The mediator of cellular immunity. X. Interaction of macrophages and specifically sensitized lymphocytes.

Specifically sensitized lymphocytes have a focusing influence on mononuclear phagocytes that is expressed in the local accumulation and division of macrophages in bacteria-induced exudates. This was demonstrated by injecting Listeria monocytogenes into the peritoneal cavity of normal rats immediately before the animals were transfused with thoracic duct lymphocytes from either Listeria-immune donors or donors that had been infected with the unrelated parasite, Francisella tularensis. Sensitized lymphocytes originally present in the intravenous inocula were found later in the exudates. The arrival in the inflamed peritoneal cavity of specifically sensitized lymphocytes was associated with an exuberant influx of newly formed host cells and a local proliferative response that involved both immunoblasts and macrophages. These cytokinetic observations provide a plausible explanation of the delayed inflammatory response induced by the parasite, and imply that sensitized lymphocytes contribute to the host's defence by encouraging the prompt and purposeful deployment of monocyte-derived macrophages in centers of infection. In addition to their focusing influence on mononuclear phagocytes, specifically sensitized lymphocytes or their products can enhance the metabolic and microbicidal activity of macrophages. This activation process was revealed in the ability of rats infected with L. monocytogenes or BCG to control the growth of F. tularensis at a challenge site in the testis. But resistance was expressed only when the challenge organisms were injected with killed bacteria against which the recipients had been specifically immunized. The results accord with the view that macrophages are functionally activated by an immunological mechanism, and imply that the process is triggered locally by sensitized lymphocytes which are recruited from the blood.     

Dissociation of macrophage accumulation and local chemotactic activity in delayed inflammatory sites.

A dissociation between the in situ generation of lymphocyte-dependent macrophage chemotactic activity (MCA) and the accumulation of macrophages in peritoneal inflammatory exudates was demonstrated in rats stimulated intraperitoneally with a saline suspension of killed Listeria monocytogenes (LM). Treatment of specifically immunized animals with cobra venom factor (CoVF) erased the hemolytic activity of serum complement (C) and the generation of peritoneal MCA. Such C-deficient rats nonetheless marshaled a substantial number of monocyte-derived macrophages into LM-induced exudates. The results suggest that MCA does not have an obligatory role in the attraction of macrophages into lesions in which there is a delayed inflammatory component. CoVF not only abrogated lymphocyte-dependent MCA in antigen-induced exudates but also decreased MCA of fresh and of heated normal rat serum. The serum of venom-treated animals could not be rendered chemotactic by C activation. It remains to be determined whether lymphocyte-dependent MCA is a product of antigen-stimulated T cells or is generated extracellularly by the interaction of T-cell factors with a humoral precursor. In any event, lymphocyte-dependent MCA differs from C-dependent MCA insofar as it is inactivated by heating (30 min at 56 °C).     

The mediator of cellular immunity. VIII. Effect of mitomycin C on specifically sensitized lymphocytes.

The antimitotic agent, mitomycin C, was used to probe cellular events underlying the expression of acquired resistance to L. monocytogenes in the rat. It was found that thoracic duct lymphocytes from donors primarily immunized with this organism can protect normal recipients against a Listeria challenge and that their ability to do so is inhibited by treatment of the lymphocytes with mitomycin C. Concentrations of the drug which are effective in this regard also inhibit lymphocyte proliferation, but have little if any effect upon RNA or protein synthesis in cells tested at the time of transfer. Yet mitomycin C probably has irreversible effects on lymphocytes in addition to its capacity to block cell replication, for inhibitor-treated immunoblasts fail to localize in peritoneal exudates induced in recipient rats. The inability of mitomycin-treated immunoblasts to extravasate in inflammatory foci is associated with a diminished capacity of specificially sensitized lymphocytes to promote cell division in peritoneal exudates induced by L. monocytogenes. The results illustrate the importance of lymphocyte-macrophage interactions in cellular resistance to infection and give credence to the view that these interactions occur locally at sites of microbial invasion.     

Allogeneic restriction of the delayed inflammatory reaction in the rat.

Delayed-type hypersensitivity (DTH) to Listeria monocytogenes was measured in rats that were recipients of syngeneic, semisyngeneic, and allogeneic immune thoracic duct lymphocytes (TDL). DTH could be transferred only to recipients that shared at least one haplotype with the TDL donors. The restriction was expressed in an inability of sensitized lymphoblasts to localize efficiently at antigen injection sites in the pinna of the ear and peritoneal cavity. Failure of allogeneic lymphoblasts to extravasate in more than trace numbers into Listeria-antigen-induced exudates was reflected in an absence of other lymphocyte-mediated expressions of DTH. Thus, lymphocyte-dependent MCA was not detected in Listeria-antigen-induced peritoneal exudates borne by recipients of allogeneic immune TDL and blood monocytes were not recruited in increased numbers into such exudates as they were in exudates borne by syngeneic rats. But allogeneic restriction of the delayed inflammatory response to Listeria antigen was overcome, at least in part, when antigen-presenting macrophages of the same MHC type as the immune TDL donors were implanted in the peritoneal cavity. The results encourage the belief that the observed failure of immune TDL to transfer DTH to allogeneic recipients is related to the inability of sensitized donor T cells to recognize antigen displayed by allogeneic macrophages.     

The mediator of cellular immunity: XI. Origin and development of MIF producing lymphocytes

Thoracic duct lymphocytes obtained from rats infected with Listeria monocytogenes were characterized with respect to size, turnover and their capacity to release macrophage migration inhibitory factor (MIF). Cells responsive to Listerial antigens (LMA) in the MIF assay were identified in lymph during the first week of an immunizing infection. These were immunoblasts or large lymphocytes, as evidenced by their sedimentation with S phase lymphocytes at unit gravity. When labeled cells from the lymph of Listeria-infected donors were infused into similarly infected recipients, donor S phase lymphocytes localized rapidly, and in substantial numbers, in peritoneal exudates induced by the unrelated organism, F. tularensis. Within this immigrant population were cells which conferred immunity against L. monocytogenes and released MIF in cultures containing LMA. Exudates harvested 36 hr or 61 hr after stimulation contained labeled lymphocytes that were smaller than the S phase cells recovered during the early post-induction period. The observed shift of radioactivity from large to smaller lymphocytes was parallelled by a shift MIF production to exudate fractions containing smaller cells. The MIF producing cells in exudates of advancing age also exhibited increasing resistance to inhibition by vinblastine. These findings suggest that MIF is released by a family of lymphocytes—large, medium and small. LMA-responsive lymphocytes are delivered to the thoracic duct soon after their formation, at a stage in development when they can be stimulated to release only low levels of MIF. These mediator producing cells circulate briefly in the blood and differentiate fully only after they extravasate into inflammatory foci.     

The bronchoalveolar lymphocyte: Studies on the life history and lymphocyte traffic from blood to the lung

Although recent work has shed some light on the identity and function of lymphocytes that reside in the bronchoalveolar air space (lung lymphocytes), little is known about the origin and life history of these cells. To determine the proportion of recently divided lung lymphocytes, DA-strain rats were labeled in vivo for 3 days with tritiated thymidine ([³H]dTR). Autoradiographs of lavaged lung and peritoneal cells indicated that a large fraction (44–77%) of lung lymphocytes was labeled and that these values were comparable to the proportion of labeled lymphocytes in peritoneal exudates (61–74%). To determine if some newly divided lung lymphocytes might come via the blood, additional experiments were performed in which rats were labeled ([³H]dTR) in vivo for 7 days. Lymphocytes were then obtained in labeled rats by thoracic duct drainage and were adoptively transferred (by vein) into syngeneic recipients. The percentage recovery of labeled lymphocytes in lung aspirates of recipient rats was determined from cell counts and autoradiographs. These results demonstrate that blood may be a source of recently divided lymphocytes but they do not indicate the relative contribution blood makes toward these cells in the lung.     

Nonrecirculating memory T lymphocytes in cellular resistance to infection

Acquired resistance of rats to intracellular infection with Listeria monocytogenes rests on the cooperation between sensitized mediator lymphocytes and effector macrophages. Large numbers of specific T lymphoblasts, capable of transferring resistance to recipients, appear in central lymph shortly (3–6 days) after subcutaneous infection of rats. In contrast, late-phase immunity is poorly transferrable with thoracic duct lymphocytes (TDL) despite high levels of specific resistance observed in spleen, liver, and testes of actively immunized animals. That late-phase immunity is mediated partly by resident, nonrecirculating T cells is attested to by the ineffective transfer of resistance from preinfected to normal partners of parabiotic rats. Transfer studies with thoracic duct cells and peritoneal cells from the stimulated and unstimulated peritoneal cavity seem to suggest that resident T cells mediating late-phase resistance are the progeny of lymphoblasts that extravasated during early phase. Assays measuring the proliferative response upon antigen stimulation in vitro support the concept of a gradual redistribution within the animal of memory T cells.     

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the goblet-cell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

The immunological response of CBA mice to Trypanosoma musculi: Elimination of the parasite from the blood

Agglutinating antibodies against Trypanosoma musculi could not be demonstrated in sera from parasitaemic, immune or immunized mice.Immune, non-adherent cells from peritoneal exudates of mice which had recovered from infection, accelerated the elimination of parasites when adoptively transferred to infected mice in which the parasitaemia was stable (plateau phase) and the parasites had reached the adult (non-dividing) stage. This effect was not influenced by the simultaneous administration of immune adherent cells and/or immune serum and, although the mechanism by which the blood infection is e radicated has not been established, the action of the sensitized cells does not appear to be due to formation of antibody which has a direct, trypanocidal effect.     

Polyester treatment in rats with a dorsal air pouch: chemotaxis in lavage fluid from the pouch]

Minced polyester threads introduced into peritoneal cavity of rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they are released locally by cells involved in inflammation. In this paper the chemotactic effect of the peritoneal and subcutaneous air pouch fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The fluids were obtained by washing the cavity of untreated rats or rats injected with polyester, 7 days after the injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear cells from normal rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrate that a chemotactic activity is present in peritoneal and subcutaneous air pouch fluids following the inflammatory process. In conclusion the chronic inflammation determines the appearance of chemotactic factors for PMN cells, in the peritoneal cavity and in the air pouch, and the air pouch is a very convenient experimental system with the particular advantage that it permits easy repeated sampling of exudate during the course of an inflammatory response.     

Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells.

After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.     

Prophylactic immunization against experimental leishmaniasis. III. Protection against fatal Leishmania tropica infection induced by irradiated promastigotes involves Lyt-1+2- T cells that do not mediate cutaneous DTH

Protective immunity against fatal L. tropica infection in genetically vulnerable BALB/c mice can be induced by prophylactic immunization with irradiated promastigotes even when heat-killed. Such immunity is adoptively transferable transiently into intact or durably into sub-lethally irradiated (200 or 550 rad) syngeneic recipients by splenic T but not B cells. The effector T cells are of the Lyt-1+2- phenotype, devoid of demonstrable cytotoxic activity. The immune splenic T cell population expresses specific helper activity for antibody synthesis. A causal role for helper T cells in this capacity, however, seems unlikely, because it was shown in the accompanying paper that antibody does not determine the protective immunity against L. tropica. The immunized donors show no detectable cutaneous DTH or its early memory recall in response to live or killed promastigotes or a soluble L. tropica antigen preparation. Spleen, lymph node, and peritoneal exudate cells from protectively immunized donors similarly fail to transfer DTH locally or systemically. These cells also lack demonstrable suppressive activity against the expression or induction of DTH to L. tropica. Thus, protection against L. tropica induced by prophylactic i.v. immunization with irradiated promastigotes appears to be conferred by Lyt-1+2- T cells that are distinguishable from T cells mediating either both DTH and T help, or cytotoxicity.     

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.     

Antigen-mediated macrophage adherence inhibition

Antigen-mediated macrophage adherence inhibition (MAI) was studied in inbred rats immunized with various transplantation, tumour-specific and protein antigens. A macrophage-rich suspension of peritoneal cells (PC) was obtained from the peritoneal cavity of immunized and control animals by washing. The adherence to glass of PC was specifically inhibited by the addition of the antigens used for sensitization of PC donors or by related (cross-reacting) antigens but not with unrelated antigens. The MAI seems to be due to the direct interaction of the respective antigen with a corresponding PC receptor and not due to the humoral factor released from immune lymphocytes of PC population upon contact with the specific antigen.     

Mitogenic factor from inbred guinea pigs. II. Properties of the factor

Thymectomized adult rats which have been heavily irradiated and reconstituted with syngeneic bone marrow cells rapidly regain the ability to defend themselves against a primary infection with the intracellular bacterial parasite, Listeria monocytogenes. They do so by a cell-mediated immunological mechanism as evidenced by the protective immunity transferred adoptively by thoracic duct lymphocytes or peritoneal exudate cells from donors infected with this organism. But peritoneal exudate cells from thymus-derived donors convey only a fraction of the immunity transmitted by exudate cells from similarly infected intact rats. Since thymectomized irradiated animals can mobilize their cellular defenses more effectively when they are injected with a modest number of thoracic duct lymphocytes, an effect that cannot be duplicated with a massive infusion of bone marrow, it is argued that thymusdependent lymphocytes or T cells have an influential role in the development of cellular resistance to infection.     

Inflammatory process caused by intraperitoneal injection of polyester in the rat: chemotactic activity in lavage fluid from the cavity

Minced polyester threads introduced into peritoneal cavity of guinea pigs or rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they may be exogenous and/or endogenous. These are released locally by the cells involved in inflammation. In this paper the chemotactic effects of the peritoneal fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The peritoneal cavity fluids were obtained by washing the cavity of untreated rats or rats intraperitoneally injected with polyester, 1, 3, 7, 14 days after the intraperitoneal injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear leukocytes from normal or treated rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrated that the peritoneal fluids taken 3 and 7 days after the intraperitoneal polyester injection, elicit an evident chemotaxis response greater than that showed by peritoneal fluids from control rats. It is suggested that chemotactic factors can be produced and released by mononuclear cells involved in the inflammatory process.     

Measles virus nucleocapsid protein protects rats from encephalitis.

Lewis rats immunized with recombinant vaccinia virus expressing the nucleocapsid (N) protein of measles virus were protected from encephalitis when subsequently challenged by intracerebral infection with neurotropic measles virus. Immunized rats revealed polyvalent antibodies to the N protein of measles virus in the absence of any neutralizing antibodies as well as an N protein-specific proliferative lymphocyte response. Depletion of CD8+ T lymphocytes did not abrogate the protective potential of the N protein-specific cell-mediated immune response in rats, while protection could be adoptively transferred with N protein-specific CD4+ T lymphocytes. These results indicate that a CD4+ cell-mediated immune response specific for the N protein of measles virus is sufficient to control measles virus infections of the central nervous system.     

Cell-mediated immune response to syngeneic ultraviolet-induced tumors. II. The properties and antigenic specificities of cytotoxic T lymphocytes generated in vitro following removal from syngeneic tumor-immunized mice.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill ⁵¹Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia^?. The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. I. Activation of Listeria antigen-responsive T cells

Soon after rats are infected with Listeria monocytogenes (LM), Listeria antigen- (LMA) responsive lymphocytes are delivered to the animal's thoracic duct. These LM-responsive lymphocytes can be restimulated in vitro by LMA to generate cells that have a potent cytolytic capability. The activation of LMA responsive lymphocytes is immunologically specific and dependent upon the activity of histocompatible accessory cells. Neither cell-free LMA nor LMA-pulsed allogeneic accessory cells can promote a significant cytotoxic response by negatively selected responder lymphocytes. LM-dependent cytolytic lymphocytes differ from natural killer (NK) cells inasmuch as their activation is not facilitated by interferon (IF). Likewise, supernatants from cultures containing specifically sensitized thymus-derived (T) lymphocytes and histocompatible LMA-pulsed accessory cells fail to augment (day 2 and day 3 supernatants actually inhibit) the activation process. The results imply that the successful activation of LM dependent cytolytic lymphocytes requires the cooperative interplay of responder T cells and specific-antigen-pulsed accessory cells.