Immune responses against allogeneic and syngeneic tumors in aged C57BL/6 mice

Aged C57BL/6 (B6) mice could reject allogeneic BALB/c RL male 1 tumor as efficiently as young B6 mice. However, in vitro analysis showed impaired generation of cytotoxic T cell response in aged B6 mice against allogeneic tumor. The reaction could be augmented by the addition of recombinant interleukin-2 (rIL-2). Enzyme-linked immunospots (ELISPOT) produced by CD8+ T cells purified from spleen cells showed no reduction in aged mice. The findings suggested that the number of CD8+ T cells capable of reacting against allogeneic H-2 antigens was similar in young and aged B6 mice. Low cytotoxic T lymphocyte (CTL) responsiveness in aged B6 mice appeared to have resulted from low responsiveness of CD4+ T cells producing IL-2. Although CTL generation was apparently impaired, strong multiple antigenicity of allogeneic tumor evoked a rejection response in aged B6 mice. On the other hand, no rejection response was observed against syngeneic EL4 tumor in aged B6 mice even after depletion of CD4+ CD25+ immunoregulatory cells. Depletion of CD4+ CD25+ cells caused rejection of EL4 tumor in young B6 mice. The findings suggested that aged B6 mice were incapable of inducing effector cells against weak tumor antigens. Only marginal CTL response and small number of ELISPOTs were generated in young but not aged B6 mice against EL4. Addition of rIL-2 to the culture augmented EL4 killing and ELISPOTs in spleen cells from young and aged B6 mice.     

The effects of cyclophosphamide on in vitro cytotoxic responses to a syngeneic tumour

Summary We have studied the effects of treating DBA/2 mice with high doses of cyclophosphamide upon their subsequent ability to generate cytotoxic cells in vitro against syngeneic tumour antigens or alloantigens. High doses of cyclophosphamide (100–200 mg/kg body weight) eliminated the response to both antigens. The addition of normal DBA/2 thymocytes into these cultures restored the response to allogeneic cells but not to tumour cells. The anti-tumour response could be restored by the addition of interleukin 2 to the cultures. Treatment with high doses of cyclophosphamide decreased the number of anti-tumour cytotoxic cell precursors in the spleen, but did not affect the capacity of bulk cultures of spleen cells to produce interleukin 2 when stimulated with the mitogen concanavalin A.Abbreviations CY Cyclophosphamide - CTL cytotoxic T cells - CTLp precursor cytotoxic T cells - IL2 interleukin 2 - Con A concanavalin A - FCS fetal calf serum     

Formation of specific antitumor cytotoxic T-lymphocytes in monoculture]

A new model for the generation of specific antitumor cytotoxic T-lymphocytes (CTL) was proposed. In contrast to other models, it allows to generate effector CTL without immunization in vitro. C57BL/10 mice or/and C57BL/6 mice were immunized by injection with gamma-irradiated syngeneic tumor cells into the footpads. For estimation of cytotoxic activity, chromium-51 release assay was used. It has been shown that effector CTL were absent in the lymph nodes in 1-fold as well as 2-fold immunization. Cytotoxic cells have not been found in 1-fold immunization even after maturation of the lymphocytes in monoculture. Specific CTL were detected only after secondary immunization and subsequent cultivation in vitro. Effector cells had Thy1.2+, Lyt2+, L3T4- phenotypes. Presence in vitro of exogenous IL-2 was needed for the generation of CTL against MX-11 sarcoma but not against EL4 lymphoma. We suggest that the release of IL-2 from lymphomas cells could stimulate generation of the effector cells through activation of the endogenous production of IL-2, or due to some other factors.     

The immunological mouse mutants nude (nu) and rhino (hr rh ) generate cytotoxic effector cells following adoptive immunotherapy but fail to reject a transplanted tumor

Summary Adoptive immunotherapy, consisting of cyclophosphamide injection and the i. v. transfer of tumor-sensitized T cells, resulted in rejection of the immunogenic fibrosarcoma, MCA/76-9, by syngeneic C57BL/6J (B6) mice. The same treatment of tumor-bearing congenic immunodeficient mice, homozygous for the deleterious mutations nude (nu) and rhino (hr ^rh ), did not result in tumor rejection. Paradoxically, the intratumor and intrasplenic changes taking place in each of the three strains after therapy were indistinguishable. There was an increase in Thy-1⁺, Lyt-2⁺, or L3T4⁺ cells at the tumor site 8 days after adoptive immunotherapy and a similar increase in Thy-1⁺ cells in the spleen. Moreover, the T cells isolated from the tumors or spleens from each genotype were shown to be specifically cytotoxic in vitro as well as in an in vivo Winn assay. Further evidence that immune amplification had occurred in the immunological mutant mice was provided by experiments showing (a) the ability of spleen cells from tumor-bearers and those tested after therapy to produce IL-2 in response to Con A stimulation and (b) an increase in class II-MHC antigen expression by tumor-associated macrophages. The data suggest that, although amplification of antitumor immune responses occurred in the immunological mutants, the absence of a critical host factor limited the potency of the antitumor response. Abbreviations used: CY, cyclophosphamide; TAM, tumor-associated macrophages; TAL, tumor-associated lymphocytes; TAC, tumor-associated cells; B6, C57BL/6J; class II MHC antigens: Ia; con A, concanavalin A; IL-1, IL-2, interleukin 1 and 2     

Specificity of adoptive chemoimmunotherapy of established syngeneic tumors

To examine the specificity of adoptive chemoimmunotherapy (ACIT) of established syngeneic tumors, two noncross-reactive C57BL/6 tumors were studied: a Friend virus-induced tumor (FBL-3) and a chemically induced virus-negative tumor EL-4(G-). In vitro studies confirmed that these tumors are antigenically distinct by demonstrating that the cytotoxic responses of spleen cells from mice immunized in vivo and reexposed to tumor in vitro are immunologically specific. Studies of ACIT with cells from mice immunized in vivo demonstrated similar specificity. Mice receiving 5 x 10(6) FBL-3 on day 0 all died by day 13. Treatment on day 5 with cyclophosphamide (CY), 180 mg/kg, prolonged the median survival time (MST) to day 23. Treatment on day 5 with CY plus 2 x 10(7) normal nonimmune C57BL/6 cells or CY plus cells sensitized to EL-4(G-) had no additional effect on survival whereas 2 x 10(7) C57BL/6 cells sensitized to FBL-3 in vivo prolonged MST to day 64 and cured 13 of 32 mice. Similarly, mice given 2 x 10(5) EL-4(G-) on day 0 all died by day 16, and CY on day 5 prolonged the MST to day 22. As an adjunct to CY, 2 X 10(7) normal cells or cells sensitized to FBL-3 had a modest effect, prolonging the MST to days 37 and 36, respectively. However, treatment with CY plus 2 x 10(7) cells immune to EL-4(G-) cured 22 of 32 mice. The results demonstrate the immunologic specificity of ACIT of syngeneic tumors treated with immune syngeneic cells.     

Augmented induction of antitumor cells in vivo by cyclophosphamide fails to benefit antitumor resistance of the host

Summary The present study was designed to examine whether cyclophosphamide augmented induction of antitumor cells and antitumor resistance in C57BL/6 mice pretreated with mitomycin-C-treated EL4 cells (EL4_MMC) plus OK-432, a streptococcal preparation. C57BL/6 mice were pretreated with EL4_MMC (10⁷) plus OK-432 (2.5 KE) i.p. twice at 1-week intervals. When the mice received an i.p. injection of cyclophosphamide at 200 mg/kg 2 days before the last treatment, the antitumor activity of their spleen cells and peritoneal exudate cells (PEC) was effectively augmented 7–8 days after the last treatment. Splenic antitumor activity disappeared 15 days after the last treatment whereas augmented antitumor activity of the PEC was detected even 28 days after the last treatment. This cyclophosphamide effect was dose-dependent and 200 mg/kg was the most effective among the doses tested. If the EL4_MMC plus OK-432 treatment was injected at a s.c. site, it was also effective in combination with cyclophosphamide. The antitumor activity of the PEC from s.c.-pretreated mice, however, was lower than that from i.p.-pretreated mice. Despite the fact that cyclophosphamide effectively augmented induction of antitumor cells in C57BL/6 mice pretreated with EL4_MMC plus OK-432, it diminished rather than augmented, under all conditions tested, the ability of the mice to resist a challenge of live EL4 cells. Reduction of antitumor resistance by cyclophosphamide was also observed in an experimental system of a semi-syngeneic host (BDF₁) tumor (EL4). These results indicate that augmentation of in vivo induction of certain kinds of antitumor cells does not necessarily result in a beneficial augmentation of the host's ability to resist tumor growth.     

Immunity to melanoma in mice immunized with transfected allogeneic mouse fibroblasts expressing melanoma-associated antigens

Summary Transfection of genomic DNA from B16 mouse melanoma into LM(TK^–) fibroblasts led to the generation of several clones of transfected cells that strongly expressed B 16 melanoma-associated antigens (MAA). The transfected cells retained their H-2^k markers and served as allogeneic cells with expressive MAA in C57BL/6 mice, syngeneic with the melanoma. The cells were capable of eliciting primary anti-B16 immune responses in vitro in spleen cells from C57BL/6 mice. Immunization of C57BL/6 mice with the transfected cells led to the generation of anti-B16 cytotoxic activity in spleen cells, and C57BL/6 mice immunized with the MAA-positive transfected cells were partially resistant to a lethal challenge with B16 melanoma cells. Under similar conditions, B16 cells were nonimmunogenic. Therefore, transfected allogeneic LM(TK^–) fibroblast cells expressing MAA served as more potent anti-melanoma immunogens than the parental B16 tumor cells themselves.     

Augmentation of the therapeutic efficacy of adoptive tumor immunotherapy by in vivo administration of slowly released recombinant interleukin 2

Summary Immunization of C57BL/6 mice with MMC-treated syngeneic lymphoma cells, MBL-2, caused the generation of antitumor effector cells in vivo and the immunized mice permanently rejected viable MBL-2 lymphoma cells. Both plastic nonadherent T cells and plastic adherent MØ obtained from MBL-2 immunized mouse peritoneal exudate cells revealed strong cytotoxic activity against MBL-2 lymphoma cells, whereas immune spleen cells were not highly active against MBL-2 lymphoma cells in vitro. However, systemic adoptive transfer of immune spleen cells into the MBL-2-bearing mice by i.v. infusion in conjunction with i.p. cyclophosphamide (100 mg/kg) treatment cured the mice of tumor. This therapeutic efficacy of immune spleen cells was reflected by the number of transferred effector cells and over 5×10⁷ immune spleen cells were required to cure the mice completely. The cells mediating in vivo rejection of MBL-2 lymphoma cells were Thy 1.2⁺ T cells. This ACIT was specific against MBL-2 lymphoma cells and had no effect on the growth of other syngeneic tumors, B16 melanoma or BMC6A fibrosarcoma. In vivo administration of recombinant interleukin 2 (r-IL 2) combined with ACIT greatly modulated the cure rate of tumor-bearing mice. In addition, we found that slowly released r-IL 2 administratered from an ALZET miniosmotic pump was more effective in augmenting the therapeutic efficacy of immune spleen cells in ACIT than a single injection of the same total dose of r-IL 2.     

Isolated soluble fractions from the murine B16 melanoma induce primary in vitro syngeneic antitumor responses

Summary This paper extends our previous studies, which documented our ability to isolate immunogenic entities from nonimmunogenic or weakly immunogenic tumors.B16 melanoma cells failed, in our in vitro experimental system, to induce anti-B16 cytotoxic responses in spleen cells derived from normal syngeneic C57BL/6 mice. The B16 melanoma cellular homogenate was fractionated on an Ultrogel AcA 34 column, and the various fractions were tested for their ability to induce anti-B16 cytotoxic responses under the same conditions as those used for intact B16, the nonimmungenic tumor cells. Certain fractions, some of them with relatively low protein concentrations, induced anti-B16 cytotoxic responses in spleen cells of normal C57BL/6 mice, whereas others, some of them with relatively high protein concentrations, failed to induce such responses. One fraction (Fr.), designated Fr. 5/6, was examined in detail. It was found that in normal syngeneic spleen cells this fraction induced effector cells that efficiently killed (at various E : T ratios) the relevant B16 target cells and RBL5 syngeneic tumor cells, but not the YAC allogeneic tumor cells or C57BL/6 lymphoblasts. Furthermore, an excess of unlabeled B16 cells most efficiently blocked the ability of these anti-B16 effector cells to kill radiolabeled B16 target cells. RBL5 tumor cells, YAC tumor cells, or C57BL/6 lymphoblasts failed to block these effector cells efficiently. A significant fraction of the effector cells induced with Fr. 5/6 was characterized as thymus-derived cells (Thy-1⁺, Thy-2⁺3⁺ cells). It was suggested that another fraction of the cellular population was natural killer cells, which cytolyzed the RBL5 target cells. Various theoretical and practical aspects of these findings are discussed.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. II. Development of syngeneic cytotoxicity in the absence of specific antigenic stimulation

Two out of four long-term murine allospecific cytotoxic T lymphocyte (CTL) clones tested could develop high levels of cytotoxicity against syngeneic target cells when cultured under appropriate conditions. All CTL clones maintained strict allospecificity so long as they were cultured with both appropriate allogeneic stimulator cells and growth factor (supernatant from secondary mixed lymphocyte cultures). In two of the clones, syngeneic reactivity rapidly developed when the allogeneic stimulator cells were replaced with syngeneic or third party stimulator cells, and when the supernatant from EL4 thymoma cells stimulated with phorbol ester was used as growth factor. In addition to killing the appropriate allogeneic target, clones with syngeneic reactivity could kill both syngeneic C57BL/6 targets and H-2-congenic BALB.B targets but not third party unrelated targets, suggesting that the self structure recognized was coded for within the major histocompatibility complex. Such clones did not kill the natural killer (NK) target YAC. The results obtained from cold target inhibition and from subcloning at limiting dilution of clones with syngeneic reactivity suggested that both allogeneic and syngeneic reactivity could be expressed by the same individual cell in the CTL clone. The specificity for syngeneic H-2 as opposed to third party H-2 and NK-sensitive target cells, and the observation that both allospecific and syngeneic killing could be partially blocked by anti-Lyt-2 antibody treatment of the CTL, strongly suggested that different recognition structures are involved in CTL-mediated syngeneic cytotoxicity and NK cytotoxicity.     

Natural cell-mediated cytotoxicity in vitro and inhibition of tumor growth in vivo by murine lymphoid cells cultured with T cell growth factor (TCGF)

Summary Lymphoid cells obtained from the spleen, thymus, bone marrow, peripheral blood, and peritoneal exudate of normal mice (BALB/c, BALB/c nude, C57BL/6, C3H) and from spleens of mice bearing a transplantable lung carcinoma or primary mammary carcinoma were expanded in culture for 1–9 months, with an increase in cell number of 10⁵- to 10⁶-fold per month, in crude or lectin-depleted medium containing T cell growth factor (TCGF). All these cultured lymphoid cell (CLC) lines exhibited strong cytotoxic activity in vitro (assessed by ⁵¹Cr-release assays) toward a variety of freshly harvested and cultured syngeneic, allogeneic, and xenogeneic tumor target cells, both lymphoid and solid (including metastatic growths) in origin. Extensive killing was observed against tumor targets that were resistant to lysis by natural killer (NK) cells as well as to NK-sensitive tumor lines. Low levels of cytotoxic reactivity were also demonstrated against fresh and cultured normal lymphoid cells. The CLC had some characteristics of NK cells but also expressed some typical T cell markers. In local Winn-type neutralization assays, CLC delayed or completely inhibited the growth of lymphomas and carcinomas in syngeneic and allogeneic recipients. In mice with metastatic growth of a second-generation transplant of mammary carcinoma, CLC were shown to have some therapeutic effect when administered IV 1 day after cyclophosphamide. No significant beneficial action of IV administered CLC was observed in the absence of chemotherapy in mice implanted with a lung carcinoma. The possibilities of employing TCGF-propagated cytotoxic effector cells in adoptive immunotherapy of human malignancies are discussed.     

Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen

In order to develop immunotherapy strategies that are based on eliciting immune responsiveness to the self-antigen, human carcinoembryonic antigen (CEA), we examined whether cytotoxic T lymphocyte (CTL) activity against CEA could be elicited in CEA-transgenic and nontransgenic mice. CEA-transgenic [C57BL/6-TGN(CEAGe)18FJP] and nontransgenic mice were primed with CEA-transfected syngeneic fibroblasts in combination with Corynebacterium parvum. Spleen cells from immunized mice were cultured with irradiated syngeneic MC-38 colon carcinoma cells transfected with CEA (MC-38.CEA) as stimulators prior to the measurement of CTL activity. Primed nontransgenic spleen cells showed augmented CTL activity against MC-38.CEA cells as compared with control parental MC-38 cells, nontransfected or transfected with vector only. Moreover, primed CEA transgenic spleen cells showed augmented CTL activity against MC-38.CEA cells that was similar to that observed in nontransgenic mice. All CTL clones derived from either transgenic or nontransgenic mice showed cross-reactivity with MC-38 cells expressing the CEA-related antigen, nonspecific cross-reacting antigen, but not biliary glycoprotein. CEA-specific CTL clones were not identified. Adoptive transfer of cloned CTL resulted in inhibition of MC-38.CEA but not MC-38.BGP tumor growth. Tumor cures were elicited in mice treated with a combination of cloned CTL and cyclophosphamide. Histopathological examination of CEA-expressing colons from either immunized mice or recipients of cloned CTL did not reveal any autoimmune reactions. These studies demonstrate that CTL recognizing cross-reactive class I epitopes on the CEA molecule can be induced in transgenic mice. The expression of these epitopes on tumor cells creates effective targets for CTL in vivo without inducing adverse reactions in CEA-expressing normal tissues. Since anti-CEA CTL have been generated in humans, CEA-transgenic mice may be a useful model to study vaccines that are based on CTL effector mechanisms. Received: 7 January 2000 / Accepted: 8 March 2000     

In vitro studies of the natural humoral antitumor reactivity of BALB/c and C57BL/6J mice

Summary Antitumor antibodies were produced in vitro by spleen cells harvested from 12- to 18-month-old C57BL/6J and BALB/c mice with a naturally occurring complement-dependent serum cytotoxicity for tumor cells. The antibodies were of the IgM class and had a complement-dependent cytotoxic reactivity on EL4 cells, which was not absorbed by normal thymus cells. No natural antibodies were produced by untreated spleen cells from 1- or 3-month-old mice of the same two strains, which had no natural serum reactivity. However, after a treatment with an anti-Thy-1 serum and complement before culturing, spleen cells from 3-month-old mice, but not 1-month-old mice, produced the natural antitumor cytotoxic antibodies in vitro. When antibody-producing spleen cells from 12-month-old mice were cultured in vitro together with spleen cells from the unreactive 3-month-old syngeneic mice, inhibition of the antibody production occurred. This inhibiting capacity increased between 1 and 6 months of age and was abrogated by a treatment with an anti-Thy-1 serum and complement or with an anti-Ly-2 serum, whereas the passage of the inhibiting spleen cells on an anti-IgG column did not modify the inhibiting capacity. The in vitro data confirm previous in vivo findings on the nature, specificity and systems of regulation of the natural antitumor cytotoxic antibodies.     

Amniotic Fluid Enhances Allogeneic Cytotoxic T Cell Responses,Whereas It Suppresses Mitogen-Stimulated Lymphocyte Proliferation

Mouse amniotic fluid has been shown to suppress T lymphocyte proliferation and suggested to be important in regulating immunity during pregnancy. In allogeneic pregnancy, cytotoxic T cells in pregnant lymphocytes against paternal transplantation antigen are impaired. We examined the effect of amniotic fluid to the alloreactive CTL responses. Although the amniotic fluid suppressed Con A or LPS stimulated lymphocyte proliferation as previously reported, the amniotic fluid taken from syngeneic C57BL/6 pregnant mice or allogeneic C57BL/6 × BALB/c pregnant mice enhanced the anti-H-2^d or anti-H-2^k CTL responses dose-dependently. We speculate that amniotic fluid contains not only immunosuppressive factors but also immunoenhancing factors which upregulate the allogeneic CTL responses.     

Evidence for Generation of Suppressor T Cells in Mycobacterium lepraemurium-Infected CBA/J Mice,a Mouse Strain Highly Susceptible to the Infection

Susceptibility to Mycobacterium lepraemurium (MLM) infection markedly differed between two mouse strains, CBA/J and C57BL/6. CBA/J mice showed high susceptibility to MLM infection and developed either very weak or no delayed-type hypersensitivity (DTH) to MLM antigen after the injection of MLM. In contrast, C57BL/6 mice, which were resistant to MLM infection, showed significant DTH reaction to MLM antigen after the injection. Treatment of CBA/J mice with cyclophosphamide (Cy) conferred significant resistance to MLM infection on the CBA/J mice, and the treated mice developed a strong anti-MLM DTH response after the MLM injection. When spleen cells from MLM-infected CBA/J mice were transferred to Cy-treated and MLM-infected syngeneic mice, the anti-MLM DTH reaction of the recipient mice was suppressed. Treatment of the spleen cells to be transferred with anti-Thy-1.2 antibody or anti-I-J^k antiserum plus complement abrogated the suppressive activity. Thus, it is suggested that the high susceptibility of CBA/J mice to MLM infection is due to the generation of Cy-sensitive, I-J^k-positive suppressor T cells after infection with MLM.     

Effect of low-dose cyclophosphamide therapy on specific and nonspecific T cell-dependent immune responses of spleen cells from mice bearing large MOPC-315 plasmacytomas

Summary Some T cell-dependent immune parameters were examined in mice bearing a large MOPC-315 plasmacytoma before and after treatment with a low dose (15 mg/kg) of CY. Prior to CY therapy, spleen cells from mice bearing a large MOPC-315 tumor were depressed in their ability to generate an in vitro cytotoxic response to the MOPC-315 tumor, to a different syngeneic plasmacytoma, MOPC-104E, and to an allogeneic thymoma, EL4. The spleen cells of these mice were also depressed in their ability to proliferate in response to the T cell mitogen PHA. Following CY therapy, the spleen cells generated an enhanced anti-MOPC-315 cytotoxic response by day 2, and the level of this response continued to increase so that by day 7, it was greatly enhanced and was much greater than the response of normal spleen cells. The recovery of the cytotoxic responsiveness to the antigenically related MOPC-104E tumor after CY therapy followed a similar pattern. In contrast, the spleen cells of these animals remained depressed in their cytotoxic response to the antigenically unrelated EL4 thymoma for at least 11 days after CY therapy. Although the anti-EL4 response recovered by day 14, the level of antitumor cytotoxicity generated did not exceed that generated by normal spleen cells. The PHA response remained greatly depressed in CY-treated MOPC-315 tumor bearers, even 14 days after the chemotherapy. Thus, at a time following low-dose CY therapy, when potent T cell-dependent antiplasmacytoma immunity had completed the eradication of a large MOPC-315 tumor burden not eliminated through the direct effect of the drug, the T cell-dependent response to an unrelated tumor and to PHA remained depressed.Supported by United Public Health Service Research Grant #CA-30088 and by Training Grant #CA-09318 from the National Cancer InstitutePresented in part at the 77th annual meeting of the American Association of Cancer Research, May 7–10, 1986In partial fulfillment of the requirements of the Graduate College for the Doctor of Philosophy degree Abbreviations used: CY, cyclophosphamide; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PHA, phytohemagglutinin     

The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2^b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2^b) (B6) and allogeneic C3H/Hej (H-2^k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.     

Prodigiosin 25-C Suppression of Cytotoxic T Cells in Vitro and in Vivo Similar to That of Concanamycin B,a Specific Inhibitor of Vacuolar Type H+-ATPase

The effects of prodigiosin 25-C (PrG) which preferentially suppresses cytotoxic T cells (CTL), was examined in comparison with concanamycin B (CMB), a specific inhibitor of vacuolar type H⁺ -ATPase (V-ATPase). PrG and CMB directly inhibited the cytotoxic function of CTL and neutralized acidic organelles of CTL in vitro. In addition, PrG or CMB was injected in C57BL/6 mice after immunization with an allogeneic mastocytoma, P815. PrG and CMB inhibited the killing activity of CTL against the tumor and reduced the population of CD8⁺ cells without affecting CD4⁺ and B220⁺ populations in the spleen. PrG and CMB had only a negligible effect on antibody production induced by sheep red blood cells (SRBC) and mitogenic responses of lymphocytes. These results suggest that PrG and CMB have similar immunosuppressive properties at least through their inhibitory effects on acidification of intracellular organelles required for the effective function of CTL.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.