Characterization of the exogenous interleukin-2 requirements for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers

We have shown previously that thymocytes from MOPC-315-tumor-bearing mice treated with low-dose melphalan (l-phenylalanine mustard) (l-PAM TuB mice) are superior to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated normal mice or normal mice treated with low-dose melphalan in their ability to generate an antitumor cytotoxic response following 5-day in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2) [Mokyr MB, Bartik MM, Ahn M-C (1989) Cancer Res 49; 870]. Here we characterize the rIL-2 requirements for the generation of enhanced antitumor cytotoxicity byl-PAM TuB thymocytes relative to normal thymocytes upon in vitro stimulation with MOPC-315 tumor cells. Specifically, we show that delaying the addition of a low concentration of rIL-2 to 5-day in vitro stimulation cultures of thymocytes resulted in a progressive decline in the generation of antitumor cytotoxicity by both normal andl-PAM TuB thymocytes. However, even when rIL-2 was added on day 2 after culture initiation, thymocytes froml-PAM TuB mice generated a more potent antitumor cytotoxicity than did thymocytes from normal mice. In addition, when rIL-2 was added at the time of culture initiation, replacement of the conditioned medium with fresh medium lacking rIL-2 on day 3 of the 5-day in vitro stimulation culture period eliminated the ability of normal thymocytes, and reduced (but did not eliminate) the ability ofl-PAM TuB thymocytes, to generate a significant level of antitumor cytotoxicity. A low concentration of fresh rIL-2 was sufficient to restore completely the generation of antitumor cytotoxicity by normal orl-PAM TuB thymocytes when added to the stimulation cultures immediately after the removal of the rIL-2-containing conditioned medium. The same low concentration of rIL-2 was also sufficient for restoring the generation of antitumor cytotoxicity by cultures ofl-PAM TuB thymocytes, but not normal thymocytes, from which the rIL-2-containing medium was removed 1 day earlier. At the same time, conditioned medium from stimulation cultures ofl-PAM TuB thymocytes was not superior to conditioned medium from stimulation cultures of normal thymocytes in supporting the generation of antitumor cytotoxicity by either normal orl-PAM TuB thymocytes. Thus, the enhanced lytic activity generated byl-PAM TuB thymocytes, relative to normal thymocytes, upon stimulation with MOPC-315 tumor cells and a low concentration of rIL-2, does not appear to be the result of enhanced production of helper-like factors byl-PAM TuB thymocytes.Supported by research grant CA-35 761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degreeSupported by Career Development Award CA-01350 from the National Cancer Institute     

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma

We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard (l-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dosel-PAM therapy (l-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was mimicked by the membranepenetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The -adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal orl-PAM TuB spleen cells. Since the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8⁺ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.     

Phorbol ester-induced enhancement in lytic activity of CD8+ splenic T cells from low-dose melphalan-treated MOPC-315-tumor bearers

Summary We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (l-phenylalanine mustard);l-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dosel-PAM (l-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1–10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity byl-PAM TuB spleen cells exposed to PMA was found to require CD8⁺, but not CD4⁺, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulatedl-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in thel-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin,l-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8⁺ splenic T cells froml-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dosel-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells. Since the curative effectiveness of low-dose chemotherapy for MOPC-315 tumor-bearing mice requires the participation of CD8⁺ T cells that exploit a cytotoxic T lymphocyte type lytic activity for tumor eradication, it is feasible that in some situations PMA-like stimulants could be used to augment the antitumor cytotoxic activity of the CD8⁺ T cells, which in turn could improve the therapeutic outcome of low-dose chemotherapy.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01 350 from the National Cancer Institute     

Enhanced expansion of the thymic CD8+ cell subset as a potential mechanism for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers

Summary We have previously shown that thymocytes from low-dose melphalan (l-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4^–/CD8⁺ or CD4⁺/CD8^–) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4^–/CD8⁺ (but not CD4⁺/CD8^–) cells than did cultures of thymocytes from the other two sources. Since CD4^–/CD8⁺ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4^–/CD8⁺ effector cells.Supported by research grant CA-35 761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degreeSupported by career development award CA-01 350 from the National Cancer Institute     

Transforming growth factor-β-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy

We have previously shown that treatment of mice bearing a large MOPC-315 plasmacytoma with a low dose of the anticancer drug melphalan (l-phenylalanine mustard;l-PAM) results in the acquisition of a potent CD8⁺ T-cell-mediated anti-MOPC-315 cytotoxic T lymphocyte (CTL) activity by the hitherto immunosuppressed tumor bearers, and this immunity contributes to complete tumor eradication. In the studies presented here, we sought to determine how the acquisition of this antitumor immunity following low-dose chemotherapy is possible, in light of the report that MOPC-315 tumor cells produce transforming growth factor- (TGF-), an immunosuppressive cytokine that can down-regulate the generation of CTL responses. We found that the acquisition of CTL activity following low-dosel-PAM therapy is not due to a chemotherapy-induced decrease in the sensitivity of MOPC-315 tumor bearer spleen cells to TGF--mediated inhibition of CTL-generation. Moreover, even spleen cells from MOPC-315 tumor-bearing mice, which had receivedl-PAM therapy 7 days earlier and had acquired CTL activity in vivo, were sensitive to the inhibitory activity of TGF- upon culture for as little as 1 day, with or without stimulator tumor cells. However, the production of TGF- by MOPC-315 tumors decreased drastically as a consequence of the low-dose chemotherapy. Thus, the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor-bearing mice may be due, at least in part, to a reduction in TGF- production that enables the development of tumor-eradicating immunity.Work was supported by research grant IM-435 from the American Cancer Society and research grant CA54413 from the National Cancer Institute.In partial fulfillment of the requirements for the Doctor of Philosophy DegreeSupported in part by the Dorothea Fleming Cancer Research Fellowship Award     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

AKR.H-2b lymphocytes inhibit the secondary in vitro cytotoxic T-lymphocyte response of primed responder cells to AKR/Gross murine leukemia virus-induced tumor cell stimulation.

We have previously shown that AKR.H-2b congenic mice, though carrying the responder H-2b major histocompatibility complex haplotype, are unable to generate secondary cytolytic T-lymphocyte (CTL) responses specific for AKR/Gross murine leukemia virus (MuLV). Our published work has shown that this nonresponsive state is specific and not due to clonal deletion or irreversible functional inactivation of antiviral CTL precursors. In the present study, an alternative mechanism based on the presence of inhibitory AKR.H-2b cells was examined. Irradiated or mitomycin C-treated AKR.H-2b spleen cells function as in vitro stimulator cells in the generation of C57BL/6 (B6) anti-AKR/Gross virus CTL, consistent with their expression of viral antigens. In contrast, untreated viable AKR.H-2b spleen cells functioned very poorly as stimulators in vitro. Viable AKR.H-2b spleen cells were also able to cause dramatic (up to > or = 25-fold) inhibition of antiviral CTL responses stimulated in vitro by standard AKR/Gross MuLV-induced tumor cells. This inhibition was specific: AKR.H-2b modulator spleen cells did not inhibit allogeneic major histocompatibility complex-specific CTL production, even when a concurrent antiviral CTL response in the same culture well was inhibited by the modulator cells. These results and those of experiments in which either semipermeable membranes were used to separate AKR.H-2b modulator spleen cells from AKR/Gross MuLV-primed responder cells or the direct transfer of supernatants from wells where inhibition was demonstrated to wells where there was antiviral CTL responsiveness argued against a role for soluble factors as the cause of the inhibition. Rather, the inhibition was dependent on direct contact of AKR.H-2b cells in a dose-dependent manner with the responder cell population. Inhibition was shown not to be due to the ability of AKR.H-2b cells to function as unlabeled competitive target cells. Exogenous interleukin-2 added at the onset of the in vitro CTL-generating cultures partially restored the antiviral response that was decreased by AKR.H-2b spleen cells. Positive and negative cell selection studies and the development of inhibitory cell lines indicated that B lymphocytes and both CD4- CD8+ and CD4+ CD8- T lymphocytes from AKR.H-2b mice could inhibit the generation of AKR/Gross virus-specific CTL in vitro. AKR.H-2b macrophages were shown not to be required to demonstrate AKR/Gross MuLV-specific inhibition, however, confirming that the inhibition by T-cell (or B-cell)-depleted spleen populations was dependent on the enriched B-cell (T-cell) population per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-10: a novel cytotoxic T cell differentiation factor

A previous report concluded that a new cytokine, designated IL-10, is a growth cofactor for thymocytes, spleen, and lymph node cells. In this report, we have focused on the effects of IL-10 on CD8+ spleen T cells. We first observed that IL-10 enhances the growth of CD8+ T cells to IL-2. We then investigated the effect of murine rIL-10 on the induction of murine effector CTL from CTL precursors (CTL-p) using both bulk and filler cell-free limiting-dilution cultures. IL-10 alone could not induce Con A-activated FACS-sorted CD8+ T cells either to proliferate or to generate effector CTL. In combination with IL-2, however, IL-10 augmented the cytolytic activity of effector CTL generated from Con A-activated spleen CD8+ T cells in bulk cultures incubated for 5 days. In limiting-dilution cultures (using solid-phase anti-CD3 mAb as stimulus), IL-10, in combination with IL-2, substantially increased the CTL-p frequency and augmented the cytolytic activity per clone expanded from one CD8+ T cell when compared with cells cultured in IL-2 alone. Kinetic studies showed that IL-10 is required at both early and late culture stages for optimal generation of effector CTL. The potentiating effects of IL-10 on CTL function were neutralized by an anti-IL-10 mAb. These results indicate that IL-10 has direct effects on mature T cells, and suggest that IL-10 also functions as a cytotoxic T cell differentiation factor, which promotes a higher number of IL-2-activated CTL-p to proliferate and differentiate into effector CTL. In contrast, IL-10 did not enhance significantly the lymphokine-activated killer cell activity of IL-2-grown CD8+ cytotoxic T cells.     

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, 1C10) can actually promote the in vitro generation of CTL activity by CD8⁺ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220⁺ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220⁺ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220⁺ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8⁺ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220⁺ cells. Received: 23 December 1998 / Accepted: 22 February 1999     

Induction of anti-CD8 resistant cytotoxic T lymphocytes by anti-CD8 antibodies. Functional evidence for T cell signaling induced by multi-valent cross-linking of CD8 on precursor cells

The effector function of most MHC class I allospecific CTL is inhibited by anti-CD8 mAb. In the present study, we report the surprising observation that multi-valent cross-linking of CD8 molecules on precursor cells by specific antibody actively induces the generation of CD8+ class I allospecific CTL whose lytic function is resistant to anti-CD8 antibody inhibition, and actively induces down-modulation of cell surface CD8 expression on these cells. In marked contrast, bi-valent cross-linking of CD8 inhibits the generation of CD8+ CTL from precursor cells and fails to induce down-modulation of cell surface CD8 expression. These results demonstrate that CD8 can transduce net positive signals, but only when the molecule is extensively cross-linked.     

B7-2 expression on tumor cells is important for the acquisition of cytotoxic T lymphocyte activity by spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers via a mechanism that requires either B7-1 or B7-2 expression on host antigen-presenting cells

 We have previously shown that B7-2 (CD86) and, to a lesser extent, B7-1 (CD80) contribute to the curative effectiveness of low-dose melphalan (l-phenylalanine mustard) for mice bearing a large MOPC-315 tumor under conditions that lead to the acquisition of potent cytotoxic T lymphocyte (CTL) activity at the tumor site. Since B7-1 and B7-2 are expressed on both tumor cells and host antigen-presenting cells (APC), the current studies were undertaken to examine the relative importance of each costimulatory molecule on tumor cells and on host APC for the acquisition of anti-MOPC-315 CTL activity. Utilizing an in vitro system for the acquisition of CTL activity, we found that B7 expression on host APC is important for the development of CTL activity in stimulation cultures of spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers, although the expression of either B7-1 or B7-2 is sufficient. In addition, we found that B7-2, which is expressed at high levels on stimulator tumor cells, but not B7-1, which is expressed at much lower levels, is also important for the acquisition of CTL activity. However, the vast majority of the CTL activity acquired in vitro in response to stimulation with the B7-2-expressing MOPC-315 tumor cells was found to depend on B7-expressing host APC. Thus, it is likely that B7-2, which is expressed at high levels on MOPC-315 tumor cells, promotes the rapid lysis of MOPC-315 stimulator tumor cells, thereby making tumor-associated antigens more readily available for efficient presentation by B7-expressing host APC which, in turn, stimulate the acquisition of CTL activity by spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers. Received: 2 September 1999 / Accepted: 19 November 1999     

Development of antigen-specific CD8+ CTL in MHC class I-deficient mice through CD4 to CD8 conversion

CD8+ CTL are the predominant tumoricidal effector cells. We find, however, that MHC class I-deficient mice depleted of CD8+ T cells are able to mount an effective antitumor immunity after immunization with fused dendritic/tumor cells. Such immunity appears to be mediated by the generation of phenotypic and functional CD8+ CTL through CD4+ to CD8+ conversion, which we have demonstrated at the single cell level. CD4+ to CD8+ conversion depends on effective in vivo activation and is promoted by CD4+ T cell proliferation. The effectiveness of this process is shown by the generation of antitumor immunity through adoptive transfer of primed CD4 T cells to provide protection against tumor cell challenge and to eliminate established pulmonary metastases.     

Inhibition of interleukin-10 (IL-10) production from MOPC 315 tumor cells by IL-10 antisense oligodeoxynucleotides enhances cell-mediated immune responses

 Interleukin-10 (IL-10) has both inhibitory and stimulatory effects on diverse cell types of the immune system. It inhibits the antigen-presenting capacity of monocytes/macrophages and stimulates T cell proliferation. Although many tumors spontaneously release IL-10, the physiological relevance of this phenomenon to the in vivo antitumor immune response is not known. To elucidate the physiological role of tumor-released IL-10, we used IL-10-specific antisense oligodeoxynucleotides (AS-ODN) for the inhibition of IL-10 production from the tumor cells. Incubation of MOPC 315 plasmacytoma with IL-10 AS-ODN in vitro resulted in inhibition of IL-10 production and also in enhancement of the expression of major histocompatibility complex (MHC) class I, MHC class II, and B7-1 molecules. MOPC 315 cells incubated with IL-10 AS-ODN (MOPC-IL10AS) for 16 h in vitro showed reduced tumorigenicity in Balb/c mice. The mice implanted with MOPC-IL10AS effectively rejected the tumor graft, and showed strong cytotoxic T lymphocyte (CTL) activity against the parental MOPC 315 cells. In addition, MOPC-IL10AS were more effective as stimulator cells in mixed lymphocyte/tumor cell culture, and as target cells in a CTL assay. These results imply that IL-10 spontaneously released from MOPC 315 cells inhibits their immunogenicity and that the inhibition of IL-10 production by IL-10 AS-ODN may be a way to enhance the host cellular antitumor immune response. Received: 11 November 1999 / Accepted: 6 April 2000     

Melphalan-mediated potentiation of antitumor immune responsiveness of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor

Summary Administration of a low dose of l-PAM (0.75 mg/kg) to mice bearing a large SC MOPC-315 tumor and extensive metastases led to the development of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells. Such drug-induced potentiation of antitumor immune responsiveness appeared by day 2 after chemotherapy, and it could not be further enhanced but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in depleting the cells known to have inhibitory activity (i.e., macrophages and metastatic tumor cells). To establish that l-PAM can lead to selective in situ abrogation of the inhibitory effectiveness of the splenic macrophages and metastatic tumor cells, we demonstrated that incubation of immunosuppressed tumor-bearer spleen cells with a low concentration of l-PAM in vitro also resulted in augmented antitumor immune potential that could not be further augmented by depletion of glass-adherent cells. l-PAM-mediated enhancement of the antitumor immune potential of immunosuppressed tumor bearer spleen cells was due at least in part to the effects of the drug on the splenic metastatic tumor cells. Isolated tumor cells treated with a low concentration of l-PAM were not only devoid of inhibitory activity for the primary in vitro antitumor immune response by normal spleen cells, but actually manifested a strong immunostimulatory capacity. Thus, l-PAM given at a low dose enhances the development of potent antitumor immunity which brings about the eradication of a large tumorigenic load that remains after the drug has been cleared from the circulation.Presented in part at the 67th annual meeting of the Federation of American Societies for Experimental Biology in Chicago, April 10–15, 1983 Abbreviations used: L-PAM, l-phenylalanine mustard (Melphalan); CY, cyclophosphamide     

Priming of influenza virus-specific cytotoxic T lymphocytes vivo by short synthetic peptides.

Influenza virus-specific CTL were primed in vivo by immunization with short synthetic peptides representing major CTL epitopes from the nucleoprotein of type A influenza virus. The resultant CTL after in vitro boosting of primed spleen cells recognized both virus-infected and peptide-pulsed target cells. The requirement of CD4+ T cell activation was investigated in several ways. First the addition of helper epitopes to the CTL epitope did not enhance CTL generation, suggesting that helper activity was either not limiting or not required. However, in vivo depletion of CD4+ T cells completely inhibited the generation of CTL by peptide immunization. The inclusion of anti-CD4 in the in vitro restimulation with peptide also prevented the generation of CTL, whereas in vitro reactivation of virus immune spleen cells with peptide was not inhibited by anti-CD4. Thus there appears to be heterogeneity in the requirement of CD4+ T cell proliferation in CTL generation. One possibility is that virus infected cells can stimulate higher affinity T cells that are less helper T cell dependent.     

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.     

Functional involvement of E-cadherin in TGF-beta 1-induced cell cluster formation of in vitro developing human Langerhans-type dendritic cells

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-beta 1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-beta 1 costimulation (basic cytokine combination GM-CSF plus TNF-alpha, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-beta 1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a-, lysozyme+, CD14+). TGF-beta 1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-beta 1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-beta 1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.