Luigi Varesio

Immunologic Reactivity of Lymphoid Cells in Tumors

There have been many studies of immune responses against tumors and almost all of these have focused on the reactivity in the blood or spleen. From such studies, it has become clear that a wide variety of effector cells and types of immune functions may be involved in antitumor responses. Particular attention has been directed toward T cells that may be directly cytotoxic against tumor cells or may proliferate or produce lymphokines upon stimulation with tumor antigens. However, other effector mechanisms may be involved and need to be considered. These include B cells, which can produce antibodies that affect tumor cells directly or that interact with K cells or macrophages and thereby mediate antibody-dependent cell-mediated cytotoxicity; macrophages and monocytes, which are spontaneously cytotoxic or can be activated to become cytotoxic against tumor cells; and natural killer (NK) cells, a subpopulation of lymphocytes with spontaneous cytotoxic reactivity against tumor cells.

Endotoxin requirement for macrophage activation by lymphokines in a rapid microcytotoxicity assay

A short term microcytotoxicity assay is described for studying the activation of M0 by lymphokines. Proteose-peptone-induced macrophages were purified by adherence in flat-bottomed microtiter plates and then activated by MAF-containing supernatants for 18 h. 51Cr-labeled tumor target cells were added for an additional 18 h, and then the supernatants were harvested and the % isotope release quantitated. When endotoxin-free medium and FBS were used, we found that small amounts of LPS were absolutely required for macrophage activation in this assay. The advantages of this technique included (a) good reproducibility, (b) the requirement for small number of M0, and (c) the potential of standardizing the assay and thereby testing a large number of samples. Moreover, this assay may have particular value for investigations of M0 activation by exogenous stimuli, since M0 that were not pretreated with activating agents did not exhibit cytotoxicity.

Immunobiology of Picolinic Acid

Exposure of various cell types to IFN results in increased enzymatic activities leading to the breakdown of tryptophan and evidence exists to suggest that the catabolism of tryptophan may be connected to the biological effects of interferon (Carlin et al., 1989. The first and rate-limiting enzymatic step in the degradation of tryptophan is the conversion of tryptophan to formylkynurenine. Two different enzymes are deputed to this function. The tryptophan 2,3-dioxygenase (TDO) is found only in the liver and the indoleamine 2,3-dioxygenase (IDO) is found in all tissues examined so far and inducible by IFN and IFN-inducers such as LPS and viruses. Tryptophan catabolism can be demonstrated in vivo in experimental murine models as well as in human subjects (Yoshida et al, 1979). We have confirmed the expression of IDO activity in human monocytes activated by IFN-γ and in the monocytic leukemia cell line THP-1 activated by IFN-γ or by TPA (Musso et al., 1994).

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor.

Abstract The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.

Activation of Tumoricidal and/or Suppressor Macrophages: Different Stimulatory Signals Trigger Either Function both in Vivo and In Vitro

Macrophages upon in vivo and in vitro stimulation become activated and manifest functional and morphological characteristics either not present or differently expressed in non-stimulated macrophage populations (1–4). Antithetical functions can be exerted by a given macrophage population. For instance, cytotoxic macrophages could contribute to the elimination of infectious agents or to the control of neoplastic growth. On the other hand, by inhibiting certain immune responses, suppressor macrophages could impair host defense mechanisms.

A microsystem to evaluate the synthesis of [3H]leucine labeled proteins by macrophages

A method is described for evaluating protein synthesis by adherent M phi by measuring the incorporation of [3H]leucine into TCA precipitable material. By using guanidine-HCl it was possible to remove completely the radiolabeled proteins from the adherent cells that were cultured in microwells, and retain TCA precipitable material. This procedure enabled us to harvest the TCA precipitable proteins with a semiautomatic cell harvester. The guanidine-HCl treatment did not affect the recovery of the radioactive proteins and did not alter the sensitivity of the assay. This method is very simple and rapid and, since it is suitable for processing microcultures, permits detailed studies on the biology of small numbers of M phi.

Hypoxia and Gene Expression

Analysis of gene expression is a promising approach to understand tumor hypoxia and to relate biology to prognosis and response to therapy. Understanding the response to hypoxia of every component of the tumor mass is indispensable to understand the biology of the system. However, the plasticity and distribution of hypoxic areas make it difficult to gauge tumor hypoxia by combining the information of individual components. Global information on tumor gene expression revealed by the analysis of the transcriptome of the tumor mass provides a global view of the hypoxic status of the tissue. The global and analytic assessment of tumor hypoxia, revealed by the whole tumor mass or by its individual components, will be discussed with particular emphasis on tumor-infiltrating leukocytes.