A novel murine model of allogeneic vaccination against prostate cancer

Prostate cancer continues to be a major cause of death in men. Surgical and medical treatments of the disease have improved, but metastasic disease remains a significant clinical problem. Novel therapies such as whole cell vaccination offer the potential of treating disease by stimulating the immune system. To study the efficacy of a whole cell vaccine in prostate cancer two strains of mice were used: C57BL/6 (H-2Kb) and C3H/HeJ (H-2K^k) in combination with four different cell lines. Thus, a model was constructed of allogeneic and syngeneic vaccine, as well as a challenge tumour for each strain. Two novel cell lines were developed during this study. Firstly, the non tumourigeneic PMC-1 was derived from a normal mouse prostate and immortalized with HPV16. Secondly, the tumourigeneic PMC-1 C6ras1p1 was transformed with human ras gene which formed tumours in both SCID and C3H/HeJ mice. Protection, and the nature of the immune response to syngeneic and allogeneic vaccine, in males and females was examined in both strains. Vaccination with both syngeneic and allogeneic irradiated whole cell vaccines induced protection from syngeneic challenge in females. However, no protection was observed when allogeneic vaccine was given to male mice. This correlated with the immune response. Two types of cellular immune responses were generated in females. A NK-mediated response was observed in C57BL/6 mice, whilst C3H/HeJ mice developed a CTL response. Little or no cellular immune response was observed in males. The cytokine profile in C3H/HeJ females was a mixture of Th1 and Th2 whilst a mainly Th1 profile was observed in C57BL/6 mice. Male mice showed a diminished cytokine secretion compared to females which was further depressed after challenge. The difference in immunity was largely as expected, since tolerance to prostate antigens should not normally develop in female mice. However, this makes this model particularly relevant clinically since it directly mimics the human situation and thus may accelerate the development of whole cell vaccines for clinical use.     

Superior anti-tumor protection and therapeutic efficacy of vaccination with allogeneic and semiallogeneic dendritic cell/tumor cell fusion hybrids for murine colon adenocarcinoma

Cancer immunotherapy by dendritic cell (DC)/tumor cell fusion hybrids (DC/TC hybrids) has been shown to elicit potent anti-tumor effects via the induction of immune responses against multiple tumor-associated antigens. In the present study, we compared the anti-tumor effects of vaccinating Balb/c mice (H-2^d) with CT26CL25 colon carcinoma cells that had been fused with either syngeneic DCs from Balb/c mice, allogeneic DCs from C57BL/6 mice (H-2^b) or semiallogeneic DCs from B6D2F1 mice (H-2^b/d). Preimmunization with either semiallogeneic or allogeneic DC/TC hybrids induced complete protection from tumor challenge, whereas mice preimmunized with syngeneic DC/TC hybrids were only partially protected (75% tumor rejection). The average number of pulmonary metastases after intravenous tumor injection decreased significantly following immunization with semiallogeneic or allogeneic DC/TC hybrids (8.3 ± 7.9 or 16.3 ± 3.5, mean ± SD) relative to syngeneic DC/TC hybrids (67.8 ± 6.3). These data demonstrate that vaccination with semiallogeneic DC/TC hybrids resulted in the greatest anti-tumor efficacy. Anti-tumor effects showed by in vivo studies were virtually accomplished by the frequency of induced CTLs specific to both gp70 and β-galactosidase assessed by using pentameric assay. Among the fusion vaccines tested, semiallogeneic DC/TC hybrids induced the highest ratio of Th1 cytokine IFN-γ to Th2 cytokine IL-10. In addition, allogeneic or semiallogeneic DC/TC hybrids elicited a significantly stronger NK activity than syngeneic DC/TC hybrids. These findings suggest that in clinical settings, DCs derived from a healthy donor (which are generally characterized as more semiallogeneic than allogeneic) may be more capable than autologous DCs of inducing promising anti-tumor effects in vaccinations with DC/TC hybrids.     

Recruitment of inflammatory cells to a tumor deposit potentiates the immunotherapeutic effects of interleukin-2

Summary Interleukin-2 (IL-2) is a potent immunotherapeutic agent in murine models of intraperitoneal, pulmonary, and hepatic tumor implantation. Because of the systemic toxicity documented at doses of IL-2 required to control tumor growth, potentiation of the effects of low dose IL-2 is an important problem in immunotherapy. To address this problem, we attempted to recruit lymphocytes into a tumor mass. Allogeneic P185 (H-2^d) tumor was mixed with MCA-105 (H-2^b) tumor and injected s. c. into C57BL/6 (H-2^b) mice. Mice were treated with 50,000 units of IL-2 twice daily from day 0 to day 6. When IL-2 alone was used to treat s. c. tumor, there was no reduction in the size of tumor implants. When allogeneic tumor was mixed with syngeneic tumor, there was a reduction in tumor size at the high dose of allogeneic tumor but not at the low dose. When allogeneic tumor was mixed with syngeneic tumor and the mice treated with IL-2, the immunotherapeutic effects of IL-2 were markedly increased. These studies show that an immune response to alloantigens, generated within tumor tissue can augment the immunotherapeutic effects of exogenous IL-2.     

Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

Tumour-induced altered gastrointestinal steady-states: absence of MHC restriction in the paraneoplastic gastrointestine

Abstract The growth of a number of experimental rodent tumours including the Lewis lung tumour (LLca) progressively compromises the integrity of the host's gastrointestine by inducing cytokinetic alterations in the small bowel resembling those generally defining the intestinal phase of a graft-versus-host reaction (GVHR). To determine whether the induction of this paraneoplastic gastrointestine (PGI) involves, similar to a GVHR, a disparity between the MHC of the donor (LLca tumour) and the recipient (host), PGI development was evaluated in various LLca tumour-bearing murine strains that were either ‘syngeneic’[C57BL/6 and BL/10 (H-2^b)], ‘semisyngeneic’[B6D2F₁ (H-2^bd) and B6C3F₁ (H-2^bk)] or ‘allogeneic’[C3H/HeJ (H-2^k) and DBA/2 (H-2^d)] to the H-2^b LLca tumour. The temporal appearance and magnitude of a PGI developing in either LLca-syngeneic or semi-syngeneic hosts, but not the allogeneic strains, suggested that the mechanism(s) involved in PGI development, like the GVHR, was restricted by the MHC. Subsequent studies using congenic strains [B10.A (H-2^k) and B10.D2/nSn (H-2^d)], however, demonstrated that the mechanism(s) responsible for the PGI was restricted by the non-MHC loci of the C57BL mouse. These observations were supported by the appearance of a LLca-induced PGI in various B10.A congenic strains carrying mutations at the I-A or I-E/I-J loci of the MHC. Not unlike the intestinal phase of a GVHR, development of the PGI required the participation of enhanced mucosal mast cells which were limited in the WCB6F₁ (S1/S1^d) but not the (+/+) murine strains. These observations are discussed in light of the postulated premature migration of immature thymocytes that accompany tumour growth and their ability to non-specifically enhance (or suppress) cell mediated immune reactions in the host.     

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.     

Enhancing CTL responses to melanoma cell vaccines in vivo: synergistic increases obtained using IFNgamma primed and IFNbeta treated B7-1+ B16-F10 melanoma cells

Sequentially treating human melanoma cell lines by priming with interferon-gamma before adding interferon-beta was previously found to be the most efficient protocol for producing concurrently increased expression of the three surface antigens B7-1, intercellular adhesion molecule-1 and human histocompatibility leucocyte antigens Class I. The present study describes similar outcomes when the same sequential intercellular adhesion molecule-based protocol is applied to murine B16-F10 melanoma cells as well as preclinical studies using the B16-F10 model as a poorly immunogenic melanoma. Thus, treating B16-F10 cells or a highly expressing B7-1 transfected subline (B16-F10/B7-1 hi) by priming with interferon-gamma for 24 h before adding interferon-beta for a further 48 h (interferon-gamma 72/beta 48) increased expression of all three surface antigens, particularly major histocompatibility complex class I whose increased expression was sustained for several days. As a whole tumour cell vaccine, interferon-gamma 72/beta 48 treated B16-F10 cells produced greater levels of cytoxic T lymphocyte response compared to vaccines prepared from cells treated with a single type of interferon. Furthermore, B16-F10 cells expressing high levels of B7-1 and treated using the interferon-gamma 72/beta 48 protocol (interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi) produced substantially increased cytoxic T lymphocyte responses with a fivefold greater synergy than the combined results of either interferon treated or B7-1 expressing cells tested individually. The resulting CD8+ cytoxic T lymphocyte showed greater specificity for B16-F10 cells with tenfold higher killing than for syngeneic EL-4 lymphoma cells. Killing proceeded via the perforin-mediated pathway. CTL responses were induced independent of CD4+ T helper cells. The majority of mice receiving interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi vaccine in vivo remained tumour free after challenge with 5 x 105 live B16-F10 cells expressing intermediate B7-1 levels. The novel strategy described will help enhance vaccine potency when applied clinically to prepare whole cell based cancer vaccine therapies.     

Levels of Endogenous lnterleukin-1, Interleukin-6, and Tumor Necrosis Factor in Congenic Mice Infected with Borrelia garinii

This study describes the levels of interleukin-1 alpha (IL-1α), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the sera and parenchymal organs of various congenic mouse strains infected with Borrelia garinii. A significant elevation of inflammatory cytokine levels was found in the organs of C3H/HeN (H-2^k) and B10.BR (H-2^k) mice but not in those of BALB/c mice (H-2^d). Focally produced cytokines can contribute to antimicrobial defense against these organisms. High levels of IL-1α were observed in the sera of C3H/HeN, B10.BR and B10 (H-2^b) mice infected with B. garinii and they were associated with the presence of spirochetes in the skin. Thus, susceptible mice demonstrated a stronger cytokine response than resistant mice. This study presents in vivo evidence that B. garinii infection affects the immunopathogenesis of Lyme disease.     

Heated tumour cells of autologous and allogeneic origin elicit anti-tumour immunity

Vaccination with established tumour cell lines may circumvent the problem of obtaining autologous tumour cells from patients, but may also need immunological adjuvants. Up-regulation of heat shock proteins within tumour cell vaccines has resulted in increased immunogenicity in some models, but this has yet to be demonstrated for allogeneic (MHC-disparate) cell vaccines. This was investigated here using a rat model for prostate tumour cell vaccination. Heating of tumour cells (42°C, 1 h) elicited significant increases in HSP70 expression. Vaccination with heated autologous PAIII cells elicited protection against PAIII challenge in 60% of rats >50 days compared to 0% with unheated vaccine and was associated with an increased Th1 (IFN) immune response. Heated allogeneic MLL cells elicited significant protection against PAIII challenge, in contrast to unheated cells. The principle was confirmed in two mouse models, although the allogeneic melanoma vaccine K1735 elicited the best protection when heated and administered mixed with autologous dendritic cells. Thus, while heating of vaccine cells in some models is highly beneficial, and is a means of enhancing immunogenicity without genetic modification or inclusion of potentially toxic adjuvants, additional immune enhancement may be required.     

Multiple cytolytic T-cell clones with distinct cross-reactivity patterns coexist in anti-self + hapten cell lines

C57BL/10 T cells sensitized with TNBS-treated syngeneic cells and maintained in culture by repeated stimulations exhibit high cytolytic activity toward syngeneic TNBS-treated target cells with marked cross-reactivity on TNBS-treated target cells from mice of independent H-2 haplotypes (ten tested). The analysis of the reactivities of 48 T-cell clones derived by limiting dilution from such T-cell populations revealed three types of cytotoxic T-cell clones: (1) clones restricted by H-2K^b + TNP without cross-reaction on TNBS-treated or untreated target cells of other tested mouse haplotypes; (2) clones that lysed H-2^b + TNP and also TNBS-treated target cells from not more than one, two or three different H-2 haplotypes; (3) clones that lysed untreated H-2^k target cells. No T-cell clone was found to exhibit the wide pattern of cross-reactivity on any TNBS-treated mouse cell, characteristic of the original T-cell populations, indicating that these were composed of individual T-cell clones specific for TNP + private or for TNP + distinct public H-2 determinants. Correlation with described serological public H-2 specificities was possible for some cytotoxic T-cell clone reactivity, but not for others. The general pattern of T-cell reactivity as revealed by clonal analysis in this study, as well as in published work, includes cross-reactions between self H-2^a + X and allogeneic H-2ⁿ + X, between self H-2^a + X and unmodified allogeneic H-2ⁿ, or between allogeneic H-2ⁿ and allogeneic H-2^m + X, and is consistent with the hypothesis that MHC-class restriction is the main rule in T-cell recognition.Abbreviations used in this paper CTL cytotoxic T lymphocyte - TNBS trinitrobenzene sulfonate - TNP trinitrophenyl - TNCB trinitrochlorobenzene - H-2 mouse major histocompatibility complex - IL-2 interleukin 2 - Con A concanavalin A - FCS fetal calf serum - SCA IL-2 containing supernatant - LPS E. coli lipopolysaccharide - X a non-H-2 conventional antigen     

TARC and RANTES enhance antitumor immunity induced by the GM-CSF-transduced tumor vaccine in a mouse tumor model

INTRODUCTION: Transduction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene into mouse tumor cells abrogates their tumorigenicity in vivo. Our previous report demonstrated that gene transduction of GM-CSF with either TARC or RANTES chemokines suppressed in vivo tumor formation. In this paper, we examined whether the addition of either recombinant TARC or RANTES proteins to irradiated GM-CSF-transduced tumor vaccine cells enhanced antitumor immunity against established mouse tumor models to examine its future clinical application. MATERIALS AND METHODS: Three million irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated into syngeneic WEHI3B-preestablished BALB/c mice. RESULTS: Vaccinations were well tolerated. Mice treated with GM-CSF-transduced cells and the chemokines demonstrated significantly longer survival than mice treated with GM-CSF-transduced cells alone. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-gamma, and TNF-alpha, suggesting enhanced innate and adaptive immunity. Immunohistochemical analysis of tumor sections after vaccination revealed a more significant contribution of CD4+ and CD8+ T cells to tumor repression in the combined vaccine groups than controls. CONCLUSIONS: TARC and RANTES enhance the immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and may be clinically useful.     

A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor’s stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam ⁻ , AroA ⁻ ) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-γ release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8⁺ T-cell response against Legumain⁺ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.     

Immunogenicity of a Recombinant Measles-HIV-1 Clade B Candidate Vaccine

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10⁵ TCID₅₀ of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.     

Interleukin-2 secretion by KHT sarcoma cells leads to loss of their vaccine potential

 We have investigated the effect of interleukin-2 (IL-2) secretion by KHT sarcoma cells upon their vaccine potential in syngeneic C3Hf/He mice. Parental KHT tumor cells were transfected with the plasmid pBCMG-neo-mIL-2 to obtain a transfectant KHT-2-3-7 that secreted 20 units IL-2. KHT-2-3-7 cells elicited protective immunity in only 10% of the immunized mice, compared with 40% of mice immunized with irradiated parental KHT tumor colls. To minimize the contribution of potential antigenic differences between the KHT-2-3-7 transfectant and parental KHT cells, a clone of KHT cells (KHT-C21) was isolated and used in subsequent experiments. A number of transfectants secreting various amounts of IL-2, ranging from 2 units to 200 units, were obtained following transfection of KHT-C21 cells with plasmid pBCMG-neo-mIL-2. Two of the transfectants, C21-13-4 and C21-1, each secreting 200 units IL-2, elicited protective immunity in a significantly lower fraction of mice than did irradiated KHT-C21 parental tumor cells (P<0.0l). Two other transfectants C21-10 and C21-11, secreting 2 and 23 units IL-2 respectively, also showed lower vaccine potential compared with the parental KHT-C21 clone (P<0.05). To minimize further any role for potential antigenic or other molecular differences between the individual transfectants and the clonal KHT-C21 parental cells in lowering their vaccine efficacy, mice were immunized with a mixture of five transfectants, and the results again showed significantly lower vaccine efficacy of the mixture compared with the irradiated parental C21 cells (P<0.0l). In view of published studies showing enhanced or unchanged efficacy of IL-2-secreting tumor cell vaccines, our observation of the lower vaccine potential of IL-2-transduced tumor cells indicates that the vaccine efficacy of IL-2-secreting tumor cells depends on the individual tumor. Such variability/unpredictability would hamper the clinical use of IL-2-secreting tumor cells as vaccines. Received: 23 April 1996 / Accepted: 7 February 1997     

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response

 This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2–3) peak of interferon γ (INF-γ), interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-γ could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Rα and IFN-γ responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/2 mice was a delay in the latter in the IL-2, IL-2Rα and IFN-γ responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning. Received: 4 September 1997 / Accepted: 2 October 1997     

H-2 antigen expression: Loss in vitro,restoration in vivo,and correlation with cell-mediated cytotoxicity in a mouse lymphoma cell line

The susceptibility to cell-mediated cytolysis of cells of the recently developed C57BL/Ka(H-2 ^b ) lymphoma cell line, BL/VL₃, was investigated in allogeneic assays with thymus-dependent lymphocytes (T cells). Compared to EL4, the widely used C57BL/6(H-2 ^b ) lymphoma cell line, BL/VL₃ cells were found to be insensitive to T-cell-mediated lysis as detected by the use of⁵¹Crrelease methods. When used as immunogens in alloreactive combinations with BALB/c(H-2 ^d ) splenocytes as responder cells, BL/VL₃ cells failed to elicit sensitization. Serological tests showed that this cell line had profoundly reduced levels of H-2^b antigens on its surface. When BL/VL₃ cells were reinjected into C57BL/Ka and BALB/c mice, full recovery of H-2^b antigen expression at the cell surface was observed in both syngeneic and allogeneic hosts after only 11 days of in vivo growth. Concomitantly, they acquired the ability to induce cytotoxic responses in allogeneic T cells and became susceptible to their lytic activity. The expression of H-2 antigens on the surface of BL/VL₃ cells is a reversibly modulated function that depends on in vivo growth conditions and is lost in vitro in the absence of immunoselective pressure.     

Mutations in H-2K b influence the specificity of alloreactive effector cells included in the repertoire of H-2D b -restricted cytotoxic T lymphocytes against Moloney leukemia virus

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2 ^b ) responder cells and a series of H-2K ^b -mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected K ^b -mutant cells, but not B6 cells, whereas K ^b -mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the K^b molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all K ^b mutants is mainly D^b-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.     

Low-dose IL-2 induces cytokine cascade, eosinophilia, and a transient Th2 shift in melanoma patients

Purpose: To assess changes in serum cytokine levels in patients treated concomitantly with or without systemic low-dose IL-2. Vaccination targeted CTL responses to peptide antigens, and IL-2 was coadministered to expand activated CTL. Paradoxically, CTL responses were diminished in patients after 2 weeks of IL-2. We hypothesized that changes in the cytokine milieu may have contributed to this result. Experimental design: Serum samples were studied from 37 patients enrolled in two clinical trials of a melanoma peptide vaccine administered with or without low-dose IL-2 therapy. Twenty-two patients enrolled in the MEL36 trial received six weekly vaccinations with the four-peptide mixture and were randomized to receive subcutaneous IL-2 (3×10⁶ IU/m²/day) daily for 6 weeks beginning either at week 1 (upfront group) or at week 4 (delayed group) of vaccine therapy. Fifteen patients on the MEL39 trial were treated with the same vaccine without concurrent IL-2 administration. Results: Circulating levels of IL-5 peaked 1 week after starting IL-2, followed 2 weeks later by a marked eosinophilia, correlating in magnitude with peak IL-5 serum levels. Levels of IFN, GM-CSF, IL-4, IL-10, and IL-12 had no observed relationship to IL-2 administration. At the time of the IL-5 serum peak, PBL responses to mitogen suggested a transient shift to Th2-dominance. Conclusions: Low-dose IL-2 appears to have induced a transient Th2-dominant secondary cytokine cascade at the time of vaccination, for which eosinophilia is a surrogate marker. For future vaccine therapies targeting cytotoxic T-cell responses, delaying IL-2 until after initiation of immune responses may be more effective.William Chad Cragun and Galina V. Yamshchikov contributed equally to this paper.     

Alloantigen-induced T helper activity. I. Minimal genetic differences necessary to induce a positive allogeneic effect.

Addition of histoincompatible lymphocytes can influence the course of ongoing immune responses. Such allogeneic effects may either augment or diminish immune responses. We describe here the minimal genetic differences necessary to generate positive allogeneic effects (allohelp) in a humoral immune response. The antibody response to sheep erythrocytes of T cell-depleted mouse spleen cells was reconstituted by addition of syngeneic or allogeneic nylon wool column-passaged spleen T cells. T cells were pretreated with mitomycin C before culture to prevent development of allo-suppression and cytotoxic lymphocytes. Positive allogeneic effects were operationally defined as superior helper effects (to generate greater antibody forming cell responses) with T cells allogeneic rather than syngeneic to the responding B cells. Thus, addition of allogeneic T cells resulted in many more antibody forming cells than did equal numbers of syngeneic T cells, and fewer allogeneic than syngeneic T cells were necessary to generate comparable responses. With congenic, recombinant, and mutant mouse lines, genetic differences in the H-2 complex and those associated with Mls were each sufficient to provide positive allogeneic effects. With intra-H-2 recombinants, differences at either I or D were sufficient. A disparity at H-2K alone, as provided by the H-2 mutant B6.C-H-2ba against the parental line C57BL/6By, also induced helper effects. The significance of these results is discussed.     

A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes

A monoclonal antibody (mAb) TP-3 has been established by immunizing rats with the BALB/c mouse thymic epithelial cell line TEL-2. The TP-3 antigen is expressed on stroma cells of thymus, spleen, and lymph node in syngeneic BALB/c mice (H-2 ^d ). This antigen is also expressed at a low level on the cell surface of immature thymocytes, and at a high level on mature T and B cells. In allogeneic mice such as C57BL/6 (H-2 ^b ) or C3H (H-2 ^k ), no cells expressed the TP-3 antigen. Using H-2 congenic mice, reactivity with mAb TP-3 was found to map to a region of H-2D ^d L ^d or between D ^d and Qa, suggesting that TP-3 is a major histocompatibility complex (MHC) class I antigen. However, immunoprecipitation analysis indicated that this antigen is not identical to the classical mouse class I molecules in terms of molecular size, antigenicity, and tissue distribution.