Neal R. Pellis

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

Abstractu2003We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR+ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3–, CD19–, CD20–, CD14–, CD11b–, CD16–, CD56–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system.

Induction of suppressor cells by a tumor-derived suppressor factor☆

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.

Immunotherapeutic effects of tumor‐specific transplantation antigens released by 1‐butanol

Active immunotherapy with materials extracted from a murine methylcholanthrene‐induced sarcoma using single phase solutions of 2.5% 1‐butanol evoked host resistance against supralethal burdens of neoplastic cells deposited subcutaneously or delivered hematogenously. After curative resection of progressing 1 cm tumors, postsurgical immunity against subsequent subcutaneous neoplastic challenges was potentiated by weekly injections of crude butanol extracts. In a conventional therapy model, hosts bearing progressing 4 mm subcutaneous tumors displayed neoplastic regression following chemoimmunotherapy with cyclosphosphamide and tumor extracts. Therapeutic effects were demonstrated not only against subcutaneous neoplasms, but also against hematogenously disseminated pulmonary and extrapulmonary metastases introduced by intravenous inoculation of sarcoma cells. These findings suggest that tumor extracts may afford potent therapeutic adjuvants for patients bearing a small body tumor burden but at high risk of disease recurrence.

Specific and non-specific immunologic mechanisms of tumor growth facilitation.

Specific and non‐specific mechanisms of tumor growth facilitation were studied using methylcholanthrene (MCA)‐induced fibrosarcomas in C3H/HeJ mice. In early and late stages of tumor growth, mice possessed non‐specific, tumor‐facilitating cells detected by local adoptive transfer assay (LATA). These cells appeared to be macrophages; they were radioresistant (700 rads), phagocytic, and adherent to plastic. Specific tumor facilitation was induced by treatment either with crude 3M KCl extracts, or with an acidic pIEF fraction (pI 3.5). After treatment with this material, animals displayed facilitated outgrowth of only MCA‐F, but not the antigenically distinct MCA‐D or MCA‐C tumors. Thus in addition to non‐specific stimuli, which accelerate neoplastic growth, tumors bear tumor‐specific transplantation antigens (TSTA), which induce specific facilitation.

Changes in spleen morphology and lymphoid cell activity during tumor progression

Abstract Morphologic alterations in the spleen and dynamic changes in the biologic activity of its cellular components occur concomitant with the neoplastic progression of a murine methylcholanthrene induced fibrosarcoma. Spleen size, weight and cell number increased with tumor growth. Using the local adoptive transfer assay (LATA), spleens from tumor bearing mice in the early and late stages of neoplastic growth were shown to possess non-specific, tumor-facilitating cells which were radioresistant, phagocytic and adherent, presumably macrophages. On the other hand, spleens from mice in the intermediate and late stages of tumorigenesis displayed specific, tumor-neutralizing cells, which were radiosensitive and Thy 1.2 positive, presumably T-cells. Thus, a balance of tumor-facilitating and neutralizing cells may determine neoplastic progression in susceptible, syngeneic hosts.

Streptococcal immunotherapy of a chemically induced murine fibrosarcoma: Effect of dose, route, sham surgery, and splenectomy on adjuvant action

SummaryAdjuvant immunotherapy with hemolytic streptococci abates the growth of a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma propagated in C3H/HeJ mice. Interperitoneal (IP) administration of streptococci either prior to or after tumor inoculation reduced neoplastic outgrowth. While subcutaneous (SC) administration of streptococci prior to tumor inoculation did not influence tumor outgrowth, SC treatment afterwards was effective. Thus, therapeutic effects of streptococcal vaccine depend upon the route and/or timing of administration.Surgery performed shortly before tumor inoculation abrogated host response to streptococci. On the other hand, surgery performed 3 days after tumor inoculation did not alter the adjuvant action of streptococcal vaccines. The failure of attempts to achieve an immunotherapeutic effect in splenectomized hosts suggests that the spleen was essential for the action of the streptococcal vaccine.

Specific chemoimmunotherapy in tumor-bearing mice with extracted antigen, cyclophosphamide, and intrasplenic administration of interleukin-2.

The effect of daily intrasplenic (I‐SP) injection of interleukin‐2 (IL‐2) in conjunction with specific chemoimmunotherapy with extracted tumor antigens and cyclophosphamide was assessed with the use of methylcholanthrene (MCA)‐induced fibrosarcoma in C3H/HeJ mice. Injections of 80 U of human IL‐2 were delivered transcutaneously into the spleen (I‐SP), which had been relocated to the subcutis with its blood supply intact. Six daily I‐SP injections into mice bearing MCA‐F tumors activated immune spleen cells (SPC), as evidenced by specific neutralization of the MCA‐F, but not the antigenically different MCA‐D tumor, in local adoptive transfer assays. The immune cell phenotype was Thy 1.2+ Lyt 2+, based upon abrogation of tumor neutralization after depletion with monoclonal antibodies and complement. In a second series of experiments, primary hosts bearing established MCA‐F tumors underwent therapy with 1 μg 1‐butanol extracted, isoelectrophoretically purified TSTA injected subcutaneously, a single intraperitoneal (IP) dose of 20 mg/kg cyclophosphamide (CY), and/or either IP or I‐SP IL‐2 injection. Triple chemoimmunotherapy with I‐SP, but not IP, IL‐2 retarded tumor outgrowth more effectively than single or double treatment protocols. Furthermore, in a third series of investigations, the triple therapy group showed both decreased numbers and incidence of spontaneous metastases from a subcutaneous implant of clone 9–4, a highly metastatic variant of MCA‐F. Indeed, 35% of hosts that received triple therapy, but none of the animals treated with other regimens, were free of lung metastasis (P < 0.02). Thus, tumor resistance engendered by chemoimmunotherapy with TSTA and CY is potentiated in vivo by I‐SP administration of IL‐2.

The effect of immunotherapy with extracted tumor antigens on sinecomitant immunity.

The fate of hosts after tumor resection depends upon the level of their sinecomitant immunity, which can destroy residual neoplastic cells. In addition, active specific immunotherapy can stimulate the resistance of mice incompletely resected of large tumors and therefore destined to develop recurrent disease. The studies presented herein, assessing host resistance by in vivo tumor challenge and an in vitro growth inhibition assay (GIA) measuring the effect of splenic lymphocytes on 3H‐thymidine incorporation into monolayers of sarcoma cells, revealed an antagonistic relationship between sinecomitant immunity and tumor antigen immunotherapy. Treatment of hosts bearing high levels of endogenous sinecomitant immunity with Fraction 15 pI 5.95 partially purified from 3 M KCI extracts by preparative isoelectric focusing, decreased their resistance to tumor challenge and the capacity of their spleen cells to perform in the in vitro GIA. When sinecomitant immunity was not demonstrable, therapy with tumor antigen induced host resistance. If therapy was delayed until sinecomitant immunity had naturally waned, hosts displayed improved resistance toward secondary challenge. These findings suggest that not only does antigen therapy effect tumor resistance by a mechanism independent of sinecomitant immunity, but also these two mechanisms are mutually antagonistic. Therefore, the design of active specific immunotherapy protocols utilizing tumor antigens may depend on the level of the host endogenous sinecomitant resistance. Cancer 52:64‐69, 1983.

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40-55% Lyt 2+ and 40-60% L3T4+ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2+ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4+ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.

The role of the spleen in tumor bearing host: II. The influence of splenectomy in mice.

The effect of splenectomy on neoplastic outgrowth was examined prior to and after implantation of methylcholanthrene-induced C3H/He murine tumors. Splenectomy performed 12 days before tumor inoculation did not affect the tumor outgrowth, however, both splenectomy and sham operation performed shortly before tumor inoculation resulted in significant tumor facilitation compared with the non-operated group, suggesting that this accelerated tumor was not related to the presence or absence of splenic tissue, but rather to systemically-induced immunosuppression. While splenectomy performed 6, 9, 12 days after tumor inoculation did not alter the tumor growth, splenectomy performed early (3 days) or late stage (20 days) after tumor cell challenge revealed a retarded neoplastic outgrowth, compared with the sham operated group. These results suggest that splenectomy in very early and late stages of tumor-bearing host may be effective for tumor treatment.