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     

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     

Production of T cell differentiation factor in syngeneic lymphocyte macrophage culture for cytotoxic T lymphocyte generation

This study demonstrated that T cell differentiation factor (TCDF) was produced in syngeneic lymphocyte-macrophage cultures. Conditioned medium containing TCDF and interleukin 2 (IL 2) induced the differentiation of leukoagglutinin (LA)-activated cytotoxic T lymphocyte precursors (CTLp) into cytotoxic T lymphocyte (CTL) effectors. The production of TCDF and IL 2 peaked at day 4 to 5 in cultures containing normal spleen cells, syngeneic peritoneal macrophages, and indomethacin. Macrophages and T cells with Thy-1+, L3T4+, and Lyt-2- phenotype were needed for TCDF production. There was no requirement for xenogeneic serum in the culture medium; thus, TCDF could be produced in a syngeneic system. Recognition of self Ia molecules appeared to be essential for TCDF production, which was completely abolished by the addition of monoclonal anti-Ia antibody. In our experiments, removal of IL 2 from conditioned medium containing TCDF abolished its ability to generate LA-activated CTL. However, the cytotoxic response could be restored by the addition of a small amount (5 U/ml) of purified human recombinant IL 2 (HRIL 2), which alone was unable to generate LA-activated CTL at this dose. The generation of LA-activated CTL by high dose HRIL 2 (greater than 50 U/ml) was likely due to the endogenous production of TCDF. The bulk of TCDF could be separated from IL 2 by gel filtration in a Sephadex G-100 column. The peak of TCDF activity was concentrated at a m.w. of 16K dalton, and there was very little IL 2 activity in these fractions. When added alone to the LA-activated lymphocyte cultures, these active fractions were unable to induce CTL; supplementation of exogenous IL 2 was needed to restore the cytotoxic responses. Our findings indicate that both IL 2 and TCDF, which are needed in CTL generation. are produced in syngeneic cultures in the absence of antigenic or mitogenic stimulation.     

Presence of an enlarged pool of MOPC-315-specific cytotoxic T lymphocyte precursors in the thymuses of mice that eradicated a large MOPC-315 tumor as a consequence of low-dose melphalan therapy

Summary We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard) treatment, thymocytes from mice bearing a large, day-10 MOPC-315 tumor, but not thymocytes from normal mice, acquire the ability to generate an enhanced level of antitumor cytotoxicity upon in vitro stimulation with MOPC-315 tumor cells plus low concentrations (9.0–90 IU/ml) of exogenous interleukin-2 (IL-2) [23]. Here we show that the time interval between tumor inoculation and low-dose melphalan therapy as well as the magnitude of tumor burden at the time of the chemotherapy are important for the ability of the drug to render thymocytes more responsive to in vitro stimulation with MOPC-315 tumor cells plus low concentrations of recombinant IL-2 (rIL-2). Specifically, the chemotherapy was found to be effective in enhancing the thymic antitumor reactivity only if the mice bore a large, late-stage tumor. Comparison of thymocytes from untreated mice bearing a large, late-stage tumor to thymocytes from normal mice revealed that tumor-bearer thymocytes contained approximately a three-fold higher frequency of cytotoxic T lymphocyte precursors (CTLp) for MOPC-315-associated antigens. Following curative low-dose melphalan therapy of mice bearing a large, latestage MOPC-315 tumor, the frequency of CTLp for MOPC-315-associated antigens increased further, reaching a level approximately tenfold higher than that found among thymocytes of normal mice. At the same time, the frequency of CTLp for an antigenically unrelated allogeneic tumor (EL4) as well as the overall percentage of mature cells was not increased. The cells responsible for the exertion of the enhanced antitumor cytotoxicity following in vitro stimulation of thymocytes from mice treated with low-dose melphalan when they have a large, latestage MOPC-315 tumor are of the CD8⁺/CD4^– phenotype. Thus, the enhanced level of antitumor cytotoxicity generated by thymocytes from mice that are treated with low-dose melphalan when they have a large, late-stage MOPC-315 tumor is due, at least in part, to the presence of an enlarged pool of CTLp specific for MOPC-315-associated antigens, which mature into CD8⁺/CD4^– effector cells upon stimulation with MOPC-315 tumor cells plus low concentrations of rIL-2.supported by Research Grant CA-35761 from the National Cancer Institute and Research Grant EY-05945 from the National Eye Institute. This work is in partial fulfillment of the requirements for the Doctor of Philosophy degree of M. M. B. M. B. M. was supported by a Career Development Award CA-01350 from the National Cancer Institute.     

In vitro generation of secondary cell-mediated cytotoxic response against a syngeneic Gross virus-induced lymphoma in rats.

Cell-mediated cytotoxic responses against a syngeneic Gross virus-induced lymphoma, (C58NT)D, in W/Fu rats were generated in vitro by using mixed lymphocyte-tumor cell cultures. The source of responding cells was either spleens from normal rats or spleens from rats carrying or having rejected (C58NT)D tumors. Mitomycin C-treated (C58NT)D tumor cells were used as stimulating cells. The secondary anti-tumor cytotoxic response occurred more rapidly and reached higher levels than the primary response, and it was antigen specific. T cells, but nor B cells or macrophages, were essential for both the induction and the effector phases of the secondary anti-tumor responses. These data suggest that specific memory T cells persist for long periods of time in the lymphoid organs of (C58NT)D immune rats, which can rapidly become cytotoxic upon re-exposure to antigen.     

The difference between 5-fluorouracil and melphalan in their ability to promote antitumor immune response against MOPC-315 plasmacytoma

Summary The anticancer chemotherapeutic drugs melphalan (L-phenylalanine mustard; L-PAM), 5-fluorouracil (5-FU), methotrexate (MTX), and daunorubicin (DAU) were tested for their toxic activity against MOPC-315 tumor cells in vitro. L-PAM, 5-FU, and DAU had a marked toxic effect whereas MTX did not affect the rate of thymidine incorporation in the tumor cells. L-PAM (7.5 mg/kg) induced permanent regression of large s.c. MOPC-315 plasmacytoma tumors, 5-FU (200–250 mg/kg) induced transient regression of MOPC-315 tumors with reappearance starting on the 6th day after the 5-FU injection and DAU (5 mg/kg) was not effective. L-PAM treatment restored the cytotoxic potential of spleen cells of tumor-bearing mice against target MOPC-315 tumor cells whereas spleen cells from tumor-bearing mice treated with 5-FU were unable to mount a cytotoxic response.L-PAM and 5-FU were also assayed for their effect in vitro on induction of suppressor T cells by ConA. L-PAM treatment in vitro markedly reduced the induction of suppressor T cells by ConA whereas 5-FU had no effect. It is suggested that anticancer chemotherapeutic drugs can be classified in immunopromoting (L-PAM as prototype) and nonimmunopromoting (5-FU as protoype) on the basis of their effect in vivo on established tumors and their effect on induction of suppressor T cells by ConA.     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

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.     

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     

Limited importance of CD40/CD40L interaction in the B7-dependent generation of anti-MOPC-315 cytotoxic T lymphocyte activity by tumor bearer splenic cells stimulated in vitro in the presence of tumor necrosis factor

 We have previously illustrated the importance of B7-2 expression for the enhanced generation of cytotoxic T lymphocyte (CTL) activity by stimulation cultures of tumor bearer splenic cells to which tumor necrosis factor α (TNFα) has been added. Here we show that the B7-1 molecule is also important for CTL generation by such stimulation cultures, although to a much lesser extent than the B7-2 molecule. In addition, we show the importance of CD40/CD40L interaction for the expression of the B7-2 molecule, but not the B7-1 molecule, by tumor bearer splenic cells stimulated in vitro in the presence of TNF. The CD40/CD40L interaction is also shown to be important for the generation of CTL activity by tumor bearer splenic cells stimulated in vitro in the presence of exogenous TNF. However, the CD40/CD40L interaction is less important for the generation of enhanced CTL activity than for the expression of an elevated level of B7-2. Specifically, blockade of CD40/CD40L interaction, which reduced the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the presence of TNF to the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the absence of exogenous TNF, failed to reduce the level of CTL generated to the level generated by tumor bearer splenic cells stimulated in the absence of exogenous TNF. Finally, blockade of CD40/CD40L interaction was inferior to blockade of B7-2/CD28 interaction in inhibiting the generation of CTL activity by tumor bearer splenic cells stimulated in the presence of exogenous TNF. Thus, although CD40/CD40L interaction is important for the generation of enhanced CTL activity by stimulation cultures of tumor bearer splenic cells to which TNF has been added, TNF also mediates its potentiating effect for CTL generation by such stimulation cultures via other mechanisms that are independent of CD40/CD40L interaction but dependent on B7-2 expression. Received: 31 December 1997 / Accepted: 27 March 1998     

Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes.

A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.     

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     

T cells with FC receptors in myeloma; suppression of growth and secretion of MOPC-315 by T alpha cells

We have previously demonstrated that 1) BALB/c mice and patients with IgA myeloma developed a marked expansion of T cells with surface IgA-Fc receptors (T alpha cells), 2) the FcR alpha were induced by direct interaction of soluble myeloma IgA with T cells, and 3) the T alpha cells induced in IgA myeloma were Lyt-1-2+, IgA isotype-specific suppressor cells in normal immune responses. These findings established that the host with IgA myeloma responds to the large amounts of Ig produced by the tumor by activating an otherwise normal IgA isotype-specific suppressor circuit. In the present studies, we extend our previous observations and show that T alpha cells can suppress both the growth and secretion of MOPC-315 myeloma tumor cells. Thus, the isotype-specific immunoregulatory circuit activated in myeloma is capable of suppressing tumor cells as well as normal cells.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Induction by DNA immunization of a protective antitumor cytotoxic T lymphocyte response against a minimal-epitope-expressing tumor

 In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2K^d-restricted NP(147–155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147–155)-specific CTL response within 2 weeks after the boost. When challenged with 10⁴ P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4⁺ or CD8⁺ T cells and then challenging with 10⁴ P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8⁺ CTL-mediated antitumor response. Received: 8 May 1997 / Accepted: 28 August 1997     

Augmentation of generation of human allospecific cytotoxic T lymphocyte by PPD in in vitro sensitization culture

Summary PPD augmented human lymphocyte blastogeneic response to allogeneic lymphocytes in the mixed lymphocyte reaction (MLR) and generation of human cytotoxic lymphocytes against allogeneic human lymphocytes in in vitro sensitization (IVS) culture. The augmenting effect of PPD in the MLR was unequivocally synergistic at its lower concentrations (0.05 and 0.01 g/ml). The augmentation of MLR was observed following addition of a supernatant of culture medium of lymphocytes which had been precultured with PPD for 24 h then washed free of PPD and recultured without PPD for another 24 h. PHA and Con A, in contrast, suppressed both MLR and the generation of alloreative cytotoxic cells. The alloreactive cytotoxic lymphocytes whose generation was augmented by PPD belonged to the SRBC-rosette forming fraction and passed through a nylon-wool column. The NK cell-like activities of the alloreactive cytotoxic lymphocytes were not augmented by PPD. Analysis of the alloreactive cytotoxic lymphocytes whose generation was augmented by PPD by competitive inhibition assay with unlabeled cells indicated that the same allogeneic lymphocytes used as sensitizing cells in IVS culture inhibited the cytotoxicity, while MOLT-4 cells, which are frequently used as target cells for the human NK-cell assay, did not. When lymphocytes with known HLA-A and HLA-B were used in the IVS culture and the cytotoxicity assay, PPD was found to augment the cytotoxicity only against the target lymphocytes that possessed the same HLA as the sensitizing lymphocytes in IVS.     

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.