Pathogenesis of mouse hepatitis virus infection. The role of nasal epithelial cells as a primary target of low-virulence virus, MHV-S.

The pathogenesis of mouse hepatitis virus (MHV-S) infection in suckling and weanling mice was comparatively studied after intranasal inoculation. In sucklings, infectious virus as well as specific antigen was first detected in the nasal mucosa at 12 hr, then in the nerve cells of the olfactory bulbs. At this stage viral particles were demonstrated both in the supporting cells and olfactory cells of the nasal mucosa. In the posterior part of the brain and spinal cord, virus was detected on days 3 to 4 postinoculation when viral growth was clearly demonstrable in the liver, spleen and intestines. In weanlings too, infection was first established in the nasal mucosa, shedding infectious virus in the nasal washing until day 6 postinoculation, and later infection spread to the brain and spinal cord. In weanling mice, however, neither infectious virus nor viral antigen was detected in the liver or other visceral organs, while serum neutralizing antibody became detectable on day 5 postinoculation, increasing in titer thereafter. Histopathologically degenerative and necrotic changes were observed in the nasal mucosa and central nervous system of both age groups of animals coincidentally with the presence of viral specific antigen, while inflammatory response was much less prominent in sucklings. In the liver, spleen and intestines, however, some lesions were observed only in sucklings.     

The effect of radiation therapy combined with natural killer cells against spontaneous murine fibrosarcoma

The effect of radiation therapy combined with lymphoid cells against spontaneous murine fibrosarcoma (FSa-II) was investigated bothin vivo andin vitro. In thein vivo experiment, syngeneic C3H mice were divided into 3 groups. Animals in the first group were injected with 1 x 10⁵ tumor cells into the right hind leg. Animals in the second and third groups were injected with 1 x 10⁵ tumor cells mixed with 1 x 10⁷ normal lymphoid cells (NLC) or effector lymphoid cells (ELC), respectively. ELC were obtained from spleen and lymph nodes of FSa-II-bearing mice and incubatedin vitro for 40 hr to eliminate suppressor T cell function. NLC were obtained from normal mice and incubated in the same way. Irradiation was given using¹³⁷Cs unit 3 days after cell inoculation. 12 out of 14 mice (85.7%) inoculated with tumor cells mixed with NLC did not show any tumor growth at 60 Gy local irradiation. 12 out of 21 mice (57.1 %) inoculated with tumor cells alone and 6 out of 10 (60%) with tumor cells mixed with ELC rejected tumors at the same radiation dose. This synergistic effect with NLC was not observed when NLC was inoculated after irradiation, indicating that lymphoid cells should be in contact with tumor cells before irradiation. In the⁵¹Cr release assay, lymphoid cells obtained from whole body irradiated (WBI) mice showed 17.8% lysis without irradiation and 28.8% lysis at 5 Gy irradiation. Untreated NLC showed almost no cytotoxic effect at the same radiation dose. This synergistic effect disappeared when WBI lymphoid cells were treated with anti asialo GM1 and complement. These results suggested that NK cells might be important in this synergistic effect with irradiation. To obtain a sufficient level of synergistic effect by in vitro combined treatment of mixed tumor cell - NLC culture and irradiation - incubation for more than 12 hrs and 8 hrs appeared to be necessary before and after irradiation, respectively.     

Reversal of SV40 tumor-mediated suppression of spleen cell cytotoxicity by antibody.

Spleen cells obtained from hamsters bearing PARA-7 tumors greater than 1.0 cm were not reactive in microcytotoxicity assays unless preincubated overnight. The events occurring during in vitro incubation which lead to reversal of tumor-mediated suppression of cellular immunity were investigated. After 24 hr of incubation, supernatants overlying spleen cells from tumor-bearing hosts contained a factor which blocked cytotoxicity of simian virus 40 (SV40)3-sensitized spleen cells at the PARA-7 target cell level but not at the effector cell level. The preparations did not mediate antibody-dependent cellular cytotoxicity (ADCC). Opposite results were obtained in assays of culture medium overlying spleen cells from hosts with a tumor burden less than 0.1 cm. Although ADCC activity was present, no significant blocking was detectable. Treatment of inactive spleen cells with anti-hamster gamma-globlin in the presence of complement (anti-HGG + C) prevented activation and formation of blocking factor but did not impair the cytotoxic activity of already activated cells. Addition of SV40 antiserum to anti-HGG + C-treated cells led to effector cell activation, whereas heterologous virus-immune sera did not. Control studies established that the antibody-mediated recovery of cytotoxicity was not due to arming. Further studies showed that PARA-7 tumor antigen extract blocked at the effector cell level, not at the target cell level. Addition of PARA-7 extract to spleen cell supernatants mediating ADCC resulted in formation of a factor which blocked at the target cell level but not at the effector cell level. These data are compatible with the following interpretation. Spleen cell unresponsiveness is due to antigen blockade. Recovery of cytotoxicity occurs because antibody synthesized during the incubation period promotes elution of antigen from the effector cell surface. Thus, activation is accompanied by the generation of tumor antigen-antibody complexes.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Tumor-immunotherapy with the use of tumor-antigen-pulsed antigen-presenting cells

Summary Our previous study demonstrated that the administration of antigen-presenting cells (APC) pulsed with tumor cell membrane fraction to naive syngeneic mice results in effective induction of tumor-specific protective immunity in vivo. The present study examined whether tumor-antigen-pulsed APC can produce the inhibitory effect on the growth of tumor cells when administered to tumor-bearing hosts. Naive BALB/c mice were inoculated with viable tumor cells. Five days later, these mice started to receive the relevant tumor-antigen-pulsed APC at 3- to 4-day intervals. The administration of tumor-antigenpulsed APC induced the rejection or growth inhibition of a growing tumor in approximately half of the recipient mice. Moreover, it was demonstrated that tumor-specific immunity was induced in such tumor-regressed mice. These results indicate that tumor-antigen-pulsed APC are effectively applicable to the tumor-specific immunotherapy.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture, Japan     

Early antigen elimination by cytotoxic T cells results in diminished cellular immune responses to allogeneic tumor

Previous reports have suggested that repeated alloantigenic challenge increases humoral responses to alloantigens, but may cause decreasing cellular responses. We stimulated BALB/c (H-2^d) mice with intraperitoneal EL-4 tumor (H-2^b) and serially assessed cytotoxic responses in spleen and peritoneal lymphocytes using ⁵¹Cr-labeled EL-4 target cells. We observed that cytotoxicity generated in the spleen of nonimmune BALB/c mice was much greater than that in immunized mice; similar peak responses were generated in peritoneal lymphocytes in normal and immunized hosts. Complement-mediated cytotoxicity was not necessary for diminished splenic responses in hyperimmune hosts, for the same phenomenon was seen in the Hzl anti-BL/6 system which is free of humoral responses. Irradiation (2000 rad) of the EL-4 tumor challenge prevented tumor cell proliferation and markedly reduced splenic responses in nonimmune mice. We suggest that cytotoxic cells suppressed further generation of cyto-toxicity; by effecting an early elimination of tumor inoculum, tumor proliferation was abrogated and dose of cellular antigen was, consequently, markedly reduced.     

Direct processing and presentation of antigen from malaria sporozoites by professional antigen-presenting cells in the induction of CD8 T-cell responses

Irradiated malaria sporozoites induce better protection than viable untreated sporozoites. We observed early differences between irradiated and viable untreated sporozoites in priming responses in vivo to a protective CD8 T-cell epitope, pb9, of the circumsporozoite protein of Plasmodium berghei. Sporozoites were processed for MHC class I presentation by dendritic cells (DC) to prime pb9-specific IFN-gamma-producing CD8 T cells. DC pulsed with untreated and irradiated sporozoites were similarly capable of priming central memory T-cell responses, detectable by the IFN-gamma cultured ELISPOT assay. However, irradiation significantly enhanced sporozoites' ability to prime effector T-cell responses detectable by the IFN-gammaex vivo ELISPOT assay. Irradiation also enhanced the ability of splenic APC to process and present sporozoites in order to re-stimulate pb9-specific polyclonal and clonal T-cell responses. Sporozoites did not stimulate T cells in the absence of APC. Over-irradiation decreased the sporozoites' T-cell stimulating capacity in vitro at high parasite doses, which may indicate that an optimal irradiation dose is necessary to induce protective immunity by sporozoite inoculation. The induction of sporozoite-specific CD8 T-cell responses without the need for liver stage infection identifies a potentially important mechanism in the development of pre-erythrocytic immunity.     

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor-specific idiotype vaccines. III. Induction of T helper cells by anti-idiotype and tumor cells

In a previous report, we have demonstrated the induction of tumor-specific immunity by monoclonal anti-idiotype antibodies generated against a monoclonal anti-tumor antibody, 11C1, that also cross-reacts with mouse mammary tumor virus envelope glycoprotein gp52. Also, we showed that whereas one anti-idiotype antibody, 2F10, could induce protective immunity, another anti-idiotype antibody, 3A4, induced nonprotective immunity. Here we demonstrated the existence of T helper cells which recognize anti-idiotypes that exert differential controls on tumor growth. The qualitative nature of idiotype recognizing T cells generated in response to 2F10, 3A4, irradiated tumor, and progressively growing tumor was compared. The reactivity pattern of idiotype recognizing T cells obtained from 2F10 and irradiated tumor immunized mice were similar in nature in the sense that Lyt-2- T cells obtained from these immunized mice responded to both 2F10 and 3A4 as antigen, although T cells from tumor immunized mice responded better to 3A4 antigen. On the other hand, the idiotype-recognizing T cells obtained from 3A4-immunized mice showed a similar reactivity pattern to T cells isolated from mice during the early phase of tumor growth (within day 4 to 5 after the inoculation of 10(4) live tumor cells). Lyt-2- T cells isolated from mice immunized with 3A4 or during the early phase of tumor growth responded only to 3A4 antigen. The inability of Lyt-2- T cells, isolated from 4- to 5-day-old tumor in mice, to cooperate with 2F10-TNP is not due to the absence of 2F10 idiotype recognizing T cells as 2F10 id recognizing T cells are present when examined at the precursor level. These data on the idiotype specificity of T helper cells show a correlation with the presence of anti-tumor immunity. This information will help in the design and application of idiotype vaccine in tumor immunotherapy.     

Cell-mediated immunity to friend virus-induced leukemia. IV. In vitro generation of primary and secondary cell-mediated cytotoxic responses.

Primary and secondary cell-mediated cytotoxic responses to FBL-3 cells, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be generated by in vitro techniques as tested by the 125IUdR release assay. The specificity of the cytotoxic reactions appeared to be directed against the Friend type-specific antigen and the FMR (Friend, Moloney, Rauscher) antigen which were also the major antigens for transplantation immunity to FBL-3. In comparison to the primary cytotoxic response, the secondary cytotoxic response was accelerated (detected at an earlier time after sensitization), enhanced (gave much higher levels of cytotoxicity), was also longer lasting, and could be induced by a wide dose range of tumor cells. The secondary response could only be induced with lymphocytes obtained from regressors that were resistant to FBL-3 challenge; lymphocytes from mice with progressive tumor growth had no detectable secondary response. It was found that both induction phase and the effector phase of cytotoxic responses were T cell dependent. The characteristics of these reactions were thus very similar to those obtained with in vivo immunization or challenge, providing a good correlation with in vivo tumor immunity.     

Antibody formation and transient immune complex glomerulopathy in A-Strain mice with C1300 neuroblastoma tumors

One to three-month-old A-strain mice, inoculated subcutaneously with 2 x 10(6) viable syngeneic C1300 neuroblastoma cells (clone NB9R) developed a palpable tumor within 9-12 days and died within 28-30 days. A transient glomerulopathy developed after 16-24 days. Despite a normal histologic appearance, the nephropathy was clearly demonstrated by electron microscopy and was classified as a focal mesangiopathic glomerulonephritis. Deposits of host 7S-G immunoglobulins and C3 complement fragments were detected in these same kidneys by immunofluorescence. Radioimmunoprecipitin determinations on sera obtained from mice at different intervals from tumor cell inoculation, revealed that untreated mice contained circulating antibodies capable of reacting with 125I-labeled gp69-71 glycoprotein from Gross murine leukemia virus (MuLV). Antibodies to p30 MuLV antigen and to crude membrane antigen (s) (CMA) solubilized from NB9R cells were found in sera only after tumor cell inoculation. Circulating immune complexes formed by host 7S-G immunoglobulins were clearly detected from day 16 to 22. Antibodies eluted from kidneys with nephropathy were shown to react with NB9R cells in vitro and to react specifically with CMA and the p30 MuLV antigen.     

Requirement for p53 in ionizing-radiation-inhibition of double-strand-break rejoining by human lymphoblasts.

Ionizing radiation (IR) triggers apoptosis, cell-cycle arrest, and DNA-repair induction in mammalian cells. These responses are mediated by proteins, including p53, which are activated or induced by IR. To determine the role of p53 in double-strand break (DSB) repair following irradiation of mammalian cells, we compared the abilities of unirradiated and irradiated TK6 human lymphoblast line and its derivatives TK6-E6-20C and TK6-E6-5E to repair restriction-enzyme-linearized shuttle pZ189 and the luciferase-reporter plasmid pGL3-control. TK6-E6-20C expresses wild-type p53 like the parental TK6 line, while TK6-E6-5E is p53 null. DSB-rejoining capacity was determined from the ratio of viable progenies arising from DSB-containing plasmids (linDNA) to the number of viable progenies from undamaged, supercoiled plasmids (scDNA). The ratio from the p53wt hosts was two- to three-fold higher than that from the p53null host, using either pZ189 or pGL3-control plasmid. After exposure of both hosts to 0.5 Gy gamma-radiation, DSB-rejoining capacity of p53null increased two-fold compared to unirradiated null controls, if transfection occurred immediately after irradiation. In contrast, the DSB-rejoining capacity of p53wt was unaffected by irradiation. If transfection was delayed for 2 h following irradiation, however, DSB-rejoining declined in both p53wt and p53null hosts. Irradiation also altered DSB-rejoining fidelity, measured from the mutation frequencies, among progenies of pZ189 linDNA. But, unlike rejoining capacity, changes in DSB-rejoining fidelity were similar in p53wt and p53null hosts. Changes in cell-cycle distribution in p53wt and p53null hosts were also similar following irradiation. These findings show that IR increases DSB-rejoining capacity in mammalian cells without functional p53, suggesting that p53 participates in suppressing DSB-rejoining following exposure of mammalian cells to IR.     

The proangiogenic factor ephrin-A1 is up-regulated in radioresistant murine tumor by irradiation

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3'], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.     

Development of a cellular immune response in a highly cancer-susceptible C3H/He strain of mice and its nonfactor substrain C3H/f after the transplantation of a syngeneic mammary gland tumor]

Syngeneic mammary gland tumor (MMT-1) grew slower in non-factor C3H/f (MTV-L) mice than in wirus-positive C3H/He (MTV-S) mice. The immunization of mice with tumor (MMT-1), inoculated and later excised, revealed a higher immune reactivity in the mice of the non-factor substrain. In studying the dynamics of the cytotoxic activity of lymphocytes in regional lymph nodes cytotoxically active lymphocytes were found to appear in C3H/f mice on day 6 after the inoculation of tumor (MMT-1), while the areactive state of lymphocytes was observed in C3H/He mice on days 3 and 24 and in C3H/f mice on day 24 after the inoculation of tumor (MMT-1). Spleen lymphocytes in C3H/f mice had no cytotoxic effect on tumor cells.     

Modulation of F1 cytotoxic potentials by GvHR. Host- and donor-derived cytotoxic lymphocytes arise in the unirradiated F1 host spleens under the condition of GvHR-associated immunosuppression

As an approach to dissect complex cellular events that lead to GvHR-associated immune disorders, we followed cytotoxic activities, including NK cytotoxicity, in the spleens of unirradiated F1 hosts undergoing GvHR induced by parental spleen cells. Spleen cells of (B10 X DBA/2)F1 or (B10 X AKR/J)F1 hosts undergoing GvHR induced by parental B10 spleen cells displayed a prompt and marked increase in NK cell activity within 36 hr, and the heightened activity lasted until day 8. The activity then declined abruptly and disappeared on day 12 of GvHR. Inversely, donor B10-derived CTL specifically directed to the opposite parental alloantigens of the F1 hosts emerged in these F1 host spleens on day 8, and the CTL activity reached a peak on day 12 when the host NK cell activity disappeared. During the period that the donor-derived anti-host CTL were present, these F1 host spleen cells lost not only NK cell activity but also the ability to mount in vitro CTL responses. In contrast, the respective F1 strain mice undergoing GvHR induced by the parental DBA/2 or AKR/J spleen cells showed only transient but marked increases in NK cell activity during the initial 36 hr, and then the activity decreased gradually to return to the normal level on day 10. In such GvHR F1 host spleens, donor-derived CTL could never be detected, and the spleen cells showed normal in vitro CTL responsiveness during the entire observation period of 16 days. These results are discussed from the viewpoint of genetically defined cellular events that lead to the GvHR-associated immune disorders.     

Graft-versus-host reaction-like phenomenon induced by BCG in mice lethally irradiated and transferred with syngeneic bone marrow cells.

Heat-killed BCG in paraffin exerted a lethal effect on CS7BL/6 mice irradiated lethally and transferred with syngeneic bone marrow cells. Such an effect was not detectable when mice were subjected to adult thymectomy and used as the hosts. Lymphoid cells from such nonthymectomized mice exhibited cytotoxicity to syngeneic tumor cells but not to allogeneic tumor cells in an in vivo cytotoxicity test and induced splenomegaly in sublethally irradiated syngeneic recipients after systemic transfer. The cytotoxicity of such lymphoid cells was abolished by a treatment with anti-θ serum and complement. In the bone marrow of mice irradiated and transferred with bone marrow cells, the number of nucleated cells, the ratio of myeloid to erythroid cell series, and the percentage of lymphocytes were increasd by BCG injection. These results suggest the possibility that self-tolerance may be broken by BCG stimulation in the process of reconstitution of lymphoid cells in the irradiated mice.     

Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

Immunotherapy of murine leukemias by monoclonal antibody. I. Effect of passively administered antibody on growth of transplanted tumor cells

The objective of the present study was to establish a model system for the evaluation of passive immunotherapy of murine leukemias. Monoclonal antibodies directed at T lymphocyte differentiation antigens (Thy 1 and Lyt 2) were tested for their effect on tumors that were grown in hosts congenic for the target antigen. Tumor challenges were selected that were at least 500 times the dose that was lethal in 50% of untreated controls. The A strain leukemia, ASL.1, was transplanted subcutaneously into a/Thy 1.1 congenic hosts. Intraperitoneal administration of anti-Thy 1.2 monoclonal antibodies of the IgG3 and IgM classes reduced tumor growth. Up to 90% of the mice receiving antibody of the IgG3 subclass failed to develop tumors, whereas IgM antibodies prolonged survival time, but the mice eventually died of tumors. Antibody was most effective if administered within 24 hr of tumor inoculation; delay of antibody injection for 48 hr prolonged host survival but did not eradicate cells at the injection site or prevent metastases. The C57BL/6-derived tumors, ERLD and EL4, were evaluated for susceptibility to treatment with antibody directed at the Lyt 2.2 alloantigen using the protocol that was effective in treating aSL.1. Monoclonal antibody of the IgG2a subclass was effective in the case of C57BL/6/Lyt 2.1 congenic mice bearing ERLD, but caused a decrease in survival time of mice bearing the transplanted EL4 tumor. Thus, antibody of the appropriate immunoglobulin subclass can be effective in controlling tumor growth if administered in the optimal treatment regimen, but inherent features of the tumor cell ultimately determine whether abrogation or enhancement of growth will occur.     

Modification of radiation induced damage in mouse intestine by WR-2721

Intestinal protection in mice against radiation injury by WR-2721 (300 mg/kg body wt, i.p., 30 min before irradiation) was studied after whole body gamma irradiation (0.5, 1.5, 3.0, 4.5, or 6.0 Gy). Crypt survival and induction of apoptosis, and abnormal mitoses in crypt cells in the jejunum were studied on day 1, 3 and 7 after irradiation. Irradiation produced a significant decrease in crypt survival, whereas apoptosis and abnormal mitoses showed a significant increase from sham-treated control animals. Maximum changes in all the parameters were observed on day 1 after irradiation and the effect increased linearly with radiation dose. There was recovery at later intervals, which was inversely related to radiation dose. WR-2721 pre-treatment resulted in a significant increase in the number of surviving crypts, whereas the number of apoptotic cells in the crypts showed a significant decrease from respective irradiated controls on day 1 after exposure. The recovery was also faster in WR-2721 pre- treated animals. It is concluded that WR-2721 protects against gastrointestinal death by reducing radiation induced cell death, thereby maintaining a higher number of stem cells in the proliferating compartment.     

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.