody, 3D8. Both T-cell lines and primary cultured human T lymphocytes were transduced with the chimeric anti-PSMA IgTCR construct and were analyzed for IgTCR expression, specific activation by PSMA, cytotoxicity against PSMA-expressing tumor cells in vitro, and retardation of tumor growth in an animal model. RESULTS: The IgTCR was incorporated into the TCR-CD3 complex and formed a functional chimeric complex. The IgTCR-modified T cells were specifically activated through the chimeric receptor with PSMA as measured by IL-2 production and increased CD25 expression and specifically lysed the PSMA-expressing prostate cancer cells in vitro as well as retarded tumor growth in an animal model. CONCLUSIONS: The anti-PSMA designer T cells exhibit an antibody-type specificity that can recognize PSMA expressing tumor cells in a MHC-independent fashion, resulting in T-cell activation, target cell lysis in vitro and inhibition of tumor growth in vivo.

Animals↗

PubMed1995

Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene.

The mouse pre-T-cell receptor alpha (pT alpha) chain is a 33,000 M(r) glycoprotein expressed on the surface of immature thymocytes as a disulfide-linked heterodimer with the T-cell receptor beta (TCR beta) chain, and in association with proteins of the CD3 complex. The cDNA for pT alpha, isolated previously, encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) superfamily. Here we report the complete nucleotide sequence, the exon/intron structure, and the chromosomal location of the pTa gene. The gene spans about 8.4 kilobases (kb) and consists of four exons. Exon 1 encodes the 5' untranslated region, the leader peptide, and the first three amino acids of the mature protein. This exon is followed by a relatively long intron of 4.9 kb that contains many short interspersed repeats (SINEs) of the B1 and B2 family. The second exon encodes the extracellular Ig-like domain and exon 3 with just 45 base pairs the connecting peptide (CP), including the cysteine required for heterodimer formation. A similar exon/intron structure encoding corresponding parts of the mature polypeptide is found both in the Tcra and Tcrd constant region genes. The last exon encodes the transmembrane portion, the cytoplasmic tail, and about 540 nucleotides of 3' untranslated sequence, including a B2 repetitive element. In situ hybridization maps the pTa gene to the D/E1 region of mouse chromosome 17.

Animals↗

PubMed1993

Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells.

In a pilot clinical study carcinoma patients with malignant ascites or pleural exudates have been treated locally with autologous lymphocytes activated ex vivo and redirected towards tumour cells with bispecific monoclonal antibodies. BIS-1, the bispecific monoclonal antibody used in this study, combines specificity against a tumour-associated antigen, AMOC-31, present on carcinomas, with a specificity against the CD3 complex on T lymphocytes. Patients selected for treatment had malignant pleural or peritoneal effusions. Treatment consisted of isolating autologous peripheral blood lymphocytes, ex vivo activation, incubation with bispecific monoclonal antibodies and injection at the effusion site of these BIS-1-redirected lymphocytes. To evaluate the effects of the bispecific monoclonal antibody, five patients received treatments with activated lymphocytes without bispecific antibodies. Effusion samples taken before and at various times after treatment were analysed by immunocytology and for the presence of the soluble factors carcinoembryonic antigen (CEA), interleukin-6 (IL-6), tumour necrosis factor (TNF), C-reactive protein and soluble CD8. In this way both immune activation and anti-tumour activity could be monitored. Conjugate formation between tumour cells and activated lymphocytes was seen as soon as 4 h after injection of BIS-1-redirected activated lymphocytes, followed by a disappearance or reduction of tumour cells after 24-48 h. In parallel with this, the soluble tumour marker CEA decreased in the effusion fluid following injection with the BIS-1-redirected lymphocytes. Furthermore, a steep increase in local granulocyte numbers was observed in the effusion fluid, which reached a maximum 24-48 h after the start of the treatment. Also levels of IL-6 and TNF were greatly elevated. The data suggest that the treatment induces both antitumour activity and a strong local inflammatory reaction. This is accompanied by no or only minor local and systemic toxicity, i.e. mild fever, which disappeared as the local inflammatory reaction diminished 48-72 h after treatment.

Abdominal Neoplasms↗

PubMed1989

Nature of the thymocytes associated with dendritic cells and macrophages in thymic rosettes.

Thymic rosettes, structures consisting of 3-30 thymic lymphoid cells attached to a central macrophage or dendritic cell, were released from mouse thymus tissue by collagenase digestion. They were shown to be preexistent structures within the thymus, but to be subject to extensive exchange with free thymocytes under certain conditions. An isolation procedure was developed, using a new technique of zonal unit-gravity elutriation, which minimized exchange and produced a completely pure sample of the larger rosettes. The rosette-associated thymocytes were analyzed by two- and three-color immunofluorescent staining and flow cytometry. The dominant cell type was a small, CD4+CD8+, cortical-type thymocyte. However, all of the established thymus subpopulations defined by CD4 and CD8, including CD4-CD8+ and CD4+CD8- mature thymocytes and CD4-CD8- early thymocytes, were also present in rosettes. Very few of the cells present were of an intermediate or transitional phenotype. Rosette-associated thymocytes were somewhat enriched in large dividing thymocytes, in CD4-CD8- thymocytes, and in mature thymocytes expressing the T-cell antigen receptor-CD3 complex. Their most striking characteristic was a marked depletion in small thymocytes lacking surface H-2K expression, a major population among free thymocytes. The physiological role of the rosette structure is discussed, and it is suggested that the heterogeneity of the associated thymocytes in part reflects the existence of different types of rosettes in different areas of the thymus.

Animals↗