Regulation of the immune response to tumor antigens. II. The nature of immunosuppressor cells in tumor-bearing hosts.

Immunosuppressor (IS) cells were found to be generated in tumor-bearing animals (TBA) within 24 hr after inoculation of tumor cells of the methylcholanthrene-induced Sarcoma 1509a and appeared to persist in the hosts as long as the tumor was progressing. However, IS cells disappeared with 5 days after extirpation of the tumor. Increasing doses of thymus cells of TBA increased the degree of suppression of tumor rejection in immune syngeneic animals. Ten million thymus cells of TBA were capable of suppressing significantly the tumor rejection. The IS cells were detected in the thymus, spleens, and draining lymph nodes, as well as in bone marrow of TBA, but could not be detected in the peripheral circulating blood. Since immunosuppressive activity of bone marrow cells from TBA was entirely abolished by the in vitro treatment of the cells with anti-theta serum and complement, the observed immunosuppression appears to be mediated by the T cell population.     

Regulation of the immune response to tumor antigens. III. Characterization of thymic suppressor factor(s) produced by tumor-bearing hosts.

Immunosuppressor T cells (IST),6 capable of inhibiting the rejection of a methylcholanthrene-induced fibrosarcoma (S1509a) in A/Jax mice immune to this tumor, produced soluble factors with similar suppressive activity. The immunosuppressive factor(s) (ISF) has been shown to be immunologically specific, as demonstrated by the complete loss of suppressive activity after absorption with S1509a cells, but not with cells of an unrelated syngeneic tumor. From its behavior on gel filtration, the size of ISF was deduced to be less than 70,000 daltons. The ISF was not removed by passage through a rabbit anti-mouse F(ab')2 reverse immunosorbent and, hence, it was concluded that ISF was not likely to be an immunoglobulin. The (immunosuppressive) activity of ISF was destroyed by treatment with Pronase, but not with RNase. The ISF was found to share the antigenic determinant(s) of the product(s) of the K end of the major histocompatibility complex of the mouse. Moreover, antibodies to ISF were induced by immunization with ISF-tumor cell complexes. Thus, IST and their factor(s) appear to play an important role in the regulation of the immune response to tumor antigens.     

Evidence for the functional binding in vivo of tumor rejection antigens to antigen-presenting cells in tumor-bearing hosts

Spleen cells of BALB/c mice bearing a syngeneic CSA1M fibrosarcoma were treated with anti-Thy-1.2 antibody plus C, yielding a T cell-depleted, APC-containing fraction. The APC-containing fraction was first tested for its capacity to present exogenous modified-self or another tumor (Meth A) Ag after in vitro pulsing. The results showed comparable Ag-presenting capacities to those obtained by APC-containing fraction from normal spleen cells, indicating that APC function is not affected in tumor-bearing mice. We next examined whether APC from CSA1M-bearing mice bind endogenously generated CSA1M tumor Ag onto its surfaces to stimulate tumor-specific T cells. Five rounds of inoculation of APC-containing fraction from CSA1M-bearing mice without further in vitro pulsing resulted in the induction of potent anti-CSA1M immune resistance. The involvement of anti-CSA1M T cells in the induction of anti-CSA1M immunity was excluded by the fact that the in vivo immunity was excluded by the fact that the in vivo immunity was delivered by Thy-1+ cell-depleted, but not by Thy-1+ cell-enriched fractions of spleen cells from CSA1M-bearing mice. Moreover, the failure of Sephadex G10-passed spleen cells to deliver anti-CSA1M resistance demonstrated the absolute requirement of APC for inducing the in vivo immunity. Finally, this in vivo resistance was found to be tumor specific, because APC fractions from CSA1M-bearing and Meth A-bearing BALB/c mice induced immune resistance selective against the corresponding tumor cell challenge. These results indicate that APC from tumor-bearing hosts can not only exert unaffected APC function against exogenous Ag, but also function to present tumor Ag generated endogenously in the tumor-bearing state and to produce tumor-specific immunity in vivo.     

Computational modeling of the immune response to tumor antigens

Vaccination protocols designed to elicit anti-cancer immune responses have, many times, failed in producing tumor eradication and in prolonging patient survival. Usually in cancer vaccination, epitopes from one organism are included in the genome or linked with some protein of another in the hope that the immunogenic properties of the latter will boost an immune response to the former. However, recent results have demonstrated that injections of two different vectors encoding the same recombinant antigen generate high levels of specific immunity. Systematic comparison of the efficacy of different vaccination protocols has been hampered by technical limitations, and clear evidence that the use of multiple vectors has advantages over single carrier injections is lacking. We used a computational model to investigate the dynamics of the immune response to different anti-cancer vaccines based on randomly generated antigen/carrier compounds. The computer model was adapted for simulations to this new area in immunology research and carefully validated to the purpose. As a matter of fact, it reproduces a relevant number of experimental observations. The model shows that when priming and boosting with the same construct, competition rather than cooperation develops amongst T cell clones of different specificities. Moreover, from the simulations, it appears that the sequential use of multiple carriers may generate more robust anti-tumor immune responses and may lead to effective tumor eradication in a higher percentage of cases. Our results provide a rational background for the design of novel strategies for the achievement of immune control of cancer.     

Regulation of the immune response to tumor antigens. X. Activation of third-order suppressor T cells that abrogate anti-tumor immune responses

The experiments described further define the suppressor T cell pathway in the S1509a tumor system. We demonstrated previously that S1509a-induced Ts1, TsF1, and Ts2 specifically suppress in vivo Ly1+2- T cell-dependent responses to S1509a and that Ts1 suppress in vivo Ly-1+2- T cell-mediated proliferative responses to S1509a. We have now shown that in vivo administration of either S1509a-induced TsF1 or TsF2 suppresses both in vivo and in vitro Ly-1+2- T cell-mediated responses to S1509a. Furthermore, we revealed the existence of Ts3, which are activated by S1509a tumor antigen and TsF2, in this murine tumor system. Finally, we demonstrated that cyclophosphamide abrogates the suppressive effect of TsF2 but not that of Ts3. These results are discussed with respect to T cell-mediated suppression in other murine tumor systems and the possible pivotal role for a tumor antigen-presenting cell in activating Ts3 in the S1509a tumor system.     

Eradication of tumor cells after injection into immunized hosts compared with the eradication of tumor cells after transfer of immune peritoneal exudates into tumor-bearing recipients

Summary DBA/2 mice were immunized against the syngeneic SL2 lymphoma by two or five injections with irradiated lymphoma cells given IP or SC, respectively. The antitumor efficacy induced in immunized mice was tested by (a) IP injection of the immunized mice with nonirradiated tumor cells and (b) transfer of the total immune peritoneal exudate, the cellular fraction only, or the cell-free fraction only, IP into tumor-bearing recipients, or (c) tumor neutralization tests (Winn assay). It was shown that immunized mice were able to reject 5×10⁷ SL2 tumor cells (8 of 14 mice survived >100 days), while in most transfer experiments 2×10⁵ SL2 cells could be eradicated. In the tumor neutralization experiments a number of 10⁶ SL2 cells were eradicated. When the immune exudates were given before the inoculations of SL2 tumor cells the number of survivors increased significantly. Further, it was shown that the cellular fraction is the major contributor to the antitumor effect in the transfer experiments, since there was no significant difference in tumor eradication after injection of a complete immune exudate and after injection of the isolated cellular fraction. Injection of the noncellular fraction had no measurable antitumor effect. An increase in the number of injections with total peritoneal exudates from immunized mice did not result in an increase in tumor eradication in the tumor-bearing recipients.Extra stimulation (IP) of immunized mice with 10⁴ nonirradiated cells 6 days after the last immunization resulted in an increase of the antitumor efficacy of these peritoneal exudates of these mice when collected 4–24 h after this stimulation. Extra stimulation with 10⁶ irradiated cells had no measurable effect.     

Induction of cell-mediated immunity to chemically modified antigens in guinea pigs. I. Characterization of the immune response to lipid-conjugated protein antigens.

Bovine serum albumin (BSA) conjugated with a lipid, dodecanoic acid, is capable of inducing strong delayed-type hypersensitivity (DTH) in guinea pigs. This paper reports experiments on the nature and specificity of this hypersensitivity. The response to lipid-conjugated BSA (L-BSA) was found to be classical DTH, as evidenced by its ability to be transferred passively by immune cells, but not by serum. In addition, special histologic examination of skin test sites demonstrated the characteristics of DTH rather than cutaneous basophil hypersensitivity. Similar results were obtained when lipid-conjugated purified protein derivative of tubercle bacilli (L-PPD) was used. The increased immunogenicity of L-BSA was not caused by the presence of protein aggregates, but seemed to be related to the hydrophobic nature of the conjugated side chains. A series of cross-reacting serum albumins was used for a study of the specificity of the antibody and DTH responses to BSA. It was found that the degree of enhancement of immunogenicity for DTH caused by lipid conjugation varied for different antigenic determinants on BSA.     

Directing the immune response to carbohydrate antigens

Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At present, the structural and immunological aspects of antigenic mimicry, which translate into immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine design approach to identify functional mimeotopes. By using concanavalin A (ConA), as a recognition template, peptide mimetics reactive with ConA were identified. Designed peptides were observed to compete with synthetic carbohydrate probes for ConA binding, as demonstrated by enzyme-linked immunosorbent assay and isothermal titration calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form of one particular mimetic binds to ConA with similar affinity as does trimannoside. Splenocytes from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell-dependent nature for the mimetic. As ConA binds to the Envelope protein of the human immunodeficiency virus, type 1 (HIV-1), we observed that mimeotope-induced serum also binds to HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-cell line adapted HIV-1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is based more upon functional rather than structural determinants that regulate mimeotope-induced T-dependent antibody responses to polysaccharide and emphasize that rational approaches can be employed to develop further vaccine candidates.     

MHC class I antigens,immune surveillance,and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T-lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T-cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T-lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T-cell immunoselection of HLA class I-deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.     

Role of dendritic cells in the immune response to T-independent antigens of type 2

Dendritic cells (DC) belong to the most effective antigen-presenting cells. Their role in the presentation of thymus-dependent antigens and initiation of primary immune response is well known. At the same time, participation of DC in the immune response to T-independent antigens of type 2 (TI-2 antigens) is poorly explored. In this work, the ability of DC to initiate the immune response to a TI-2 antigen α(1→3) dextran (Dex) is investigated. Mouse bone-marrow-derived DC were generated by culturing the precursors with GM-CSF and then DC were pulsed by TI-2 antigens. The pulse induced DC activation, as was verified by an increase in the number of CD80 and CD86 positive cells. Uptake of FITC-labeled Dex was examined by flow cytometry. At a concentration of FITC-Dex of 100 μg/10⁶ cells, the number of DC binding the antigen (Ag) reached “plateau”. DC charged by TI-2 antigens were mixed with normal mouse splenocytes and cultivated in RPMI-1640 medium for 4 days. The numbers of antibody- and immunoglobulin-forming cells were determined by ELISPOT method. The mixtures of splenocytes and naïve DC not charged by the Ag were used as control. It was shown that the increase in the numbers of AFC and IFC under the influence of naïve DC did not exceed 20%. On the contrary, the addition of DC pulsed by the Ag increased specific immune response more than twofold. The data obtained point to the direct interactions of DC with TI-2 antigens. Pulsed DC present TI-2 antigens to mouse splenocytes and induce specific and polyclonal B-cell activation, i.e., possess immunostimulating activity.     

Activation of antigen-enriched b cells. I. Purification and response to thymus-independent antigens

The development of a procedure for the selection of a large number of functional antigen-binding B cells is described. Forty to 70% of the cells in the enriched population bind antigen, and the antigen-binding can be inhibited by pretreatment with either the free hapten (TNP-Lys) or anti-immunoglobulin. The enriched cells express both slgM and slgD and respond to mitogenic stimuli by proliferating and developing into antibody-forming cells. In contrast to normal spleen cells, however, the enriched cells also proliferate in response to submitogenic levels of TNP-Brucella abortus and TNP-LPS.     

Mouse immune response to meningococcal antigens

The mouse immune response against Neisseria meningitidis was studied by using an extract from group Y (Slaterus) known to contain protein antigens common to other meningococci. By using a solid-phase radioimmunoassay, high titers of specific IgM and IgG class antibodies were measured which lasted over 2 months after immunization. These antibodies cross-reacted with similar extracts from other meningococci groups. Bactericidal antibodies directed against protein antigens were also elicited after immunization and they belonged to IgM, IgG2a, and IgG2b isotypes. Cellular immunity, expressed as delayed type hypersensitivity under the conditions tested, could be detected neither in homologous nor heterologous reactions.     

The in vitro antibody response to cell surface antigens. I. The xenogeneic response to human leukemia cells.

An in vitro spleen fragment culture system has been developed for the production and analysis of xenogeneic antibody responses to cell surface antigens. Depending on the methods of immunization and in vitro stimulation employed, mouse spleen fragments can produce antibody of both IgG and IgM classes directed against human cell surface antigens for more than 30 days in culture. A saturation binding analysis of the antibody products indicates that their range of specificities was more restricted than that of serum antibody. Approximately 5% of the in vitro antibody products raised against a homogeneous population of human leukemia cells could distinguish between the antigens present on the leukemia cells and those present on normal human lymphocytes. Methods previously employed to influence the range of serum antibodies expressed against complex immunogens, such as suppression of certain responses by passive administration of antibody at the time of immunization, were tested in the in vitro spleen culture system and resulted in successful modulation of the antibody response patterns observed.