Jian-Ping Zou

Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD4+ T cell activity

Splenic CD4+ T cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 2 to 3 wk after the inoculation with CSA1M cells produced IL-2 and macrophage-activating factor upon in vitro cultures. This lymphokine production was achieved without stimulation of these T cells with exogenous stimulating tumor Ag. However, elimination of APC from spleen cells resulted in almost complete abrogation of the capacity of CD4+ T cells to produce IL-2/macrophage-activating factor. The lymphokine production was regained when APC from CSA1M-bearing mice were added back to cultures. APC from normal or another syngeneic tumor (Meth A)-bearing mice failed to regain the lymphokine production. These observations demonstrated that the lymphokines were produced by CD4+ T cells from CSA1M-bearing hosts through their collaboration with APC binding CSA1M tumor Ag in the tumor-bearing state. The lymphokine-producing capacity of whole spleen cells from tumor-bearing mice reached the maximal level around 2 to 3 wk after tumor implantation but gradually decreased with the progress of tumor-bearing stages. Importantly, tumor-bearing stage-related changes were observed in a different fashion in the capacities of anti-CSA1M CD4+ T cells vs CSA1M tumor Ag-binding APC. The capacity of APC increased with the progress of tumor-bearing stages as demonstrated by the stimulation of CSA1M-immunized T cells with APC from different CSA1M-bearing stages. In contrast, the reactivity of anti-CSA1M T cells to APC from a given CSA1M-bearing stage decreased with the tumor-bearing stage. These results demonstrate a stage-related increase tumor Ag-binding APC function, as well as a reciprocal reduction in tumor Ag-responsive CD4+ T cell activity.

Transforming Growth Factor‐β CD4+ T cells Tumor‐bearing state Immunosuppression Enhanced Production of TGF‐β and a Progressive Increase in TGF‐β Susceptibility of Anti‐tumor CD4+ T Cell Function

The present study deals with the effect of transforming growth factor‐β (TGF‐β) on anti‐tumor immune responsiveness at various stages of the tumor‐bearing state. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 wk after inoculation with CSA1M cells produced interleukin‐2 (IL‐2) and macrophage‐activating factor (MAF)/interferon‐γ (IFN‐γ) upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti‐CSA1M CD4+ T cells and antigen‐presenting cells that had been pulsed with CSA1M tumor antigens in vivo in the tumor‐bearing state. The IL‐2‐producing capacity of CD4+ T cells reached the maximal level as early as one week after tumor implantation but decreased with the progress of tumor‐bearing stages. In contrast, the capacity of CD4+ T cells to produce MAF/IFN‐γ was not affected but was maintained at high levels even late in the tumor‐bearing state. The addition of recombinant TGF‐β (rTGF‐β) to cultures of spleen cells from various tumor‐bearing stages resulted in the suppression of lymphokine production. However, the magnitude of the TGF‐β‐induced suppression varied depending on which tumor‐bearing stages of splenic cells were tested as a responding cell population; it was slight in cells from early (1–3 wk) tumor‐bearing stages but increased in cells from donor mice at later tumor‐bearing stages. Thus, spleen cells from late tumor‐bearing stages with weak but significant IL‐2‐producing and considerable MAF/IFN‐γ producing capacities failed to produce these lymphokines when rTGF‐β was present in cultures. A progressive increase in the TGF‐β susceptibility was also observed for IL‐4‐producing Th2 as well as IL‐2/MAF‐producing Th1 cells. In addition, increased levels of TGF‐β were detected in plasma from tumor‐bearing mice at late stages. Taken together, these results indicate that tumor‐bearing mice exhibit enhanced production of TGF‐β as well as a progressive increase in the susceptibility of anti‐tumor CD4+ T cells to TGF‐β‐induced suppressive mechanisms.

Tumor-immunotherapy with the use of tumor-antigen-pulsed antigen-presenting cells

SummaryOur previous study demonstrated that the administration of antigen-presenting cells (APC) pulsed with tumor cell membrane fraction to naive syngeneic mice results in effective induction of tumor-specific protective immunity in vivo. The present study examined whether tumor-antigen-pulsed APC can produce the inhibitory effect on the growth of tumor cells when administered to tumor-bearing hosts. Naive BALB/c mice were inoculated with viable tumor cells. Five days later, these mice started to receive the relevant tumor-antigen-pulsed APC at 3- to 4-day intervals. The administration of tumor-antigenpulsed APC induced the rejection or growth inhibition of a growing tumor in approximately half of the recipient mice. Moreover, it was demonstrated that tumor-specific immunity was induced in such tumor-regressed mice. These results indicate that tumor-antigen-pulsed APC are effectively applicable to the tumor-specific immunotherapy.

Thymic stromal cells eliminate T cells stimulated with antigen plus stromal Ia molecules through their cross-talk involving the production of interferon-gamma and nitric oxide.

We previously established a thymic stromal cell clone capable of inducing differentiation of immature thymocytes and described a clonal elimination model in which T cell clones are killed on the monolayer of this stromal clone by stimulation of their T cell receptors (TCR) with antigen plus stromal Ia molecules. This study investigated molecular mechanisms underlying this phenomenon. Antigenic stimulation on thymic stromal cells produced large amounts of interferon-gamma (IFN-gamma) and small amounts of tumor necrosis factor-alpha (TNF-alpha). Addition of anti-IFN-gamma monoclonal antibody (mAb) to these cultures largely prevented death of TCR-stimulated T cells. T cell death was also induced when cultures were treated with recombinant IFN-gamma (rIFN-gamma) or rTNF-alpha instead of the relevant antigen, showing that these lymphokines are involved in the process of T cell death. It was further demonstrated that these lymphokines, especially IFN-gamma, induced the expression of mRNA for the inducible type of nitric oxide (NO) synthase in thymic stromal cells and that enhanced levels of NO were produced by stromal cells cultures with T cells plus antigen or stimulated with rIFN-gamma or rTNF-alpha. NO was found to be critically responsible for inducing T cell death on the stromal cell monolayer following stimulation of T cells with antigen or of stromal cells with rIFN-gamma or rTNF-alpha, because T cells death was completely prevented by addition of NG-monomethyl-L-arginine (L-NMMA), which is capable of inhibiting NO production. These results indicate that elimination of TCR-stimulated T cells on thymic stromal monolayers with the capacity to support thymocyte differentiation is induced by the cross-talk between IFN-gamma/TNF-alpha-producing T cells and stromal cells capable of producing NO in response to these lymphokines.

Recovery of antitumor CD4+ T cell responsiveness, suppressed in the tumor-bearing state, by release from tumor burden

The present study investigates the recovery of antitumor CD4+ T cell responsiveness, suppressed in the tumor-bearing state, following release of tumor burden. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 weeks after the inoculation with CSA1M cells produced interleukin-2 (IL-2) and IL-4 upon in vitro cultures without addition of exogeneous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4+ T cells and antigen-presenting cells (APC) that has been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The lymphokine-producing capacity gradually decreased as the tumor-bearing period increased, and spleen cells from mice at late (8–10 week) tumor-bearing stages produced reduced levels of lymphokines. Because APC in these cells exhibited enhanced capacities to present tumor antigens, the reduced responsiveness was ascribed to the dysfunction of CD4+ T cells themselves. However, removal of a tumor after 8 weeks resulted in a remarkable recovery of the lymphokine-producing capacities of whole spleen cells. In contrast to the reduction in CSA1M-antigen-presenting activity of APC following tumor resection, CD4+ T cells exhibited a reciprocal increase in their responsiveness to CSA1M antigens. The recovery of antitumor responsiveness was also observed in the in vitro cultures free from tumor burden; when spleen cells from mice at late tumor-bearing stages were pre-incubated for 1–2 days and re-cultured in fresh medium. They produced potent amounts of IL-2 and IL-4. These results indicate that the immunodysfunction of antitumor CD4+ T cells induced in the tumor-bearing state is not irreversible, and release from tumor burden results in almost complete recovery of the potent antitumor responsiveness they previously expressed.

Suppression of Anti‐tumor CD4+ T Cell Responsiveness in the Tumor‐bearing State and Its Recovery in in vitro Culture Free of Tumor Burden

We investigated whether the responsiveness of anti‐tumor CD4+ T cells suppressed in the tumor‐bearing state is reversed in conditions free of tumor burden. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 wk after inoculation with CSA1M cells produced interleukin‐2 (IL‐2) and IL‐4 upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti‐CSA1M CD4+ T cells and antigen‐presenting cells (APC) that had been pulsed with CSA1M tumor antigens in vivo in the tumor‐bearing state. However, spleen cells from late (8–10 wk) tumor‐bearing stages produced reduced levels of lymphokine production despite the presence of comparable proportions of CD4+ T cells. Because APC in these cell populations exhibited enhanced capacities to present tumor antigens, reduced responsiveness was ascribed to the dysfunction of CD4+ T cells themselves. When spleen cells from early tumor‐bearing mice were preincubated for 1–2 days and recultured in fresh medium, the magnitude of lymphokine production by these cells was not changed. In contrast, the same protocol of preincubation and reculture for cells from late tumor‐bearing mice resulted in the recovery of anti‐tumor lymphokine‐producing capacity. The recovered capacity was comparable to or slightly higher than that expressed by cells from early tumor‐bearing stages. Since the CD4+ T cell content did not significantly differ before and after preincubation, enhanced lymphokine production was due to the recovered responsiveness of anti‐tumor CD4+ helper T cells. The recovery of anti‐tumor responsiveness was also induced in vivo by tumor removal at the late tumor‐bearing stage: spleen cells from mice 2–4 wk after tumor resection efficiently produced IL‐2 and IL‐4. These results indicate that the immunodysfunction of anti‐tumor CD4+ T cells induced in the tumor‐bearing state is reversible because release from tumor burden either by preincubation in vitro or by tumor removal in vivo results in almost complete recovery of the potent anti‐tumor responsiveness initially expressed.

Antigen-presenting cells constitutively bind tumor antigens in the tumor-bearing state in vivo to construct an effective immunogenic unit.

Antigen‐presenting cells (APC) constitntively process endogenous (self) proteins to bind the processed peptides to la molecules. In the present study, we investigated whether the same associative recognition also holds true for tumor‐associated antigens (TAA) that are regarded as “self” molecules in tumor‐bearing hosts. The following results were obtained: (i) an APC‐depleted splenic T cell population from CSA1M tumor‐immunized hosts was stimulated to produce interleukin 2 in vitro when co‐cultured with APC from CSA1M‐bearing mice, but not from normal mice; and (ii) a Thy‐l+ cell‐depleted APC population from CSA1M‐bearing mice could produce CSA1M tumor‐specific protection in vivo when inoculated into naive syngeneic mice. These results provide evidence for the functional binding in vivo of TAA to APC in the tumor‐bearing state. The results are discussed in the context of the paradox that tumor‐bearers fail to reject their own malignancy despite the formation on APC of an effective immunogenic unit which is capable of stimulating tumor‐specific T cells.