In vivo elimination by specific effector cells of an established syngeneic rat moloney virus-induced sarcoma.

BN rats immunized subcutaneously with a viral induced tumor (MST) or with a chemical-induced fibrosarcoma (BC5) were donors of immune spleen cells. Samples of immune spleen cells were tested in vitro against MST and BC5 in a 51Cr release assay before culturing and after 7 days of culture with mitomycin C-treated MST and/or BC5 tumor cells (MSTMit, BC5Mit). These spleen cells were infused in vivo i.v. into x-rayed (400 R) and nonirradiated BN recipients that bore a vascularized and progressive (1 to 1.5 cm in diameter) subcutaneous MST or BC5. Spleen cells from untreated BN donor rats were also tested in vitro and in vivo as controls. Established MST were specifically eliminated by spleen cells immune to MST after culture with MSTMit, but not by spleen cells immune to MST without further culture nor by cultured or uncultured BC5 immune spleen cells and control spleen cells. Also, the growth of BC5 was not affected by MST immune spleen cells cultured for 7 days with MST and/or BC5. Elimination of Moloney sarcoma (MST) in vivo occurred in less than 35 days and was correlated with the generation of cytotoxicity in vitro since only MST immune spleen cells cultured with MSTMit were able to augment significantly their cytotoxic capability in vitro.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

In vitro induction of polyclonal killer T cells with 2-mercaptoethanol and the essential role of macrophages in this process.

Killer T cells against allogeneic and syngeneic tumor cells were generated in vitro by the addition of 2-mercaptoethanol (2-ME) to the murine spleen cell culture in the absence of any antigenic stimulation. The maximum activity of T cell-mediated cytotoxicity (CMC) induced with 2-ME was observed on day 4 of culture and the induction of CMC was completely inhibited by the addition of inhibitor of DNA synthesis, hydroxyurea, or cytosin arabinoside. CMC induced with 2-ME was specifically inhibited by the addition of unlabeled target cells to the 51Cr-release assay system. These results indicated that killer T cells were generated in the presence of 2-ME as a result of nonspecific polyclonal activation of precursors into cytotoxic effector cells and that they recognized target cells with antigen-specific recognition receptors. Spleen cells deprived of adherent cells showed impaired induction of CMC with 2-ME. The addition of peritoneal exudate macrophages to splenic T cells restored this response. The result indicated that macrophages were essential for the induction of CMC with 2-ME. The possibility that the function of macrophages was mediated by soluble factor(s) released from macrophages was demonstrated by the separate culture of splenic T cells and macrophages in double-chambered, Marbrook-type vessels and by the addition of supernatants from macrophage cultures to splenic T cells. 2-ME and soluble factor(s) released from macrophages seemed to be required for the activation of precursors into killer cells.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).     

Cellular basis of immunologic interactions in adoptive T cell therapy of established metastases from a syngeneic murine sarcoma

The adoptive transfer of specifically sensitized T lymphocytes can effectively mediate the regression of established local and metastatic tumors. Previous experiments using the weakly immunogenic MCA 105 sarcoma indicated that cellular interactions between transferred L3T4+ helper and Lyt-2+ cytotoxic immune T cells were necessary for mediating tumor regression. In this study, the kinetics of T-T cell interactions were analyzed by in vivo depletion of T cell subsets with mAb. The anti-tumor efficacy of transferred immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb on the day of cellular therapy. However, if mAb were given 3 days after the transfer of immune cells, depletion of Lyt-2+ but not L3T4+ cells abrogated anti-tumor efficacy. T cell depletion on day 6 after transfer of immune cells had no adverse effect on tumor regression, indicating the period required for T cell reactivity in vivo. Furthermore, depletion of Ia+ cells by in vivo mAb treatment abrogated the anti-tumor efficacy of immune cells. It is thus hypothesized that there are two distinct but sequential phases of in vivo T cell interactions leading to the regression of established tumors after adoptive immunotherapy. An initial "helper/inducer" phase apparently requires the interaction of L3T4+ immune cells and the tumor Ag involving Ia+ cells. The inducement of L3T4+ cell activation is to provide helper function via the secretion of IL-2. The second phase designated as an "effector phase" involves differentiation of immune Lyt-2+ cells under the influence of IL-2 secreted during the helper/inducer phase for generation of mature Lyt-2+ effector cells. To further support the hypothesis of a two-phase process we have examined the phenotype and kinetics of tumor regression mediated by effector cells generated by secondary in vitro sensitization (IVS). Although the IVS cells were generated from fresh MCA 105 immune spleen cells, their anti-tumor efficacy was mediated solely by Lyt-2+ lymphocytes. Kinetic studies revealed that the in vivo requirement of IVS Lyt-2+ effector cells to mediate tumor regression was less than 3 days, and the anti-tumor reactivity of these cells was not affected by in vivo depletion of Ia+ cells. Thus, the IVS reaction is likely representative of the in vivo counterpart of the helper/inducer phase leading to the generation of mature Lyt-2+ immune effector cells. Tumor regression after transfer of Lyt-2+ cells generated in IVS therefore required a relatively shorter period of time than that required after the transfer of fresh noncultured MCA 105 immune spleen cells.     

Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities

The susceptibility of cytotoxic effector lymphocytes and their induction to in vivo or in vitro treatment with rabbit anti-neutral glycolipid ganglio-N-tetraosylceramide (anti-ASGM1) antiserum was investigated. Intravenous injection of anti-ASGM1 antiserum eliminated measurable natural killer (NK) cell activity in spleen cells of mice infected for 5 days with Vaccinia virus, or for 8 days with lymphocytic choriomeningitis virus (LCMV) if injected 24 hr prior to testing. In addition, this treatment lowered measurable virus-specific cytotoxic T cell activity by 60 to 95%. Virus-specific cytotoxic T cell and NK cell activity generated during a primary infection in vivo was also sensitive to treatment in vitro with anti-ASGM1 antiserum (1/300 to 1/600 dilution) plus rabbit complement at a dilution of 1/15 (20 to 50% cell death, more than 30-fold decrease of cytotoxic activity); in vitro treatment with rabbit complement alone often enhanced NK and cytotoxic T cell activity slightly. In vivo treatment with anti-ASGM1 before primary immunization decreased generation of primary CTL only if high doses of anti-ASGM1 antiserum were injected twice. Antiviral T cells generated during secondary stimulation in vitro and alloreactive cytotoxic T cells from a mixed lymphocyte culture were resistant to treatment in vitro with anti-ASGM1 plus complement at the end of the culture period. Treatment in vitro of in vivo-primed responder spleen cells with anti-ASGM1 plus complement before their addition to a secondary restimulation culture resulted in complete inhibition of a secondary antiviral cytotoxic T cell response. In vivo treatment with anti-ASGM1 24 hr before their spleen cells were harvested and restimulated in vitro significantly reduced the virus-specific T cell activity of mice that had been immunized with virus several weeks previously. A cloned T cell line exclusively exerting NK-like activity was resistant, and two cloned virus-specific cytotoxic T cell lines were susceptible to treatment with anti-ASGM1 plus complement in vitro. These results caution the general use of rabbit anti-ASGM1 as a marker to distinguish NK from CTL cells; they indicate a possible relationship between NK and CTL cells and suggest that in vitro culture of lymphocytes may alter or select the cell surface expression or availability of the ASGM1 marker(s).     

Evidence for cytostatic T cell activity in the effector mechanism against syngeneic TMT mammary tumor cells in mice

The effector mechanism of immune spleen cells against syngeneic TMT mammary tumor cells was analyzed in vitro. C3H/He mice were first inoculated with TMT tumor cells, and then the tumors were x-irradiated with 2000 rad 1 wk after the inoculation. Spleen cells from these treated mice inhibited the growth of tumor cells in vitro when assessed by (3H)-TdR incorporation by tumor cells (cytostatic activity). The same spleen cells did not have any cytotoxic activity on TMT tumor cells detected by a 51Cr-release assay. The cytostatic activity was mediated by Lyt-1+23- T cells. The purified T cells alone could not inhibit the growth of tumor cells, but accessory cells were required for the induction of cytostatic T cell activity. The accessory cells were Ia-positive, macrophage-like adherent cells. Furthermore, both T cells and macrophages were also required for the inhibition of tumor growth even after the spleen cells were activated in vitro. These results suggest T cells and macrophages play an important role in the effector mechanism against TMT mammary tumor cells. The mechanism of cytostasis by T cells and macrophages was discussed from the standpoint of the cellular interaction.     

Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.     

Activation of rat B lymphocytes. I. Characterization of anti-immunoglobulin responses and isotype density of rat B cells

Spleen cells of two rat strains, Lewis and Brown Norway (BN), have been activated by lectins and by antibodies specific for immunoglobulin isotypes embedded in their cell membranes. Optimal concentrations of antibodies specific for mu, gamma, or delta-chains of rat augments in vitro incorporation of 3H-TdR 5 to 18-fold in Lewis B lymphocytes and 1.5 to 4-fold in BN B lymphocytes. In addition, F(ab')2 fragments of anti-Ig reagents induced Lewis splenic B cells but not BN B cells to incorporate 3H-TdR. Responses to LPS and dextran sulfate, B lymphocyte mitogens, measured by radioactive uptake, were five to 10 times greater in Lewis B cell populations than in BN B cell populations. Density of surface Ig isotypes and capping kinetics were similar in the two rat strains, although the percentage of T cells, T cell subsets, B cells, and Ia+ B cells differed in the spleens of these strains of rats. Both T lymphocytes and macrophages were needed in culture to effect an optimal response. IL-2 restored the response in B cell cultures depleted of T cells and macrophages, and enhanced 3H-TdR uptake in whole spleen cells of Lewis but not BN rats. The strain-dependent responsiveness of B cells to specific anti-Ig reagents or B cell mitogens appears to be associated with inherent (genetic) defects in T cells and B cells or defects in T cell to B cell cooperation in BN rats.     

An in vitro system for the generation of suppressor cells and the requirement for B cells in their induction

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.     

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)     

Augmentation of the therapeutic efficacy of adoptive tumor immunotherapy by in vivo administration of slowly released recombinant interleukin 2

Summary Immunization of C57BL/6 mice with MMC-treated syngeneic lymphoma cells, MBL-2, caused the generation of antitumor effector cells in vivo and the immunized mice permanently rejected viable MBL-2 lymphoma cells. Both plastic nonadherent T cells and plastic adherent MØ obtained from MBL-2 immunized mouse peritoneal exudate cells revealed strong cytotoxic activity against MBL-2 lymphoma cells, whereas immune spleen cells were not highly active against MBL-2 lymphoma cells in vitro. However, systemic adoptive transfer of immune spleen cells into the MBL-2-bearing mice by i.v. infusion in conjunction with i.p. cyclophosphamide (100 mg/kg) treatment cured the mice of tumor. This therapeutic efficacy of immune spleen cells was reflected by the number of transferred effector cells and over 5×10⁷ immune spleen cells were required to cure the mice completely. The cells mediating in vivo rejection of MBL-2 lymphoma cells were Thy 1.2⁺ T cells. This ACIT was specific against MBL-2 lymphoma cells and had no effect on the growth of other syngeneic tumors, B16 melanoma or BMC6A fibrosarcoma. In vivo administration of recombinant interleukin 2 (r-IL 2) combined with ACIT greatly modulated the cure rate of tumor-bearing mice. In addition, we found that slowly released r-IL 2 administratered from an ALZET miniosmotic pump was more effective in augmenting the therapeutic efficacy of immune spleen cells in ACIT than a single injection of the same total dose of r-IL 2.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Altered levels of mononuclear leukocytes in tumor-bearing rats: decrease of helper T lymphocytes and increase of suppressor cells

In the spleen and peripheral blood of BN rats with progressive tumors, W3/25+ T helper cells were significantly reduced and OX8+ T suppressor/cytotoxic cells were significantly increased. The ratio of helper to suppressor elements was decreased to 1.6 from a ratio of 3 in normal BN rats without tumors, and this decreased ratio correlated with tumor growth. When tumors were eliminated in vivo by infusion of effector cells (W3/25+ T lymphocytes), the levels of W3/25+ and OX8+ T cells returned to normal and the ratio of helper to suppressor/cytotoxic cells in the spleen and peripheral blood reverted to 3.0 or higher. Macrophages and null cells, T-sIg-, were also elevated in the spleen and peripheral blood of rats bearing expanding tumors and returned to normal levels after cure. Assays of spleen cells for cell-mediated cytotoxicity in rats with large tumors revealed little or no specific cytotoxicity. Cytotoxic activity was high in spleen of rats cured of their neoplasms by infusion of helper cells.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Killer cells of feline leukemia virus- and feline sarcoma virus-infected transformed cells: the role of NK, ADCC, and in vitro generated cytotoxic cells

Feline white blood cells (WBC) manifested a primary in vitro mixed lymphocyte tumor cell culture (MLTC) proliferative response to feline leukemia virus-feline sarcoma virus (FeLV-FeSV)-infected transformed target cells, which reached a peak at Day 15. Furthermore, primary in vitro MLTC cultures generated cytotoxic killer cells capable of killing a variety of targets in non-major histocompatibility gene complex restricted fashion, and effector cells were capable of killing targets introduced repeatedly into cultures over a 49-day period. The presence of feline fibrosarcoma (f-sarc) stimulators was the primary driving force for proliferation and generation of killing because exogenous IL-2 conditioned medium did not appreciably increase the yield of killer cells generated in vitro. WBC cultured without f-sarc stimulators with or without IL-2 supplementation also generated killer (K) cells but at a low level. The killer cell population was composed of approximately 50% lymphocytes and 50% monocytes. Cats had K cells functional in antibody-dependent cell-mediated cytotoxicity against FeLV-coated chicken red blood cells but not against any FeLV-FeSV-infected transformed targets tested. Natural killer (NK) cell activity to any targets tested was not found. Although no evidence was found for K or NK cell activity against FeLV-FeSV-infected transformed cells, feline WBC were readily able to generate killer cells in vitro and it is probable that this cell-mediated immune potential is functionally important in vivo.     

Interleukin 2 administered in vivo induces the growth of cultured T cells in vivo

The capacity of exogenous IL 2 to induce the growth of antigen-activated T lymphocytes in vivo was evaluated. The in vivo growth of adoptively transferred T lymphocytes that had been previously cultured long-term with IL 2 was initially examined, because in vitro such T cells are exquisitely dependent upon exogenous IL 2 for proliferation and survival. Daily administration of IL 2 in vivo, beginning on the day of cell transfer, induced these IL 2-dependent long-term cultured T lymphocytes to proliferate in vivo, and the magnitude of in vivo growth was proportional to the dose of IL 2 administered. The capacity of IL 2 to induce the in vivo growth of antigen-activated T cells not previously exposed in vitro to exogenous IL 2 was similarly studied. T lymphocytes from the spleens of immune mice, activated by 5-day culture with tumor antigen before transfer, survived poorly in vivo when injected with antigen alone, but demonstrated marked proliferation in vivo in response to antigen and exogenous IL 2. By contrast, immune spleen cells transferred with antigen, but without prior culture, proliferated without supplementary exogenous IL 2. Moreover, the growth of noncultured donor T cells was not augmented by the administration of exogenous IL 2, implying that noncultured spleen cells immune to tumor antigens can produce sufficient amounts of endogenous IL 2 in vivo to sustain maximal T cell growth over the time period examined. Importantly, the ability of exogenous IL 2 to induce donor T cell growth in vivo correlated with its ability to function in vivo to augment the anti-tumor efficacy of specifically immune donor T cells in models for the adoptive therapy of disseminated antigenic murine leukemia. Thus, the current studies highlight the potential of exogenous IL 2 to induce T cell growth in vivo and suggest that the administration of IL 2 in vivo may be useful for augmenting T cell responses that are relatively deficient in the production of endogenous IL 2.