Virus-augmented delayed hypersensitivity skin tests in gynecological malignancies

Summary Cultured human tumor cells of various histologic origins were infected with PR8/A/34 influenza virus. Nonviable crude membrane extracts were derived from the infected and uninfected cells. The extracts were coded and tested for their ability to produce delayed hypersensitivity skin reactions (DHSR) in allogeneic patients with squamous uterine cervical carcinoma, epithelial ovarian carcinoma, and malignant melanoma. Augmented antigen sensitivity to the virus-modified extracts compared with virus alone or to the unmodified extracts was observed in all patient groups. There was insufficient specificity to delineate a response by individual tumor type and related tumor extract, but some of the observed responses suggested tumor or organ site associations. Cervical carcinoma patients reacted more frequently to the virus-modified cervix extract, which also produced a high frequency of response in patients with ovarian carcinoma and melanoma. Ovarian carcinoma patients demonstrated increased sensitivity to both virus-modified ovarian carcinoma extracts, although 14 of 21 patients also showed responsiveness to one of the unmodified ovarian extracts. Malignant melanoma patients showed increased sensitivity to all virus-modified extracts except one of two derived from the ovarian carcinoma, and demonstrated a significantly augmented response to the virus-modified melanoma extract when the response to this extract was compared with that in ovarian carcinoma patients.The augmented reactions appear to be due to an association of the PR8 virus and as yet undetermined cellular components rather than to the virus alone. The possible involvement of tumor-associated determinants and the clinical significance of this phenomenon require further investigation.     

Studies on delayed hypersensitivity to protein antigen. Induction of delayed hypersensitivity by chemically modified antigen.

it was shown in our previous paper that mice primed with chemically modified bacterial alpha-amylase (BaA), which was neither cross-reactive with anti-BaA antibody nor able to induce a humoral anti-BaA response, developed enhanced responses to a subsequent challenge with native BaA and that the magnitude of the immunological memory was closely related to the priming dose of modified BaA. This paper describes the experimental conditions for induction of delayed hypersensitivity (DH) by modified BaA in relation to the development of immunological memory for antibody response to native BaA. Mice primed with either an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of modified BaA in complete Freunds adjuvant (CFA) developed enhanced anti-BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to a subsequent challenge with BaA. In contrast, when mice were immunized with an s.c. injection of the modified BaA only, a significant level of DH to native BaA could be induced, as measured by the footpad reaction (FPR). The highest degree of DH was observed in mice given 50 micrograms of modified BaA. DH was detectable within 5 days and persisted for 25 days after immunization. In the reciprocal combination of native BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to DH was examined. The primary anti-BaA responses induced by an i.p. injection of large doses of BaA was markedly higher than those induced by an s.c. injection, while DH was exhibited only in mice given s.c. injection of BaA in CFA. With respect to DH to native BaA induced by the modified BaA, it was shown that C3H/He mice were high and C57BL/6 mice were low responders.     

Studies on delayed hypersensitivity to protein antigen. Antigen-specific suppression of delayed hypersensitivity by chemically modified antigen.

An extensively modified protein antigen (methylated bacterial α-amylase, M-BαA) which was neither reactive with anti-BαA antibody nor able to induce a humoral anti-BαA response, retained the ability to prime native BαA-specific T cells which were responsible for the enhanced anti-BαA response to subsequent immunization with BαA and delayed hypersensitivity (DH). The splenic T cell-rich fraction from mice primed with M-BαA collaborated with a native BαA-primed B cell-rich fraction to give a good adoptive IgG anti-BαA response in syngeneic irradiated mice, whereas M-BαA-primed B cell fractions failed to cooperate with native BαA-primed T cell fractions. Splenic T cells from mice given a subcutaneous (s.c.) injection of M-BαA in complete Freund's adjuvant (CFA) exhibited DH in syngeneic cyclophosphamide-treated mice. In the present study, native and methylated BαA were tested for their ability to generate suppressor T cells capable of inhibiting the development of DH. An intraperitoneal (i.p.) injection of either native or methylated BαA in incomplete Freund's adjuvant (IFA) interferred with the development of DH to M-BαA by an s.c. injection of the same antigen in CFA. Transfer of spleen cells from mice given an i.p. injection of either of these antigens 5 days previously, suppressed antigen-specifically induction and expression of DH in the syngeneic recipient mice. The suppressive activity was sensitive to treatment with anti-θ antiserum plus complement. These results indicate that the early phase of inhibition of DH after an i.p. injection is in part mediated by suppressor T cells and that M-BαA cross-reacts with native BαA at the suppressor T cell level as well as the level of effector T cells in DH.     

Biologic activity of extracts of delayed hypersensitivity skin reaction sites

Extracts obtained from skin sites of delayed hypersensitivity reactions show chemotactic activity for monocytes and lymphocytes but not neutrophils. The soluble extractable factors present at these sites have in vivo activity as well; they promote the accumulation of monocytes in peritoneal exudates and cause inflammatory reactions in the skin of nonimmunized animals. The skin inflammatory infiltrates are predominantly mononuclear and are similar to those of delayed hypersensitivity reactions in actively immunized guinea pigs. The extracts which produced these effects had no detectable MIF activity, nor permeability inducing activity in excess of that obtainable from normal skin.These monocyte and lymphocyte chemotactic factors were analyzed by sucrose density ultracentrifugation. By this technique the distribution of monocyte factors corresponded rather closely with that of the monocyte chemotactic factors obtained from an antigen-activated lymphocyte culture. Similar correspondence was obtained for the bulk of the lymphocyte chemotactic activity present in skin extracts and in culture supernatants. This suggests the possibility that the lymphokine-like substances in the skin extracts might in fact represent lymphokines. Further documentation of this point will provide a link between in vitro and in vivo manifestations of delayed hypersensitivity.     

Nonantibody-mediated suppression of delayed hypersensitivity to human gamma-globulin in mice.

Development of delayed hypersensitivity (DHS) to human γ-globulin (HIgG) in mice was documented by histological analysis, by the kinetics of footpad swelling in animals exhibiting humoral or delayed responses, and by the failure of sera to transfer delayed reactions to normal, syngeneic recipients. Since cyclophosphamide (CY) treatment resulted in diminished humoral and augmented delayed reactions, we used this as a tool to explore the nature of the regulatory mechanisms which affect expression of this type of cell-mediated immunity. In order to evaluate the effect which the presence or absence of antigen-specific cells might exert on expression of DHS, we subjected mice to experimental regimes which would result in lymphocyte proliferation or depletion, respectively (see Bachvaroff, R., and Rapaport, F. T., Cell. Immunol. 15, 336, 1975). Cell proliferation was induced by injection of 80 μg of aqueous antigen on Day ?4; this was followed by sensitization with HIgG-CFA (Freund's adjuvant) on Day 0, and footpad challenge on Day 13. These mice exhibited strong humoral reactivity; three of six died of anaphylaxis following footpad challenge, and the remaining three showed a diminished delayed response. Similarly treated mice that, in addition, received 6 mg of CY 3 days after injection of aqueous antigen and, therefore, would have antigen-specific cells present showed greatly diminished humoral reactivity, due to B-cell depletion. However, they also exhibited a marked diminution in delayed responsiveness. The data clearly demonstrate that a nonantibody-mediated, possibly cell-directed, regulatory influence is exerted on DHS where cell proliferation has occurred. We next examined the impact which the depletion of proliferating cells would exert on the expression of DHS. Cell depletion was attempted by giving one injection of aqueous antigen (Day 0) early in a regime of chronic CY administration (Days ?1 through +3) ; antigen-induced proliferating cells would be susceptible to CY and, therefore, depleted under these conditions. The results show that mice receiving both aqueous antigen and CY have depressed humoral and markedly diminished delayed reactivity compared to animals that were injected with CY alone. Thus, the augmenting effect which CY exerts on DHS is abrogated by stimulation with aqueous antigen. One interpretation is that CY removes a regulatory cell population in the normal animal, thereby allowing enhanced expression of delayed responsiveness. Clearly, regulatory function cannot be attributed solely to bumoral antibody production.     

The formation of delayed hypersensitivity to mycobacterial antigens

In experiments on guinea pigs and BALB/c mice delayed hypersensitivity to mycobacterial antigens was induced by the sensitization of the animals with live BCG or killed Mycobacterium bovis or M. avium in incomplete Freund's adjuvant. In the study of the dynamics of the development of skin reactivity to tuberculin some advantages of the sensitization of guinea pigs with live mycobacteria were revealed, while after the revaccination of the animals no development of secondary cell-mediated immune response was observed. The immunization of guinea pigs with atypical mycobacteria prior to their sensitization with BCG was found to lead to the development of higher skin reactivity to allergen prepared from atypical mycobacteria than skin reactivity to tuberculin.     

Murine transfer factor. II. Transfer of delayed hypersensitivity to synthetic antigens

Synthetic polyaminoacid antigens were used to examine the specificity of transfer of delayed-type hypersensitivity with spleen cell dialysates in mice. Dialysates from GAT10-sensitized donors sensitized recipients to GAT10, but not GLA5 or cytochrome c. Dialysates from GLA5-sensitized donors sensitized recipients to GLA5, but not GAT10 or cytochrome c. We interpret these findings as consistent with the concept that passive transfer of delayed hypersensitivity with dialyzable materials is an immunologically specific event.