Kikuyoshi Yoshida

Role of the regional lymph node in cancer metastasis

At an early phase of tumor growh, T-cell responses, i.e. the proliferation of T cells and the generation of cytotoxic T cells or killer-augmenting T cells are induced in the regional lymph node depending upon the immunological properties of the tumor cells. A small number of tumor cells seems to be rejected in the regional lymph node in situ. A progressive tumor induces suppressor activity in the regional node. Suppressor cells facilitate tumor growth and lead to lymphatic metastasis. Thus, the regional lymph node operates only as a temporary barrier to tumor growth. Experimental studies have demonstrated that cradication of the suppressor cells and effective immunization, or both, lead to tumor rejection by augmenting the immunological activity of the regional lymph node.

Development of antigenic heterogeneity in the splenic meshwork of severe combined immunodeficient (SCID) mice after reconstitution with T and B lymphocytes

Recently, we produced monoclonal antibodies reacting specifically with the reticular meshwork (RM) of lymphoid tissues, and demonstrated that, in the splenic white pulp of normal mouse, the antigenic heterogeneity of RM was associated with the segregation of the T and B lymphocytes. In the present study, we attempted to visualize further the interaction between splenic RM and T and B lymphocytes transferred into severe combined immunodeficient (SCID) mice. The splenic white pulp of naive SCID mice, containing a few T and B cells, showed little tendency for T-B segregation and antigenic diversity of RM. Transfer of spleen or bone marrow cells from normal mice resulted in complete recovery of lymphocyte populations, showing not only a clear segregation of T and B lymphocytes but also a remarkable antigenic diversity of RM. The same results were obtained following the transfer of spleen or bone marrow cells from the nude mouse. Next, we transferred purified T lymphocytes to one group of SCID mice and B cells to another. In mice given T cells, a few B cells were observed in the white puop; T lymphocytes lodged not only in the inner periarterial lymphatic sheath (PALS) but also in the outer PALS and follicles. In the animals to which B cells were transferred, T cells were few and the homing of B cells occurred only into their proper compartments, such as the outer PALS, follicles and marginal zone, but not in the inner PALS. Thus, B cells can home into their proper compartments of the splenic white pulp independently of T lymphocytes.

Antigenic heterogeneity of the reticular meshwork in the white pulp of mouse spleen

SummaryMonoclonal antibodies against cellular components of reticular meshworks were produced by immunizing rats with heterogeneous stromal-cell population of mouse spleen. Immunohistochemical screening selected two antibodies, WP-1 and RPSC-2. WP-1 proved to immunostain the meshwork of the B area densely, leaving the marginal zone unstained; it also reacted sparsely with the meshwork of the T-cell region. In contrast, RPSC-2 selectively immunostained the meshwork of the T region. Immuno-electron microscopy clearly visualized, for both antibodies, reaction products being deposited along the cytomembrane of the fibroblastic reticulum cells, along their abundant cytoplasmic processes that were densely intertwined with lymphocytes. Double immunostaining with RPSC-2 followed by WP-1 clearly divided the white pulp into the T and the B domains. The meshwork in the T-cell region proved to be immunostainable with both WP-1 and RPSC-2. Thus, the fibroblastic reticulum cells of the T-and the B-cell areas, while indistinguishable by routine microscopy, are at least partially heterogeneous.

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4+8− T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4+ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4+ T cells admixed with irradiated MM48 tumor cells, but not by CD4+ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4+ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4+ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4+ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4+ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4+ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4+ T cells is discussed.

Cyclophosphamide-dependent lymph node modification in lymph node metastasis of MM48 tumor cells in syngeneic mice.

We investigated the role of immunosuppressive activity induced in the regional lymph nodes (RLN, popliteal lymph nodes) in the establishment of lymph node metastasis by cyclophosphamide (CY) administration. The CY treatment led to the elimination of suppressive activity with the appearance of positive immune responses, and the inhibition of lymph node metastasis of MM48 tumor cells. In CY‐treated mice, the removal of RLN together with the primary tumor lowered the survival rate compared with the mice in which the RLN remained intact. During 4 days after primary tumor resection, the proliferation of tumor cells in the RLN was significantly decreased in CY‐treated mice. These results suggested that the induction of suppressive activity in the lymph node is closely associated with the establishment of lymph node metastasis.