The role of adherent cells in the secondary cell-mediated response in vitro to a natural poxvirus pathogen.

The role of adherent cells in an in vitro secondary response to ectromelia virus infection was investigated. Spleen cells from ectromelia-primed mice ("responder" cells) depleted of adherent cells by either carbonyl iron treatment, adherence to plastic or passage through cotton wool columns had a markedly decreased capacity to produce a secondary response, as indicated by decreased T cell-mediated cytotoxicity against virus-infected target cells, when cultured with virus-infected "stimulator" cells. The secondary response was restored by the addition of peritoneal cells from either normal or ectromelia-immune mice. Small numbers of peritoneal cells completely reconstituted the response within a certain dose range but larger numbers produced a marked inhibition of the response. Spleen cells were less effective in restoring the response. The peritoneal cells were not merely acting as additional, infected "stimulator" or antigen-presenting cells, since they could be added as late as 3 days after culture. Reconstituting activity was not affected by pretreatment with anti-theta serum and complement and cell separation studies showed that the activity was associated mainly with Ig-negative cells and that the active cell probably bears Ia antigens on its surface. These results indicate that the adherent cells involved are probably macrophages and that they act non-specifically to produce optimum conditions for the specific response of T cells.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.     

The in vitro killing of syngeneic cells by peritoneal cells from adjuvant-stimulated mice.

The cytotoxicity of peritoneal exudate cells from mice which had been injected with anaerobic coryneform organisms which have adjuvant activity was assessed by measuring the release of radioactive chromium from monolayers of whole mouse embryo cells. It was found that the peritoneal cells from adjuvant-stimulated mice were more cytotoxic than cells from normal mice. The increased cytotoxicity was present as early as 2 days after injection of the organisms, and was abolished by trypsin treatment of the peritoneal cells. The cytotoxic effect requires the presence of live peritoneal cells, and is more marked as the ratio of effector to target cells is increased. The plastic adherent cells of the peritoneal cell population are more effective in the cytotoxic reaction than are the non-adherent cells. The stimulated peritoneal cells can kill both syngeneic and allogeneic mouse embryo cells. Consideration is given to the possible mechanisms by which the increased cytotoxicity might be induced.     

Cellular interactions in lymphocyte proliferation: effect of syngeneic and xenogeneic macrophages.

Secondary cell-mediated responses to ectromelia virus infection were studied using an in vitro system. Lymphoid “responder” cells from mice which had recovered from intravenous primary infection at various times prior to sacrifice, were cultured with syngeneic, virus-infected macrophages or spleen cells as “stimulator” cells at 39 °C, a temperature which prevented the virus from exerting cytopathic effects against responder cells. This restrictive temperature and medium with 2-mercaptoethanol at 10^?4M often gave viable cell yields of more than 100% of the original responder cells over 4 days of culture. Preliminary experiments showed that spleen cells from primed mice, cultured with syngeneic, infected spleen cells from normal mice gave the most powerful secondary cytotoxic cell responses as measured by ⁵¹Cr release from virusinfected H-2-compatible target cells. The cytotoxic cells were sensitive to anti-θ and complement treatment and lysed H-2-compatible, virus-infected target cells much more efficiently than infected, allogeneic target cells, thus indicating that they were T cells. Some activity against uninfected H-2-compatible target cells was also generated, but this was largely independent of the presence of virus-induced antigen, (i.e. infected stimulator cells were unnecessary) and therefore seemed to be a consequence of the cultural conditions. Cold target competition showed that this activity was the responsibility of a T cell subset separate from the virus-specific cytotoxic T cells. The peak of cytotoxic activity against virus-infected targets occurred at 4 days of culture and DNA synthesis was maximal on day 3. The concentration of cytotoxic T cells at the peak was eight-fold higher than at the peak of the splenic primary response in vivo, Memory T cells (precursors of secondary cytotoxic T cells) appeared in spleen within 12–14 days of primary infection in vivo, reached a plateau at 5–6 weeks and persisted for at least 16 months. Spleen cells appeared partly refractory to secondary stimulation in vitro at 8–10 days post-priming. This did not seem to be due to cellular migration from spleen to lymph nodes or peritoneal cavity, but its cause was not determined. Primary responses in vitro were not detectable under conditions optimal for secondary responses, thus suggesting a major quantitative, or qualitative difference between virgin and memory T cells.     

Generation of cytotoxic T lymphocytes during coxsackievirus B-3 infection. I. Model and viral specificity1.

The production of cytotoxic cells in the spleen of adult male BALB/c mice infected with Coxsackievirus B-3 has been examined.An in vitro 51Cr release assay was used to measure cytotoxic activity against virus-infected and uninfected neonatal sygeneic fibroblasts. Cytotoxicity of immune spleen cells against virus-infected targets was detected on the 3rd day after infection, reached a peak on day 7, and then declined to low levels by days 12 and 14. Spleen cells obtained 3 and 5 days after infection also exerted cytotoxicity against uninfected fibroblasts, but by the 7th day there was little or no reactivity against uninfected target cells, although activity against infected fibroblasts was maximal at this time. Reciprocal assays performed by using Coxsackie and vaccinia viruses provided evidence of virus specificity of the cytotoxic reaction. When spleen cells were obtained 7 days after infection, the Coxsackievirus-immune population was not cytotoxic for vaccinia-infected fibroblasts, and the vaccinia-immune population was not cytotoxic for Coxsackievirus-infected targets, although each immune cell preparation caused significant lysis of fibroblasts infected with the homologous virus. Additional studies showed that primary mouse or hyperimmune rabbit anti-Coxsackieviral serum could not block immune spleen cell cytotoxicity or induce complement-mediated lysis of infected targets. The findings indicate that Coxsackievirus infection results in surface membrane alterations, but no evidence was obtained that antiviral antibody could react with the infected cells.     

Mechanism and specificity of macrophage-mediated cytotoxity.

The cytotoxic effect of macrophages derived from alloimmunized mice (immune macrophages) was found to be immunologically specific. The immune macrophages killed only target macrophages carrying the alloantigens used for immunization in mixed macrophage cultures (MMC) under optimal conditions of contact between effector and target cells. T-sensitized lymphocytes, but not B cells, were capable of arming nonimmune macrophages and conferring upon them cytotoxic activity; the arming factor, which seemed to be a T mediator or T-cell receptor (membrane component) was removable by trypsin. Frequent rinsing or addition of hydrocortisone significantly decreased the cytotoxicity of the MMC. Pretreatment of peritoneal cells with anti-θ antisera and complement markedly decreased immune macrophage cytotoxic activity. It is suggested that the presence of a very small number of T-sensitized lymphocytes is required for strong cytotoxic activity to be manifested by the macrophages.     

Generation of cytotoxic T lymphocytes during coxsackievirus tb-3 infection. II. Characterization of effector cells and demonstration cytotoxicity against viral-infected myofibers1.

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing eyngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished dtheir cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition and in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.     

Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

The cell-mediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo.

The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 × 10² up to 5 × 10⁴ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a ⁵¹Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4–22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5–6 days post-infection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-δ and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days 1–4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.     

Defective spontaneous but normal antibody-dependent cytotoxicity for an extracellular protozoan parasite, Giardia lamblia, by C3H/HeJ mouse macrophages

To understand murine host responses to extracellular protozoa, the capacity of peritoneal macrophages to exhibit cytotoxicity for [3H]thymidine-labeled Giardia lamblia trophozoites was investigated. Resident peritoneal macrophages from C3H/HeN mice expressed spontaneous cytotoxicity for G. lamblia in a manner that was dependent on both time and effector cell number; this cytotoxic activity was increased with cells elicited by an intraperitoneal injection of thio-glycollate. In contrast, spontaneous cytotoxicity for G. lamblia by resident and thioglycollate-elicited peritoneal macrophages from C3H/HeJ mice was markedly reduced. In the presence of anti-G. lamblia serum (ADCC), however, peritoneal macrophages from both C3H/HeN and C3H/HeJ mice exhibited striking augmentation of their cytotoxic activity for G. lamblia to equivalent levels. We conclude that macrophages from C3H/HeJ mice express defective spontaneous cytotoxicity but normal ADCC for the extracellular protozoan parasite, G. lamblia. The dissociation between the expression of these two effector cell functions suggests that macrophage spontaneous cytotoxicity and ADCC for extracellular protozoa are mediated by separate macrophage functions.     

Effect of cAMP modifiers on the cytotoxic activity of macrophages

The effects of various cAMP modifiers on the changes in the intracellular cAMP level and on the coupling of the cAMP system with realization of macrophage cytotoxicity depending on their functional activity were studied. Nonactivated and activated by E. coli polysaccharides peritoneal macrophages of BALB/c mice and macrophage-like cells of J744 mice were incubated in the presence of cAMP modifiers and further assayed for cytolytic and cytostatic activities. Cells of tetraploid strain of Ehrlich carcinoma G10 were used as target cells. Among other modifiers only dibutyryl-cAMP caused a steady increase of the intracellular nucleotide content, whereas methylisobutylxanthine and isoproterenol in combination with methylisobutylxanthine caused only a temporary increase of the cAMP level. Isoproterenol did not induce any appreciable changes in the intracellular cAMP level. All modifiers under study suppressed the cytotoxic activity of macrophages irrespective of the nature of changes in the intracellular cAMP content. It was assumed that cAMP accomplishes a triggering function in the regulation of the cytotoxic activity of macrophages and that the cAMP system is universal in the regulation of cytotoxicity at various functional states of macrophages.     

Cell-mediated cytotoxicity against virus-infected target cells in humans. II. Interferon induction and activation of natural killer cells.

The mechanisms by which human lymphocytes lyse virus-infected allogeneic fibroblast cultures were analyzed with particular consideration of the role of anti-viral antibodies and interferon. Human cells infected with viruses were able to induce high levels of interferon upon contact with human lymphocytes. Interferon, whether produced by lymphocytes after direct infection with virus or induced upon exposure of lymphocytes to virus-infected fibroblasts, appeared to be responsible for enhancing the cytotoxic efficiency of the natural killer cell against the infected target. Activation of cytotoxic lymphocytes occurred as early as 6 hr after addition of interferon and increased up to 24 hr. Antibody-dependent cell-mediated cytotoxicity (Ab-CMC) could be easily induced by sensitization of infected target cells with antiviral antibodies and could be detected at 4 hr from the beginning of the cytotoxic test, before the effect of interferon on the natural killer cell was evident. However, the antibody-dependent effector cell was inactive after 4 hr of incubation. F(ab')2 fragments of rabbit anti-human IgG completely inhibited Ab-CMC but did not at all affect the spontaneous cytotoxic activity of the effector cells against virus-infected target.     

Effects of macrophage activity on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae: an in vitro cytotoxicity and ultrastructural study

The effect of the activity of macrophages on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae was studied in vitro. Macrophages present in peritoneal exudates from mice genetically selected for high and low antibody production (HL and LL, respectively) showed an inverse cytotoxic effect. Cells from HL mice were ineffective, whereas cells from LL mice had a very high killing capacity. Ultrastructural studies of cells after incubations of up to 36 h supported these observations. Furthermore, peritoneal macrophages from congenitally athymic (nu/nu) mice showed a higher killing potential than cells from thymus-bearing littermates (+/nu) mice. The activity of the latter cells could be increased by in vitro pretreatment of the mice with Calmette-Guérin bacillus, a well-known macrophage stimulating agent. The results indicate that macrophages, although not the only effector cells, may play an important role in the defence against T. spiralis newborn larvae.     

Effects of microwave exposure on the hamster immune system. II. Peritoneal macrophage function

Acute exposure to hamsters to microwave energy (2.45 GHz; 25 mW/cm2 for 60 min) resulted in activation of peritoneal macrophages that were significantly more viricidal to vaccinia virus as compared to sham-exposed or normal (minimum-handling) controls. Macrophages from microwave-exposed hamsters became activated as early as 6 h after exposure and remained activated for up to 12 days. The activation of macrophages by microwave exposure paralleled the macrophage activation after vaccinia virus immunization. Activated macrophages from vaccinia-immunized hamsters did not differ in their viricidal activity when the hamsters were microwave- or sham-exposed. Exposure for 60 min at 15 mW/cm2 did not activate the macrophages while 40 mW/cm2 exposure was harmful to some hamsters. Average maximum core temperatures in the exposed (25 mW/cm2) and sham groups were 40.5 degrees C (+/- 0.35 SD) and 38.4 degrees C (+/- 0.5 SD), respectively. In vitro heating of macrophages to 40.5 degrees C was not as effective as in vivo microwave exposure in activating macrophages to the viricidal state. Macrophages from normal, sham-exposed, and microwave-exposed hamsters were not morphologically different, and they all phagocytosed India ink particles. Moreover, immune macrophage cytotoxicity for virus-infected or noninfected target cells was not suppressed in the microwave-irradiated group (25 mW/cm2, 1 h) as compared to sham-exposed controls, indicating that peritoneal macrophages were not functionally suppressed or injured by microwave hyperthermia.     

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Involvement of lymphocyte mediators in the rejection of a murine tumor allograft

Macrophage migration inhibition by peritoneal leukocytes was studied in BALB/c mice bearing intraperitoneal allogeneic EL-4 lymphomas to explore the role of this immune effector function in allograft rejection. The nonadherent peritoneal leukocyte population harvested between 8 and 10 days after allograft inoculation inhibited migration of nonimmune murine macrophages as demonstrated by both direct and indirect migration assays using the agarose droplet method. This host response also contained large numbers of adherent macrophages which others have shown to be cytotoxic to EL-4 target cells. These findings provide direct evidence for lymphokine activity in allograft rejection and suggest that lymphocyte mediators may attract and activate the cytotoxic macrophages observed in this response.     

Secondary cell-mediated cytotoxic response to syngeneic mouse tumor challenge.

When C57BL/6 mice previously immunized with murine sarcoma virus (MSV) were challenged with a Rauscher virus-induced lymphoma, RBL-5, a secondary cell-mediated cytotoxic response could be detected by the Cr release cytotoxicity assay. The level and distribution of the secondary cytotoxic response was affected by the route of challenge. Animals injected i.p. demonstrated a high level of cytotoxicity in the peritoneal exudate cells 3 days after challenge and subsequently cytotoxicity was detected in most lymphoid organs, although at lower levels. However, when the animals were challenged intramuscularly in the leg, the response was not detected as rapidly and furthermore cytotoxic lymphocytes were found only in the draining lymph node and not in other lymphoid organs. Treatment of the effector cells with anti-theta and complement showed the secondary response to be predominately dependent on T cells. In addition, the cytotoxicity was specific in that cells lacking cross-reacting antigens were not killed by these attacker cells from mice undergoing a secondary response.