T and B lymphocyte populations of tumor-bearing mice treated with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) or pyran

Summary The immunostimulator pyran copolymer and the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have been studied to determine their effect on T and B lymphocytes from BALB/c mice bearing a syngeneic tumor, Madison lung 109 carcinoma. Indirect immunofluorescent and mitogenic assays revealed that the tumor-bearing controls usually had T and B cell populations lower than those observed for the normal controls. The tumor-bearing group treated with BCNU generally had T and B cell levels lower than the tumor controls. The administration of pyran resulted in an increase of T cells and a temporary increase in the B cell population. Although this increase in the B cell population of tumor-bearing mice treated with pyran is higher than that seen for the untreated tumor controls it was never higher than in the normal control mice. The experimental results indicate that pyran appears to reconstitute the depressed T cell population and has a transitory effect on the B cell population resulting from tumor burden.     

Migration of B lymphocytes in lymphoid organs of lethally irradiated,thymocyte-reconstituted mice

Summary The migration of radiolabeled intravenously injected B lymphocytes through thymus-dependent areas was studied in lymphoid organs of mice with experimentally defined T cell domains (B cell-deprived mice or T mice). In the spleen, B cells were found to enter the peri-arteriolar lymphoid sheath (PALS) by two routes: (i) via the marginal zone, and (ii) via reticulin sheaths surrounding terminal arterioles. B cells migrated through the peripheral and central PALS and initiated the formation of primary follicles in the peripheral PALS 6 h after injection. Distinct primary follicles were noted at 18 h after injection of the labeled B cells. After 24 h small numbers of labeled cells were also noted in the efferent lymphatic vessels of the spleen.The reconstitution of B cell compartments in the mesenteric lymph node was delayed compared to the spleen. B cells entered the nodal stroma across the wall of high endothelial venules in the paracortex and by 6 h were found scattered throughout the paracortex. Isolated clusters of a few labeled cells were noted in the outer cortex at 18 h after cell transfer. Defined primary nodules were seen only 24 h after reconstitution. A minority of labeled cells was found at 24 h in the cortico-medullary junctions and in medullary cords.The present study shows that B lymphocytes traverse T cell domains on their way to their own specific B cell compartments. The immunological significance of this particular migration route is discussed in view of data on the cellular cooperation of B cells, T cells and macrophages during the humoral immune response.     

Nuclear disintegration of target cells by killer B lymphocytes from tumor-bearing mice

Normal murine B lymphocytes are not known to be effectors of the Fc receptor-mediated, antibody-dependent cellular cytotoxicity (ADCC). In contrast, we report here that highly purified splenic B cells from mammary tumor-bearing mice develop the potential of lysing antibody-coated target cells. These lymphocytes are characterized by being G-10 nonadherent, nylon wool adherent, sIg+, FcR+, Thy 1.2-, asialo GM1-, and the immunoglobulin heavy-chain genes of both chromosomes are rearranged. The lytic reaction is characterized by a noninterdigitating binding and by the appearance of endocytotic vesicles in the target cells. Nuclear disintegration occurs 18 h after initial effector-target cell conjugate formation. At such time, only minor cytoplasmic membrane alterations are evident. The emergence of killer B cells in tumor-bearing hosts indicates that all lymphoreticular cell types bearing Fc receptors are capable of mediating ADCC.     

Suppressor cell activity in tumor-bearing mice. I. Dualistic inhibition by suppressor T lymphocytes and macrophages.

Spleen cells from mice bearing methylcholanthrene-induced fibrosarcomas impaired mitogen responses of normal syngeneic lymphocytes. Nylon wool column and other depletion techniques were utilized to characterize the cellular source of suppressive activity in tumor-bearing host (TBH) spleens. Evidence is presented for two distinct suppressor cell systems operating in the spleens, but not lymph nodes, of BALB/c mice bearing transplanted tumors. Spleens from TBH were shown to have greatly increased numbers of macrophages over their normal counterparts. TBH macrophages were observed to have suppressive activity at low in vitro concentrations. Anti-Thy 1 serum treatment of TBH macrophages abrogated low dose inhibition but not suppression due to high numbers of macrophages. No functional difference was detected between anti-Thy 1 serum-treated TBH and normal splenic macrophages. In a macrophage-depleted culture system, mildly nylon wool adherent, anti-Thy 1 serum, and hydrocortisone succinate-sensitive suppressor cells could be detected. Soluble supernatant products of TBH spleen and thymus cells were also found to inhibit in vitro mitogen responses, whereas TBH macrophages and lymph node cells demonstrated no soluble suppressive activity. The major source of soluble inhibitor of DNA synthesis (IDS) seems to be an anti-Thy 1 serum, hydrocortisone-sensitive population.     

RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.     

Preferential homing of tumor-infiltrating lymphocytes in tumor-bearing mice

Summary The heptanoyl tripeptide, FK-565 is a biological response modifier with potent therapeutic properties for the treatment of experimental and spontaneous metastases. Doses of FK-565 greater than 5 mg/kg are required for in vivo augmentation of natural killer cells, macrophages, and for therapeutic activity, presumably because FK-565 is a peptide small molecular mass which is rapidly degraded and excreted. Optimal therapeutic activity is observed at approximately 25–50 mg/kg FK-565, administered i.v. three times per week for 4 weeks. In addition to its therapeutic properties, which were consistently greater than the positive control at optimal doses, FK-565 had significant immunoaugmentary properties for natural killer cells, macrophages, and T cells both in vitro and in vivo, suggesting that its therapeutic activity is due to immune augmentation.This research was sponsored by the DHHS, under contract no. N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States GovernmentAbbreviations used: BRM, biological response modifier; MDP, muramyl dipeptide; poly(I,C)-LC poly(I,C)-Lys_n polyinosinicpolycytidylic acid complexed with poly(l-lysine) and carboxymethylcellulose; MLR, mixed lymphocyte reaction; MLTR-CMC, mixed lymphocyte tumor response-cell-mediated cytotoxicity; HBSS, Hanks' blanced salts solution; NK, natural killer; IFN, interferon; t. i. w., three times a week     

Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy

Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Production of specific macrophage activating factor by lymphocytes from tumor-bearing mice.

Lymphocytes from C57BL mice bearing a syngeneic UV-induced fibrosarcoma (UV-112) produced macrophage activating fatcor (MAF) when cultured with UV-112 cells in vitro. This MAF rendered normal C57BL macrophages cytotoxic in vitro to UV-112 cells. MAF production and lymphocyte-mediated cytotoxicity were detected in the early stages of tumor growth, but were absent in mice bearing large tumors. This eclipsed reatcivity was specific for the growing tumor. Lymphocytes from mice bearing a large UV-112 tumor were still able to produce MAF in response to B16 melanoma to which they had been preimmunized. In all instances, the MAF produced was specific in that it rendered syngeneic macrophages cytotoxic against only the tumor used for immunization.     

Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.     

T and B lymphocytes and serum alpha 1-antitrypsin activity in children with bronchial asthma treated with broncho-vaxom

The aim of this study was to evaluate an effect of Broncho-Vaxom treatment on T and B lymphocytes and serum alpha 1AT in children treated at "Zuch" sanatorium in Szczawno-spa. The trial involved 46 school aged children suffering from infectious or infectious-atopic asthma. The post-Broncho-Vaxom treatment values for T lymphocytes were significantly higher in infectious, and significantly lower for B lymphocytes in infectious-atopic asthma. Serum alpha 1AT activity in children suffering from infectious asthma decreased significantly after the treatment. A correlation between the efficacy of the treatment and the lymphocyte percentage was observed. In children with very effective clinical results of Broncho-Vaxom treatment, the significant increase in T lymphocyte, and decrease in B lymphocyte populations was observed. Changes in T and B lymphocyte percentage were analysed in respect to alpha 1AT pre-treatment activity. In children with high alpha 1AT value, T lymphocytes after the treatment increased significantly in infectious, and B lymphocytes decreased significantly in infectious-atopic group.     

Immunosuppression of T lymphocyte function by fractionated serum from tumor-bearing mice.

Sera from mice with transplanted 3-methylcholantrene-induced tumors have been shown previously to inhibit the function of normal lymphoid cells. When chromatographed on Sephadex G-150, the fraction eluting with immunoglobulin has been shown to inhibit the proliferative response of normal spleen cells to concanavalin A and to inhibit the in vitro antibody response to a T-dependent antigen, but has a lesser effect on the antibody response to a T-independent antigen. This paper deals with studies on the mode of action of the serum factor. The immunoglobulin containing fraction of serum from tumor-bearing mice inhibited the in vitro generation of both allogeneic and syngeneic cytotoxic lymphocytes. Time course studies demonstrate that the serum fraction inhibits the generation of antibody-producing and cytotoxic lymphocytes if added during the first 2 days of a 5-day culture. Serum fractions added after day 2 had no effect on the in vitro response. The serum factor appears to inhibit the generation of specific T cell function during the proliferative stage of development but has no effect on the differentiation stage which leads to either antibody-producing cells or cytotoxic lymphocytes.     

Comparison of the metabolic fate and tissue distribution of B700, an albumin-like melanoma-specific antigen with serum albumin in normal and tumor-bearing mice.

1. B700, a murine melanoma antigen, is a member of the serum albumin protein family, being closely related to murine serum albumin (MSA). 2. We have studied and compared the metabolic fate and anatomic distribution of radioiodinated B700 and MSA administered to semisyngeneic naive and tumor-bearing mice. 3. Labelled material from both proteins is excreted primarily into urine. 4. The rate of excretion of the two proteins is markedly different, with B700 having a shorter half-life in the body. 5. Despite their similar molecular weights, intact B700 represents approx. 30% of the radioactivity in the urine but only 4% of the MSA in the urine is intact. 6. These studies demonstrate that the host can readily distinguish between very similar normal (MSA) and tumor-associated (B700) molecules and process them differently. 7. Similar findings of differential fate and distribution have been reported in comparing other albuminoid molecules [Dueland S., Blomhoff R. and Pedersen J. I. (1990) Biochem. J. 267, 721-725].