In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57Bl/6 mice

Natural resistance has been detected in lethally irradiated C57Bl/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.     

Natural suppressor cells in spleens of irradiated, bone marrow-reconstituted mice and normal bone marrow: lack of Sca-1 expression and enrichment by depletion of Mac1-positive cells

We have recently reported the development of natural suppressor (NS) cells in lethally irradiated, bone marrow-reconstituted mice during the early weeks after bone marrow transplantation (BMT). These cells were shown to be derived primarily from the syngeneic marrow component in recipients of mixed allogeneic plus syngeneic (host type) marrow, and it was speculated that they might be responsible for the anti-GVHD effect previously described for T-cell-depleted syngeneic marrow. It was therefore of interest to look for such suppressive activity in normal adult bone marrow, which might serve as an obtainable source of such cells if they were to be isolated and used clinically. Such activity has indeed been found in normal adult bone marrow and its characteristics compared to that in spleens of early BMT recipients. Suppressive cells from both sources were similar in their specificity patterns and radiosensitivity, and were of the null (i.e., non-T, non-B, nonmacrophage) cell phenotype. Suppression from either source can be enriched by removal of Mac1-positive cells, providing a possible approach to obtaining NS-enriched populations for in vitro expansion and adoptive transfer studies. Such depletion of Mac1-positive cells was associated with a threefold enrichment of Thy1-positive cells, of which one half were CD4- and CD8-negative, similar to the reported phenotype of cultured NS cell lines. Even when enriched in this manner, the contribution of Thy1-positive cell populations did not reach statistical significance. A recent report has suggested that NS cells might actually be pluripotent hematopoietic stem cells. In contrast, we report here that depletion of Sca1-positive pluripotent hematopoietic stem cells with monoclonal antibody plus immunomagnetic beads does not remove NS activity.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.     

Immunotoxin-mediated elimination of clonogenic tumor cells in the presence of human bone marrow

An immunotoxin was synthesized with pokeweed antiviral protein and an IgG1 monoclonal antibody directed against human B and pre-B cells. The B43 murine monoclonal antibody does not react with normal human bone marrow precursor cells. The immunotoxin bound to all Burkitt's lymphoma cell lines that were tested but not to human peripheral blood T cells. The ability of antibody-toxin conjugate to inhibit human lymphoblast cell lines was checked in a clonogenic assay system. The immunotoxin in the presence of chloroquine elicited 5.8 logs of killing of Burkitt's lymphoma cells (B-ALL). The efficient inhibition of clonogenic growth of B-ALL cells was not affected by the presence of normal bone marrow cells. The immunotoxin was not very toxic to pluripotent stem cells; less than 50% of the stem cells were lost under conditions where 5.6 logs of clonogenic lymphoma cells were eliminated from a 100-fold excess of normal marrow cells. Further, when assayed by long-term human bone marrow cultures, immunotoxin treatment did not result in a significant loss of pluripotent precursor cells.     

The effect of specific anti-rat thymocyte serum on cell-mediated tumor immunity and other lymphocyte functions

A burro anti-rat thymocyte serum (ATS) was rendered specific for thymus-derived lymphocytes (T cells) by repeated absorptions with Murphy-Sturm lymphoma cells. All of the cells in this lymphoma cell line were shown to have surface immunoglobulin and it was presumed to be bone marrow-derived. Treatment of lymphocytes with ATS eliminated stimulation by phytohemagglutinin and concanavalin A, but did not reduce the proportion of cells with surface immunoglobulin or complement receptor. ATS also did not decrease the number of antibody-producing spleen cells from rats immunized against sheep erythrocytes. The ATS was used to determine the nature of the effector cells in the cell-mediated cytotoxicity of immune W/Fu rat spleen cells against a syngeneic Gross virus-induced lymphoma. This cytotoxic response was consistently inhibited, indicating that T cells were needed for reactivity.     

Different induction of adaptive response to ionizing radiation in normal and neoplastic cells

Since the beneficial effects of low-dose radiation (0.01 Gy) are usually observed in normal cells, we investigated whether the adaptive response was induced by low-dose radiation in neoplastic cells of different origin as well as in normal cells. Cell lines used in this experiment were as follows: mouse lymphocytes (NL); L929 cells established from mouse connective tissue; primary mouse keratinocytes (PK); line 308 from mouse papilloma; X-ray sensitive lymphoma cells, L5178Y-S and EL-4 cells from mouse lymphoma. The adaptive response was determined by cell survival and apoptosis. The involvement of apoptosis in the adaptive response was examined by ELISA and TUNEL assay. Adaptive response was induced by pretreatment with low-dose radiation of 0.01 Gy in normal cells such as NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. In addition, the reduction of apoptosis by pretreatment with low-dose radiation was observed in NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. These results suggested that the adaptive response could be induced by pretreatment with low-dose radiation and the phenomena were observed in normal cells, not in neoplastic cells. In addition, pretreatment with low-dose radiation reduced apoptosis, suggesting that an anti-apoptotic pathway may be involved in the adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

The lack of generalized immunosuppression in C57BL/6 mice during progressive growth of syngenic T lymphoma EL-4 and Lewis lung carcinoma 3LL

The immune competence of C57Bl/6 mice implanted with EL-4 lymphoma of Lewis Lung carcinoma 3LL was investigated during 3 weeks after implantation. Splenic lymphocyte responses to mitogens (Con A, PHA, LPS, PWM) cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2) production were assessed. A dramatic reduction in mitogenic responses to Con A and PHA was observed during tumour progression. LPS and PWM responses were less depressed. Con A-induced IL-2 production correlated with Con A and PHA responses. Allospecific CTL response to mastocytoma P 815 was not decreased in syngeneic tumour-bearing mice.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC).

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

A platelet-derived immunoregulatory serum factor with T cell affinity

A factor in mouse and human serum which enhances immune responses to SRBC and TNP-Ficoll in mice has been described. This factor completely reverses immunosuppression induced by syngeneic lymphoma (RCS) or allogeneic lymph node cells. T lymphocytes bind this factor, since the activity can be removed from serum by absorption with T cell containing spleen cells or cloned cytotoxic T lymphocytes. Furthermore, the factor is active only in the presence of mature T cells. Platelet alpha-granules seem to be the source of this serum factor because 1) the activity is lacking in plasma, in serum prepared in the absence of platelets, and in serum prepared from a patient lacking platelet alpha-granules and 2) isolated mouse or human platelets release an immunostimulatory factor with very similar properties upon incubation with thrombin.     

Lymphokine-activated killer cells are generated before classical cytotoxic T lymphocytes after bone marrow transplantation in mice

Lymphokine-activated killer (LAK) cells are demonstrable within 2 wk after syngeneic or allogeneic (H-2-compatible) bone marrow transplantation in mice. Classical cytotoxic T lymphocytes (CTL) are not active until at least 4 wk after transplant. Both LAK cells and CTL bear the Thy-1 marker and do not possess the murine natural killer cell marker asialo GM.     

Graft vs graft reaction resulting in the elimination of maternal cells in a SCID patient with maternofetal GVHd after an HLA identical bone marrow transplantation

In a young girl with a severe combined immunodeficiency, the presence of circulating maternal T lymphocytes was proven by HLA typing. Manifestations of skin graft vs host disease were associated with the persistence of maternal cells. The patient received an HLA identical bone marrow transplantation from her brother without any conditioning. The bone marrow transplantation was quickly followed by a transient and dramatic increase in skin lesions associated with fever and the finding of a high number of circulating lymphocytes and eosinophils. Lymphocytes were shown to be of donor origin and exerted a spontaneous cytotoxic activity toward maternal cells. This activity progressively disappeared within 90 days, whereas maternal cells were no longer detected in patient's blood, and skin graft vs host disease was resolved within 8 wk. Cytotoxic activity was proven to be mediated by donor T lymphocytes specific for the mother's HLA antigens. The cytotoxic activity was demonstrated to be specific for the HLA class I molecules of the mother not shared with her daughter (HLA A1, B17) as shown by the use of a series of HLA typed cells as targets. In addition, cold K562 target cells did not block the cytotoxic activity, and the kinetics of the cytotoxic activity did not correlate with that of natural killer activity emergence after the bone marrow transplantation. Patient's serum did not contain antibodies toward maternal specific HLA class I antigen. Cytotoxic activity was totally blocked by anti-T3 monoclonal antibodies and partially by anti-T8 and anti-T4. It is thus likely that donor origin cytotoxic T lymphocytes were promptly activated after bone marrow transplantation and provoked the elimination of the maternal graft after a transient exacerbation of graft vs host disease manifestations. This observation represents one of the first examples of the possible role in vivo of allogeneic cytotoxic lymphocytes in humans.     

Antilymphocyte antibodies in various human diseases as a factor of reduced functional activity of T-suppressors

A total of 157 sera from adults and children with rheumatoid arthritis, rheumatic fever, myocarditis, neurodermite, bronchial asthma, wound infections, second degree obesity without symptoms of diabetes were examined. 60% of sera contained high concentrations of antibodies possessing cytotoxicity against thymus cells, but not against bone marrow cells. Sera of healthy children and adults contained no cytotoxic antibodies. Sera cytotoxic against mouse thymus cells inhibited the suppressing activity of mouse splenocytes in experiments on syngeneic transfer, reducing the ability of human lymphocytes to form T-RFC. The latter phenomenon is associated with the decline in the number of T-theophyllin-sensitive lymphocytes, known as T-suppressors.     

Antilymphocytic antibodies in human diseases as a factor decreasing the functional activity of T-suppressors

157 sera from adults and children with rheumatoid arthritis, rheumatic fever, myocarditis, neurodermatitis, bronchial asthma, wound infections, second degree obesity without symptoms of diabetes were examined. 60% of sera contained high concentrations of antibodies possessing cytotoxicity against thymus cells, but not against bone marrow cells. Sera of healthy children and adults contained no cytotoxic antibodies. Sera cytotoxic against mouse thymus cells inhibited the suppressing activity of mouse splenocytes in experiments on syngeneic transfer, reducing the ability of human lymphocytes to form T-RFC. The latter phenomenon is associated with the decline in the number of T-theophylline-sensitive lymphocytes, known as T-suppressors.     

Activation of cytotoxic T lymphocytes requires at least two spleen cell-derived helper factors besides interleukin 2

The dependency of induction of T cell cytotoxicity on lymphokines was studied. 1 X 10(5) nylon wool-purified thymic lymphocytes or 10(4) spleen cells were cultured with TNP-haptenated syngeneic UV-irradiated spleen cells in the presence of a variety of lymphokine preparations. Concanavalin A-induced spleen cell supernatants mediated strong cytotoxic responses in this system. Three other preparations, namely, a partially purified IL 2 preparation from PMA-stimulated EL-4 thymoma cells, a Con A-induced spleen cell supernatant that was absorbed with an IL 2-dependent cell line, and a Con A-induced supernatant that was dialyzed at pH 2 were all ineffective in mediating a cytotoxic response. In reconstitution experiments, cytotoxic responses were only obtained when either the absorbed preparation or the pH 2-treated preparation was mixed with the IL 2 preparation from EL-4 cells. No reconstitution occurred after mixing of the absorbed with the pH 2-treated preparation. pH 2 treatment of the absorbed preparation did not abolish its synergistic effect when added to the IL 2 preparation from EL-4 cells. These results led to the conclusion that activation of cytotoxic lymphocyte precursors requires at least two other lymphokines in addition to IL 2. One T cell cytotoxicity-inducing factor (TCF1) remained in Con A-induced supernatants after absorption with IL 2 receptor-bearing T cell line cells. It was pH 2-resistant and was not found in EL-4 supernatants. A second T cell cytotoxicity-inducing factor (TCF2) was pH 2-sensitive and was found in Con A-induced spleen cell supernatants as well as in interferon-free supernatants of PMA-stimulated EL-4 cells. This activity co-purified with IL 2. It was absorbed by the IL 2-dependent T cell line together with IL 2. IL 2 differs from TCF2 since it is pH 2-resistant.     

IN VITRO PROLIFERATION OF MOUSE LYMPHOBLASTOID CELL LINES: GROWTH MODULATION BY VARIOUS POPULATIONS OF ADHERENT CELLS

The in vitro growth pattern of a number of mouse lymphoblastoid tumour cell lines was modified in the presence of adherent cell layers from various sources. The AVRij-1 and ST-4b cell lines exhibited a concentration—dependent growth pattern, i.e., they would only grow well when seeded at high starting cell concentrations. Better growth of these cells from low cell concentrations was observed in the presence of adherent cell layers from syngeneic or allogeneic bone marrow. Adherent cell layers derived from mouse spleen and pleural or peritoneal cavity could also promote the growth of the above tumour cells, but in a narrower range of cell concentrations and to a lower extent. Moreover, confluent adherent layers from the pleural and peritoneal cavities completely inhibited the growth of AVRij-1 and ST-4b cells, while adherent cell layers from the bone marrow did not inhibit growth at any cell concentration tested. The in vitro growth of concentration—independent cell lines was also affected by the presence of adherent cells from the bone marrow. Under syngeneic conditions, a slight increase in the growth of the ‘null’ or pre-B lymphoma cell line ABLS-8.1 was observed. On the other hand, the growth of tumour cells expressing more differentiated properties, such as the thymus T lymphoma tumour cell line ST-1.3 and the plasma cell tumour MPC-11.45.6.2.4, was inhibited in the presence of syngeneic bone marrow derived adherent cell layers. This inhibition was more pronounced under allogeneic conditions. Growth inhibition was also observed when concentration—independent cell lines were co-cultured with adherent cells from the pleural and peritoneal cavities. Thus, adherent cell layers from non-haemopoietic sources inhibited the growth of all cell lines tested. On the other hand, adherent cells from the bone marrow had a differential effect on growth of lymphoblastoid tumour cell lines. This depended on the in vitro growth properties of each tumour cell line and on some additional specific tumour cell properties. The latter could relate to the differentiation stage characterizing each tumour cell line. The culture method described here may serve as a model system for studies on interaction of leukaemic cell and the haemopoietic microenvironment.     

Adoptive immunotherapy of a BALB/c lymphoma by syngeneic anti-DBA/2 immune lymphoid cells: Characterization of the effector population and evidence for the role of the host's non-T cells

Summary It has been previously shown that the BALB/c lymphoma YC8 is susceptible to lysis by syngeneic anti-DBA/2 lymphocytes and that YC8-bearing BALB/c mice can be cured by adoptive transfer of such immune effectors. In this study in vivo and in vitro functions of the curative immune lymphocytes have been evaluated together with the role of the host immune system in the mechanism of tumor eradication. It was found that the curative anti-DBA/2 lymphocytes were not directly cytotoxic to YC8 cells although they developed into YC8-lytic cells after in vitro restimulation with YC8. In vivo, the immune lymphocytes were able to mediate a tumor-specific delayed type hypersensitivity reaction against YC8 but had a low tumor-neutralizing activity in the Winn assay. Proliferation of infused BALB/c anti-DBA/2 lymphocytes was necessary for the in vivo therapeutic effect, since irradiation of effector cells or treatment of the donor immune lymphocytes with vinblastine abolished their curative capacity. Immunodepression of the T cell compartment of the prospective tumor-bearing animals by thymectomy plus irradiation or its abrogation in B mice (thymectomized, lethally irradiated, and reconstituted with fetal liver cells) did not interfere with the therapeutic effect of the transferred anti-DBA/2 lymphocytes. Blocking the macrophage functions of the host by carrageenan, however, abolished the therapeutic effect of immune lymphocytes. These data indicate that a radiation-resistant, non-T cell is involved in the tumor eradication induced by anti-DBA/2 lymphocytes. It was also shown that cured mice, tested 90 days after therapy, become resistant to 5×10³ LD₈₀ YC8 cells and that this resistance was due to the presence of memory cells derived from the transferred and not from the host lymphocyte population.