Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [³H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC₅₀) of 4.6 nM compared with an IC₅₀ of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC₅₀ values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [³H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.     

In vitro and in vivo properties of an anti-CD5—momordin immunotoxin on normal and neoplastic T lymphocytes

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified fromMomordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC₅₀ = 1–10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC₅₀ = 10 pM). Moreover, the in vitro performances of the immunotoxin compared favourably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model ofnu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%,P <0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.     

Different cytotoxic activity and intracellular fate of an anti-CD5-momordin immunotoxin in normal compared to tumour cells

We investigated the different sensitivity of peripheral blood mononuclear cells (PBMC) and human T cell leukaemias (Jurkat and CEM) to an anti-CD5-momordin immunotoxin. In a short-term assay, the immunotoxin displayed different cytotoxic activity on normal and tumour cells: for leukaemic cell lines an incubation time of 72 h was necessary for the immunotoxin to reach the IC₅₀ of 41–53 pM, compared to the 1 h sufficient for 6 pM immunotoxin to inhibit 50% of PBMC protein synthesis. In a long-term clonogenic assay (15 days), the immunotoxin demonstrated a compareble efficacy of clonogenic cell killing for both cell types. We investigated the immunotoxin internalization pathway by a flow-cytometric method and our data seem to indicate that the molecules meet a different intracellular fate in the two cell populations. It may be assumed that the low cytotoxic activity of immunotoxins on tumour cells, detected in the shortterm assay, is due to infefficient delivery to their cytoplasmatic target, while a longer exposure of the cells to the immunotoxin promotes adequate intracellular distribution.     

An immunotoxin for the treatment of T-acute lymphoblastic leukemic meningitis: studies in rhesus monkeys

Summary Monoclonal antibody WT1 (anti-CD7), conjugated to ricin A chain, was administered intrathecally to rhesus monkeys to test its suitability for use in the therapy of leukemic meningitis. The WT1-SMPT-dgRTA conjugate was cytotoxic to CEM (T-lymphoblastic leukemia) cells in vitro with an ID₅₀ of 53 pM. Immunoperoxidase testing showed no binding of WT1 to normal human tissues other than lymph nodes. Thirteen animals received one or more intrathecal 60-g doses of WT1-SMPT-dgRTA. All monkeys receiving repeated doses developed a cerebrospinal fluid (CSF) pleocytosis (primarily eosinophils), which was generally resolving by 3–4 weeks after therapy. Pharmacokinetic studies showed a half-life of 99 min, consistent with CSF clearance by bulk flow. Peak CSF immunotoxin concentrations exceeded the ID₅₀ for CEM cells by more than 2 log units and a concentration exceeding the ID₅₀ was maintained for as long as 24 h. All eight monkeys receiving repeated doses of immunotoxin developed serum antibodies against both WT1 and ricin A chain. In six of these monkeys antibodies were also present in the CSF. Both anti-WT1 and anti-(ricin A chain) antibodies were able to inhibit in vitro cytotoxicity of the immunotoxin for CEM cells; however, only anti-WT1 antibodies could block immunotoxin binding to the cell surface. No monkey developed anti-immunotoxin antibodies fewer than 7 days after the initiation of therapy, suggesting that repeated doses could be administered for up to 1 week without inhibition of clinical activity.     

Anti-CD30 immunotoxins with native and recombinant dianthin 30

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein fromDianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30⁺ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus). L428 and L540 (both from Hodgkin's lymphoma). IC₅₀ values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30^– cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC₅₀ higher than nM).     

In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain

Summary The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab)₂ or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab)₂-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins.     

Mechanisms of transmembrane signalling in human T cell activation

Several monoclonal antibodies directed against a number of T cell surface molecules are used to elucidate the role of these molecules (cell surface molecules) in T cell activation. The activation of T cells via these molecules are both antigen-dependent (CD3/TcR complex) and antigen-independent. Irrespective of their antigen-dependency, these monoclonal antibodies activate T cells by a classical signal transduction pathway, in which the binding of monoclonal antibodies to their cell surface receptors leads to activation of phospholipase C resulting in the the depolarization of plasma membrane, hydrolysis of IP2 and IP3 and DAG, the second messengers. IP3 leads to mobilization of intracellular calcium to contribute to an increase in [Ca⁺⁺]_i, whereas DAG causes activation and translocation of PKC and an increasing apparent affinity for Ca⁺⁺. The role of IN in the mobilization of intracellular calcium is emerging. In addition, influx of extracellular calcium also contributes to increase in [Ca^–+];. The increase in [Ca⁺⁺]; following activation via some T cell surface antigen is predominantly due to intracellular mobilization of Ca^–+ (e.g. CD3/TcR complex), whereas activation via other T cell surface antigen, the increase in [Ca^+–]_i is almost entirely due to an influx of extracellular calcium (e.g. CD5 antigen). All these molecules activate autocrine system of T cell growth, namely IL-2 production, IL-2 receptor expression and T cell proliferation.     

T lymphocytes can mediate lysis of autologous melanoma cells by multiple mechanisms: Evidence with a single T cell clone

Summary The specificity analysis of a CD3⁺, WT31⁺, CD8⁺ cytotoxic T lymphocyte (CTL) clone (CTL 49), isolated from peripheral blood lymphocytes of a melanoma patient (no. 665) after mixed lymphocyte culture with an HLA-A2⁺ allogeneic lymphoblastoid cell line (VSKB-LCL), revealed that CTL 49 could lyse, in addition to HLA-A2⁺ lines, autologous HLA-A2^– melanoma (Me665/2) and K562 targets. Killing of VSKB-LCL, but not of Me665/2, could be inhibited by anti-CD3 and by anti-HLA-A2 antibodies or by modulation of the CD3 complex. Cold-target competition studies showed that K562, but not VSKB-LCL, could compete with Me665/2 for lysis by CTL 49. However, unlike K562, Me665/2 could be lysed by CTL 49 in a Ca²⁺-independent fashion in 4 h and 18 h assays. CTL 49 expressed mRNA specific for tumor necrosis factor (TNF) and, to a lesser extent, for lymphotoxin (TNF). Exposure of the clone to anti-CD3 antibodies induced the expression of interferon(IFN)--specific and the up-regulation of TNF- and TNF-specific mRNA. Antibodies to TNF, TNF and IFN reduced the lysis of Me665/2, but not of K562, by CTL 49 in 18-h cytotoxic assays. Antibodies to TNF and to IFN almost completely inhibited the lysis seen on Me665/2 (but not on K562), in 96-h assays, by supernatants isolated from VSKB-LCL- or anti-CD3-stimulated CTL 49 cells. Taken together, these data indicate that major-histocompatibility-complex-independent lysis of autologous tumor cells and of natural killer reference targets by the same alloreactive T cell clone are activities related at the level of target recognition but distinct at the level of the lytic hit. Thus, efficient lysis of autologous tumor cells results from a complex mechanism based upon direct effector-target interaction as well as on cytokine-mediated cytolytic effects.     

Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Summary Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ^–1, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.Supported in part by the NIH BRSG grant SO7RRO5712, the American Cancer Society, Mass. Div. Research Grant 1543-C-1, and by the Aid for Cancer Research (Boston) award to L. V. L., and by RO1 CA 29160 and RO1 CA 39748 grants to T. W. G.     

Near-Term Anti-CD25 Monoclonal Antibody Administration Protects Murine Liver from Ischemia-Reperfusion Injury Due to Reduced Numbers of CD4+ T Cells

Background

CD4⁺ T cell is acknowledged as a key factor in the initiation phase of liver ischemia reperfusion injury. The purpose of current study is to demonstrate the effect of antecedent near-term anti-CD25 monoclonal antibody treatment on IR-induced liver injury by modulation of CD4⁺ T cells.

Methods

70% liver warm IR was induced in male C57BL/6 mice after anti-CD25 mAb or non-specific IgG administration. Liver function, histological damage, in vitro Proliferation, FACS, cytokine production, and immunofluorescence were assessed to evaluate the impact of antecedent near-term PC61 treatment on IR-induced liver injury.

Results

After 70% liver ischemia, mice preconditioned with PC61 displayed significantly preserved liver function as characterized by less histological damage and reduced serum enzymes level. Mechanistic studies revealed that the protection effect of anti-CD25 mAb was associated with ameliorated intrahepatic inflammatory milieu and reduced CD4⁺ T lymphocytes as manifested by the decrease of proinflammatory cytokine production (less expression of TNF-α, IFN-γ, IL-2, and IL-6) and the lower CD4/CD8 proportion.

Conclusions

Our results provide first line of evidence indicating that near-term treatment with anti-CD25 monoclonal antibody might provide protection for livers against IR-induced injury by reducing CD4⁺ T cells, but not influencing functional Treg population. Therefore, our results demonstrate a potential function of anti-CD25 monoclonal antibody which was neglected in the past, and may be helpful in various clinical conditions, particularly in liver and kidney transplantations.     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

An effective chemical mutagenesis procedure for Petunia hybrida cell suspension cultures

Summary By using mercury(II)-chloride (HgCl₂) and Dl-6-fluorotryptophan (6FT) as positive selection conditions we were able to show that N-methyl-N-nitro-N-nitrosoguanidine (NG) is an effective mutagen for Petunia hybrida suspension cells.A number of the 205 calli resistant to HgCl₂ and 17 calli resistant to 6FT were isolated. The highest mutation frequency was 1.0 x 10^–5 and 2.0 x 10^–6 for HgCl₂ and 6FT, respectively. A preliminary characterization of the mutants is presented.A significant increase in the number of drug-resistant calli was only obtained at NG-concentrations (5–40 g/ml) that had no observable effect on the survival of the mutagenized cultures.     

An immunotoxin containing a rat IgM monoclonal antibody (Campath 1) and saporin 6: effect on T lymphocytes and hemopoietic cells

Summary The elimination of the cells responsible for graft-versus-host disease in allogeneic bone marrow transplantation has been attempted with a variety of methods, including the use of the ribosome-inactivating toxin ricin bound to monoclonal antibodies acting as carriers. However the high nonspecific toxicity of these immunotoxins containing the whole toxin greatly limited clinical application. Toxicity can be reduced using the A-chain of ricin or other ribosome-inactivating proteins (RIPs) which are devoid of a B-chain with lectin properties. We used saporin 6 purified from Saponaria officinalis seeds, which was conjugated with the rat IgM monoclonal antibody Campath 1 specific for mature T and B lymphocytes as well as for monocytes. The immunotoxin retained both RIP and antibody activity, inhibiting protein synthesis both in a cellfree system and in cells bearing the Campath 1 antigen; it also abolished methyl ³H-thymidine uptake in phytohemagglutinin-stimulated T lymphocytes. Myeloid progenitors were largely spared as shown by myeloid stem cell (CFUGM) growth which was scarcely affected. Toxicity of the immunotoxin to cell lines not expressing the antigen recognized by Campath 1 monoclonal antibody was not greater than the toxicity due to free saporin 6, while the immunotoxin was more toxic to mice than free saporin.Work supported by grants of Regione Emilia Romagna, delibera n. 1970, 13/5/86, by the Italian National Research Council, Roma, Finalized Project Oncologia, contracts n. 86.00589.44 and n. 86.00603.44 and by the Pallotti's Legacy for Cancer ResearchAngelo Dinota is supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy.     

Cadmium-induced apoptosis in C6 glioma cells: Mediation by caspase 9-activation

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC₅₀-value of 0.7 M, while human A549 adenocarcinoma cells were relatively resistant with an IC₅₀-value of 164 M CdCl₂. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl₂ and was concentration-dependent between 1–100 M CdCl₂, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 M CdCl₂. Furthermore, cadmium (1 M, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd²⁺, other toxic heavy metal ions (Hg²⁺, Pb²⁺, Ni²⁺, Fe²⁺, CrO₄ ^2–, Cu²⁺ or Co²⁺) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn²⁺ which caused apotosis at high concentrations (>150 M) whereas it protected against cadmium-induced apoptosis at low concentrations (10–50 M).     

Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster-5A antigen associated with human small-cell lung cancer

Summary The cytotoxic properties of a ricin A chain immunotoxin made with the mouse monoclonal antibody SWA20, recognising a family of sialoglycoprotein antigens selectively expressed by human small-cell lung cancer (SCLC), were examined using a panel of tumour cell lines in tissue culture. SWA20—ricin-A-chain was selectively toxic to the SW2, NCI-H69 and GLC-8 SCLC cell lines, inhibiting the incorporation of [³H]leucine by 50% at a concentration of 0.2–2 nM, but had no selective activity against the NCI-H23 and NCI-H125 lung adenocarcinoma or the control CEM T-lymphoblastoid cell lines. The SWA20 immunotoxin intoxicated the SW2 cell line rapidly, inhibiting [³H]leucine incorporation by 50% within 2 h compared with 0.5 h for ricin. Analysis of the effects of SWA20—ricin-A-chain on the growth of SW2 cells using a limiting-dilution clonogenic assay revealed that the immunotoxin could eliminate 95% of clonogenic malignant cells. Although SWA20—ricin-A-chain was found to be rapidly active against the majority of tumour cells, its action was limited by the presence of insensitive cells expressing low levels of the target antigen.     

T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

Protective CD8 memory T cell responses to mouse melanoma are generated in the absence of CD4 T cell help

Background

We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma.

Methodology and Principal Findings

To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (T_reg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete T_reg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision.

Conclusions and Significance

This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer.     

Correlation between the Vß4+CD8+ T-cell population and theH-2 d haplotype

The Vß4 ⁺ T-cell population was examined with a newly established antibody, KT4, specific for Vß4. Between 4.8% and 19.4% of CD3⁺ peripheral T cells from various inbred strains of mice or Fl hybrids expressed Vß4. The CD4 T-cell population had higher numbers of V4⁺ T cells (5.5%–20.6%) than the CD8 T-cell population (2.5%–10.7%). Deletion of certain Vexpressing T cells due to the presence of the Mls^a antigen and/or the absence of certainTcrb-V genes increased relative numbers of Vß4⁺ T cells. The data suggest that V4⁺ CD8⁺ T cells might be positively selected by H-2^d molecules.     

Metabolism measurements ofAurelia aurita planulae larvae,and calculation of maximal survival period of the free swimming stage

Respiration, ammonia and phosphate excretion experiments were performed with planula larvae ofAurelia aurita (Scyphozoa) from Kiel Fjord, Baltic Sea, in summer 1983. The mean respiration measured was 3.22 nl O₂ ind^–1 h^–1 (at 20 °C). Excretion experiments revealed average values of 11.41 pM NH₄-N ind^–1, and 0.92 pM PO₄-P ind^–1h^–1, respectively. The atomic C:N:P ratio of excretion products was 133:10:1. The O:N ratio of 25:1 and O:P ratio of 313:1 point to a lipid-carbohydrate-oriented catabolism of theAurelia larvae. On the basis of experimental results and of biomass determinations, the maximal survival period of the non-feeding free swimming planula stage was calculated. Typically, the value lies in the range of some days to one week.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.