Induction of immunocellular resistance to IL-2-activated lymphocytes within ovarian carcinoma cells.

The development of resistance within ovarian carcinoma cells to activated cytotoxic lymphocytes was the objective of this study. Primary ovarian carcinoma cells were obtained from the ascites of a patient. These cells were cocultured with IL-2-activated autologous tumor-associated lymphocytes (TALs) for 1 week. The resulting selected cells underwent a second coculture for 3 days with IL-2-activated autologous TALs or tumor-infiltrating lymphocytes (TILs). Phenotype analysis of the lymphocytes was performed prior to selection and 4-hr chromium release assays were used to detect resistance induction. Resistance to all effector cells could be demonstrated for the selected cells. However, selected cells maintained in culture demonstrated no difference in cytotoxic susceptibility from unselected cells. The following conclusions were made: (i) rapid immunoselection can occur for ovarian carcinoma in vitro; (ii) the resistance induced is not MHC-restricted; (iii) resistance induced by one type of cytotoxic cell results in general resistance to other types of cell from the same patient; and (iv) this resistance is not maintained during in vitro culture. These results may have direct implications on the future immunotherapy for this condition.

Induction of foetal lethality in AKR offspring after repeated inoculations into AKR females of anti-TCR/V beta 6 monoclonal antibody.

Female AKR (H-2k, Mlsa) mice were repeatedly injected with monoclonal anti-V beta 6 prior to and during syngeneic pregnancy. The offspring were born non-viable or died within 24 h. Continued injections into the mother resulted in abortions and conception eventually ceased altogether. Antisera from hyperimmunized mothers, when injected into the neonatal offspring of untreated AKR mothers, also had a lethal effect within 4 to 10 days after injection. Some mice survived for several weeks. All injected neonates developed a graft-versus-host disease (GVHD)-like syndrome characterized by runting, presence of skin lesions and weight loss. Antiserum injected in a diluted form caused similar but less severe symptoms. FACS (fluorescence-activated cell sorter) analysis of lymphocyte profiles of these mice revealed significant increases in the L3T4+ and Lyt-2+ T lymphocyte subsets; the number of V beta 6 T cells also increased. However Histopathological findings and mechanisms of the GVHD-like syndrome in these mice are discussed.

Anti-TCR-V beta 6 breaks tolerance in female AKR, Mlsa mice inducing foetal lethality.

Immunisation with anti-TCR-V beta 6 of female AKR mice prior to and during syngeneic pregnancy resulted in neonatal lethality and eventually in abortion or foetal resorption. The sera of the hyperimmunised mothers were shown to have anti-H-2k and anti-Mlsa autoantibodies and were cytotoxic to H-2k targets in vitro and also blocked Mlsa-induced mixed lymphocyte reactions. These observations are discussed herein.

Cellular mechanisms of graft-versus-host disease in a mouse model.

Female CBA/H (H-2k, Mlsb) mice alloimmunized prior to and during syngeneic pregnancy with DBA/2 (H-2d, Mlsa) splenocytes gave rise to offspring which resisted graft-versus-host disease (GVHD) following neonatal intraperitoneal inoculation of high doses of DBA/2 spleen cells. Lymphocytes from GVHD-resistant mice tested after 6 weeks of age were unresponsive to DBA/2 stimulator cells in 72 h mixed lymphocyte cultures. Isotope uptake measured 24 h after culture, however, indicated that a considerable early response was made to DBA/2 which later declined. Proliferative responses to BALB/c were also depressed but no early response to this strain was detected. FACS analysis of T-lymphocyte profiles of the GVHD-resistant CBA/H mice revealed a 100% increase in the Lyt-2+ subpopulation compared to normal CBA/H mice. Significant increases in Lyt-2+ cells were also noted in in vitro cultures of CBA/H lymphocytes responding to GVHD-resistant CBA/H stimulators. Lymphocytes from GVHD-resistant mice suppressed the proliferative responses of normal CBA/H lymphocytes to alloantigenic but not mitogenic stimulation. Suppression of alloantigenic responses were shown to be specific to DBA/2 and did not affect the response to BALB/c stimulator cells, indicating that both anergy and specific suppressor cells were operative in inducing unresponsiveness.

Tumour necrosis factor-alpha enhances the cytolytic and cytostatic capacity of interleukin-2 activated killer cells.

The cytotoxic and cytostatic responses of peripheral blood lymphocytes from eight cancer patients and splenocytes from four patients activated with rIL2 and a combination of rIL2 and rTNF-alpha were tested against two tumour cell lines. The cytotoxic response of rIL2-activated lymphocytes did not exceed the natural killer cytotoxicity values in all patients tested. In fact the killing capacity of some PBL deteriorated after rIL2 activation. The combined use of rIL2 and rTNF-alpha reversed this detrimental effect and enhanced the cytotoxic capacity of all PBL tested. In instances where high levels of killing were already achieved by rIL2 alone additional rTNF-alpha did not induce a significant change. This indicates that the role of rTNF-alpha may be to promote the response to rIL2 of PBL which react suboptimally to this lymphokine. rTNF-alpha did not only enhance cytotoxic capacity but also conferred cytostatic capacity to rIL2-activated LAK cells which were cytotoxic but unable to inhibit the growth of the surviving target cells. Natural killer cell selected K562 target cells which were less susceptible to killing by untreated lymphocytes than the parent K562 tumour cell line were killed more aggressively by rIL2 + rTNF-alpha LAK cells than by rIL2-LAK cells. No phenotypic differences were detected in these two cultures of LAK cells which indicates that the increased cytotoxic and cytostatic capacity of rIL2 + rTNF-alpha-LAK cells may be due to a higher state of activation of these cells or due to their capacity to recognise a broader spectrum of targets than rIL2-LAK cells.

Modulation and biological effects of Ly-6.2 expression on EL4 tumour cells.

EL4 tumour cells maintained in culture were separated by FACS analysis to Ly-6.2 negative and Ly-6.2 positive subsets. The Ly-6.2 negative subset gained expression of this determinant on repeated in vivo passage in C57BL/6 mice. Both subsets injected intraperitoneally or intramuscularly in syngeneic mice induced identical changes in lymphocyte profiles. There was generalised lymphocytolysis in both T- and B-cell compartments. The Lyt-1+, 2- T-lymphocytes were more susceptible to cytolysis causing an alteration of the proportional representation of the Lyt-2+ subset from 30% of splenic T-cells (in normal mice) to over 90% of remaining T-lymphocytes in tumour bearing mice. There was thymic regression in both groups of mice with a resultant thymocyte population expressing the range of phenotypes of mature medullary cells. In spite of similar rates of growth both in vivo and in vitro and identical effects on the lymphoid system the Ly-6.2 negative and Ly-6.2 positive tumour subsets were different in their metastatic potential. Mice injected intramuscularly with either subset had enlarged spleens by the second week of tumour growth caused largely by the accumulation of Ig, Lyt-1 and Thy-1 negative cells. Tumour cells were present only in the group injected with the Ly-6.2+ subset. These mice died of their tumour load a week earlier than those injected with the Ly-6.2- tumour cells.

Human cytotoxic T-cells against measles virus-infected and myelin basic protein-coated targets are cross-reactive.

Cytotoxic T lymphocytes (CTL) which recognized measles virus antigens were generated by in vitro sensitization of peripheral blood lymphocytes from normal volunteers against autologous measles virus-infected lymphocytes. Cytotoxicity of measles virus-infected targets by these effectors was considerably enhanced when the effector-target cell mixtures were incubated in presence of 10 or 100 ng myelin basic protein (MBP) for the 3-hour duration of the 51Cr release assay. In most experiments, specific release of radioisotope was doubled or tripled. Bovine serum albumin caused only slight increases in cytotoxicity. The killing of allogeneic target cells by alloimmune CTL was not affected by either of these reagents. Measles-specific CTL were also able to kill target cells that were cultured overnight in presence of MBP but washed prior to the assay. Conversely, CTL generated by culturing lymphocytes in presence of MBP for 6 days were able to kill MBP-coated and measles virus-infected target cells. The implications of these findings in the pathogenesis of multiple sclerosis are discussed.

Association of the lymphoproliferative disease inducer gene Arp with autoimmunity and resistance to tumour growth.

This report describes a biological role for the diallelic gene system, Arpa and Arpb, which codes for surface antigens on murine lymphocytes and tumour cells. Arpb is present only in a mutant strain of BALB/c which is designated BALB/c-Arpb. Normal BALB/c and all other strains of mice tested express Arpa. Cross immunizations between BALB/c and BALB/c-Arpb generated autoantibodies and autoreactive cytostatic effector cells which recognize Arp-encoded determinants on normal and tumour cells. The latter expressed quantitatively more Arp encoded products than normal cells as indicated by increased binding of autoantibodies and susceptibility to cytostasis. The anti-tumour response generated by Arp-incompatible immunizations resulted in increased resistance to challenge with syngeneic tumour cells and in some cases total suppression of tumour growth. BALB/c-Arpb mice were inherently different from BALB/c in that they generated H-2 unrestricted cytostatic effectors, produced higher levels of autoantibodies on immunization and survived longer than normal BALB/c when challenged with 10(5) Meth. A tumour cells. The role of the Arp gene in tumour immunity is discussed.

Characterization of cytostatic effector lymphocytes during the development of a syngeneic lymphosarcoma in C3H mice: use of monoclonal reagents to identify T-cell subsets.

The development of the in vitro cytostatic capacity of splenic lymphocyte subpopulations from C3H mice carrying the syngeneic Gardner tumor was examined at different times after intramuscular tumor injection. Most mice died between 3 to 6 weeks after tumor injection, while some rejected their tumors or survived longer than 3 months. Cell separation procedures and monoclonal antibodies against T-cell subsets were used to identify the cells responsible in anti-tumor immunity. Cytostatic capacity against tumor cells developed in the T-cell enriched subpopulation of splenocytes 3 days after tumor injection and was partly abrogated by anti-Lyt-1. Effector function of Lyt-2+ T cells and B cells developed later and peaked at around 10 days after tumor injection. Another cell population with cytostatic capacity which was not blocked by anti-Lyt-1, anti-Lyt-2, or anti-Ly-5 was noted to develop early after tumor injection and lacked both T-cell and B-cell markers ("null"). This subpopulation was eluted with T cells from nylon wool columns and comprised up to 50% of the T-enriched fraction of splenocytes in later stages of tumor growth. An interesting characteristic of these "null" cells was susceptibility to T-cell suppression both in early and later stages of tumor growth except in regressor mice which lacked suppressor T cells. The cytostatic capacity of the "null" cells could be restored either by removal of Thy-1+ cells from the T-enriched fraction by panning, or the addition of anti-Thy-1 or F(ab')2 fragments of anti-Thy-1 to the lymphocyte-tumor reaction mixtures. Most mice examined after 10 days of tumor growth were immunosuppressed to varying degrees. Unseparated splenocytes from these mice were not cytostatic but removal of T cells allowed the B cells to exert their cytostatic capacity. A strong underlying B-cell cytostasis was shown to be present in long survivor mice even though their unseparated spleen cells were only weakly cytostatic. T cells did not play a role in the regression of tumors or long-term survival of tumor bearer mice. Splenocytes from regressor mice were strongly cytostatic, their anti-tumor activity residing in the "null" and B-cell populations.

Alloimmune interactions of a lymphoproliferative disease-inducer gene Arp and linkage to Pep-7.

In this report we describe a new diallelic gene system, Arpa and Arpb, which codes for surface antigens on murine lymphocytes. Arpb is present only in a mutant strain of BALB/c which is designated BALB/c-Arpb. Normal BALB/c and all other strains of mice tested express Arpa. The Arpb mutation is associated with a newly discovered polymorphism of the Peptidase-7 enzyme, Pep-7b, which codes for a variant form of the enzyme with a faster anodal mobility on electrophoresis than the commonly known form. The Arp locus controls a range of alloimmune interactions between Arp incompatible lymphocytes. These include mixed lymphocyte reactivity, host-versus-graft and graft-versus-host reactions and the development of weak cytotoxic but strong cytostatic effector lymphocytes which are allo- as well as autoreactive. The association between Arp and Pep-7 and the biological significance of the Arp locus are discussed.

Molecular weight determination of two genetically linked cell surface murine antigens: ThB and Ly-6.

Various murine tumor lines were screened by FACS analysis for the surface antigens ThB and Ly-6.2. Positive cell lines were used for immunoprecipitation studies. A monoclonal ThB-specific antibody immunoprecipitated a unique acidic protein of approximately 16 000 daltons from several positive tumors and from concanavalin A (Con-A) and LPS activated splenic lymphocytes. Monoclonal Ly-6.2-specific antibody was used to immunoprecipitate a 33 500 dalton protein that was shown to exist in four similarly sized forms with different basic charges. In the course of these studies, the apparent molecular weight of the surface antigen T 30, immunoprecipitated with a monoclonal T 30-specific antibody from the cell line EL4, was found to be approximately 25 000 daltons.

H-2d-like specificities on Gardner (H-2k) tumour detected in a restricted anti-tumour reaction.

Cytostatis of the H-2d tumour LSTRA by H-2-restricted effector lymphocytes was inhibited by antisera against H-2.4 and H-2.31 but not by antisera against public specificities or non-H-2 antigens. The unexpected reaction of the same effector cells against Gardner tumour (H-2k) was also shown to be inhibited by a combination of antisera against H-2.4 and H-2.31 but not by each antiserum used separately. The inhibitory capacity of these antisera was removed by absorption with B10.D2 but not with B10 lymphocytes. This indicated the presence of H-2d-like specificities on gardner tumour which could function as self-recognition structures in an H-2Kd and H-2Dd restricted system.

Contrasting effects of H-2 and Mls immunization on the polyclonal mitogenicity of murine lymphocytes.

During the immune response to H-2 and Mls alloantigens, murine lymphocytes showed altered sensitivity to polyclonal mitogens. The reactivity to the T-cell mitogen PHA followed a similar pattern in both H-2 and Mls-immunized mice while the reactivity to the B-cell mitogen LPS was contrasting in the two groups. In the former group, the response exceeded control levels by the seventh day after immunization and then gradually dropped below control levels; the response of Mls-immunized lymphocytes dropped below control levels soon after immunization and remained so for the period of study. Nylon wool column-purified Mls-immunized B cells also showed a suppressed reactivity to LPS, while the T-enriched populations from Mls-immune mice when added to normal B cells lowered their LPS reactivity. Soluble factors derived from clutures of Mls-immune lymphocytes had a suppressive effect on normal B cells.

Modification of the anti-tumour immune response by suppressor gene products of lymphoid cells.

Immunization of mice with BALB/c spleen cells leads to the production of effector lymphocytes which are cytostatic in in vitro assays to tumours of the same haplotype or carrying cross-reacting antigens. Immunization with B10.D2, a strain H-2 identical with BALB/c, does not generate cytostatic effector cells, nor does immunization with the F1 hybrids between B10.D2 and BALB/c. Analysis of the progeny of backcrosses of the F1 hybrids to BALB/c gave evidence that the suppressive effect of B10.D2 immunization is controlled by a single gene. Spleen cells from mice immunized with BALB/c or B10.D2 cultured in vitro with the corresponding stimulator cells yielded soluble factors in the supernatants that were respectively capable of amplifying or suppressing the in vitro cytostatic effect. Such experiments revealed that the inhibition of cytostasis caused by immunization with B10.D2 is not at the sensitization but at the effector phase of the assay. Possible mechanisms of action of this suppressor gene are discussed.

Specificity of the macrophage spreading test with reference to Leishmania antigens and correlation with delayed hypersensitivity.

The inhibition of the macrophage spreading test, claimed to be an in vitro correlate of delayed hypersensitivity, was examined in guinea-pigs immunized with L. enriettii and L. tropica soluble antigens. Cells from peritoneal washings of the guinea-pigs were tested in presence of the homologous and heterologous antigens and also without antigen. Inhibition of macrophage spreading compared to control preparations was noted only in the presence of the homologous antigen when the skin test response of the donor animal was relatively small. The degree of inhibition decreased as the skin test volume increased and when skin test volumes were large there was actual stimulation of macrophage spreading, rather than inhibition. The addition of heterologous antigen to the peritoneal cell preparation always resulted in the augmentation of macrophage spreading above control levels. The possible mechanisms of this in vitro technique and its use as a taxonomic or diagnostic tool are discussed.