Delayed-type Hypersensitivity Reaction Site Research Articles

Vaccination of cancer patients with dendritic cells electroporated with mRNA encoding the wilms' tumor 1 protein (WT1): correlation of clinical effect and overall survival with T-cell response

Background & Aim Dendritic cells (DC) have the capacity to induce potent tumor antigen-specific T-cell immunity. We have completed vaccination in the adjuvant setting in 77 cancer patients (acute myeloid leukemia (AML, n=30), metastatic breast cancer (MBC, n=12), glioblastoma multiforme (GBM, n=13), malignant pleural mesothelioma (MPM, n=10) and other solid tumors (n=12)) with autologous DC electroporated with mRNA encoding the nearly universal tumor-associated antigen Wilms’ tumor 1 protein (WT1). Methods, Results & Conclusion WT1-targeted DC vaccination was feasible and safe in all patients. The majority of the patients showed a positive delayed type hypersensitivity (DTH) response to the vaccine. Objective clinical responses were observed among all tumor types, including complete (CR) and/or molecular remissions or stable disease (SD) in the AML group, partial responses (PR) and SD in the GBM group and SD in the MBC and MPM groups. In AML patients in first CR, median overall survival (OS) calculated from time of diagnosis was 56.1 months (mo). Median OS from time of diagnosis of metastasis was 41.9 mo in MBC, 43.7 mo from diagnosis in GBM and 35.7 mo from start of therapy in MPM; this compares favorably to numbers reported in the literature, respectively 24.8 mo, 14.7 mo and 22 mo. In AML, long-term OS was correlated with WT1-specific polyfunctional CD8+ T-cells in the DTH reaction sites and long-term CR with polyepitope WT1-specific tetramer+ CD8+ T-lymphocytes. In solid tumors, PR or SD was correlated with interferon (IFN)-gamma+ and/or tumor necrosis factor (TNF)-alpha+ WT1-specific CD4+ and/or CD8+ T-cells; increased OS in the GBM+MPM cohorts was correlated with IFN-gamma+ WT1-specific CD4+ T-lymphocytes. In conclusion, WT1-targeted DC vaccination is feasible, safe and immunogenic, and displays relevant anti-tumor activity in patients with hematological and solid malignancies. Most importantly, this treatment modality can confer a significant survival benefit to the patients.

T cell sensitivity to TGF- is required for the effector function but not the generation of splenic CD8+ regulatory T cells induced via the injection of antigen into the anterior chamber

The introduction of antigen into the anterior chamber (AC) of the eye induces the production of antigen-specific splenic CD8+ regulatory T cells (AC-SPL cells) that suppress a delayed-type hypersensitivity (DTH) reaction in immunized mice. Because the generation of these regulatory T cells is also induced by exposure to transforming growth factor (TGF)-β and antigen or F4/80+ cells exposed to TGF-β and antigen in vitro, we investigated (i) whether these cells are produced in dominant negative receptor for transforming growth factor β receptor type II (dnTGFβRII) or Cbl-b−/− mice whose T cells are resistant to TGF-β, (ii) whether DTH is suppressed by wild type (WT) CD8+ AC-SPL cells in Cbl-b−/− and dnTGFβRII mice and (iii) the effect of antibodies to TGF-β on the suppression of DTH by CD8+ AC-SPL cells. DnTGFβRII immunized and Cbl-b−/− mice produced splenic CD8+ regulatory cells after the intracameral injection of antigen and immunization. The suppression of a DTH reaction by CD8+ AC-SPL cells in WT mice was blocked by the local inclusion of antibodies to TGF-β when WT splenic CD8+ AC-SPL cells were injected into the DTH reaction site. Moreover, the DTH reaction in immunized dnTGFβRII and Cbl-b−/− mice was not suppressed by the transfer of WT CD8+ AC-SPL cells to the site challenged with antigen. In aggregate, these observations suggest that T cell sensitivity to TGF-β is not an obligate requirement for the in vivo induction of CD8+ AC-SPL T cells but the suppression of an in vivo DTH reaction by CD8+ AC-SPL cells is dependent on TGF-β.

Cellular Players and Role of Selectin Ligands in Leukocyte Recruitment in a T-Cell-Initiated Delayed-Type Hypersensitivity Reaction

Delayed-type hypersensitivity (DTH) reactions are characterized by a strong cellular infiltrate, including neutrophils, macrophages, and T lymphocytes. In all these cell types, both E- and P-selectin-dependent adhesion pathways play a significant role in recruitment into the inflamed skin. Accordingly, inhibition of selectin-mediated interactions (eg, by antibodies) results in impairment of acute DTH reactions. However, whether inhibition of a specific cell type is responsible for the anti-inflammatory effect or whether all leukocytes are affected remains unclear. To address this question, we used fucosyltransferase-VII knockout mice that lack functional selectin ligands as either donors or recipients in a DTH model elicited by Th1 cell and antigen transfer. We found that selectin-mediated adhesion is required by Th1 effector cells to enter the DTH reaction site and, additionally, to elicit the DTH reaction. On the other hand, elimination of selectin binding in the recipient's neutrophils and macrophages by use of fucosyltransferase-deficient mice receiving wild-type Th1 effector cells resulted in a strongly reduced infiltration of neutrophils and macrophages but unimpaired footpad swelling. These findings demonstrate a major role for both E- and P-selectin in the recruitment of different leukocyte cell types. However, only the presence of selectin ligands on T cells was critical for the inflammatory reaction. These findings reveal T cells as the predominant targets for selectin blockade that aim to suppress skin inflammation.

Vaccination of colorectal cancer patients with carcinoembryonic antigen peptide-loaded dendritic cells

2544 Dendritic cells (DC) are the most potent antigen presenting cells of the immune system, capable of inducing an effective anti-tumor immune response. They can be cultured in large quantities from peripheral blood monocytes from cancer patients, loaded with tumor antigen ex vivo and subsequently reinjected into the patients. Currently DC are employed by us and others in clinical vaccination protocols and objective clinical and immunological responses have been observed. Recent investigations indicate that carcinoembryonic antigen (CEA) is an effective target for DC-based immunotherapy. However, very little data exist concerning the induction of an in vivo immune response, in colorectal cancer patients. We have initiated a clinical study with keyhole limpet hemocyanin (KLH) and CEA-peptide loaded DC in HLA-A2.1 positive patients with resectable liver metastases of recurrent colorectal cancer. Patients are vaccinated 3 times intravenously and intradermally with autologous CEA-loaded mature DC prior to surgical resection of the metastases. Immuno-monitoring consists of T cell responses in peripheral blood, delayed type hypersensitivity (DTH) reactions, and in resected tumor and lymph node material. To date, 7 patients have completed vaccination. Treatment was well tolerated with minor side effects. Vaccination resulted in an antibody response and a proliferative T cell response against KLH in all tested patients. Furthermore, CEA-peptide specific T cells were observed in T cell cultures from positive DTH reaction sites in 5 pts. These T cells were not detected in biopsy specimens of the first vaccination site, indicating a de novo induction. CEA-tetramer positive T cells were also detected in blood (1 pt) and abdominal lymph nodes (1 pt) obtained after vaccination. Together, these results demonstrate that CEA-pulsed dendritic cells are capable of inducing specific immune reactions in colorectal cancer patients. Our previous results have shown that specific T cell responses in DTH infiltrating T lymphocytes in stage IV melanoma patients were correlated with survival (de Vries et al. submitted for publication). Therefore these observations warrant further confirmation in our ongoing study. No significant financial relationships to disclose.

Intramuscular injection of antigens and adjuvant preferentially decreases 18:2n-6 and 18:3n-3 in pig neck muscle.

Linoleic (18:2n-6) and alpha-linolenic acids (18:3n-3) have many important physiological functions including immunomodulation. We tested how immunization influences the metabolism of 18:2n-6 and 18:3n-3 in the neck muscle of pigs. At 35 d old, pigs received either an intramuscular neck injection containing hen egg white lysozyme (HEWL), killed Mycobacterium tuberculosis, and Freund's complete adjuvant (immunized) or PBS (control). At 49 d old, immunized pigs received a booster injection of HEWL and Freund's incomplete adjuvant, and the control pigs received PBS into the neck. At 56 d old, all pigs received an intradermal injection of Mycobacterium bovis into the hind leg to induce a delayed-type hypersensitivity (DTH) reaction. At 57 d old, immunized pigs had a twofold increase in serum haptoglobin, a 10-fold increase in antibodies to HEWL, and the skinfold at the DTH reaction site was 10 times thicker than the controls. Both 18:2n-6 and 18:3n-3 (% composition) were approximately 25% lower in muscle TG, 40% lower in FFA, 50% lower in phospholipids, but not different in cholesteryl esters of the neck muscle of immunized pigs. The antigens in this model induce an increased response in the innate (haptoglobin), humoral (antibodies), and cellular (DTH) immune systems as well as a preferential decrease of 18:2n-6 and 18:3n-3 in the inflamed neck muscle. It appears that 18:2n-6 and 18:3n-3 are preferentially metabolized (possibly beta-oxidized) in response to antigens.

Effects of tumor necrosis factor alpha on dendritic cell accumulation in lymph nodes draining the immunization site and the impact on the anticryptococcal cell-mediated immune response.

Cell-mediated immune (CMI) responses and tumor necrosis factor alpha (TNF-alpha) have been shown to be essential in acquired protection against Cryptococcus neoformans. Induction of a protective anticryptococcal CMI response includes increases in dendritic cells (DC) and activated CD4(+) T cells in draining lymph nodes (DLN). During the expression phase, activated CD4(+) T cells accumulate at a peripheral site where cryptococcal antigen is injected, resulting in a classical delayed-type hypersensitivity (DTH) reaction. Induction of a nonprotective anticryptococcal CMI response results in no significant increases in the numbers of DC or activated CD4(+) T cells in DLN. This study focuses on examining the role of TNF-alpha in induction of protective and nonprotective anticryptococcal CMI responses. We found that neutralization of TNF-alpha at the time of immunization with the protective immunogen (i) reduces the numbers of Langerhans cells, myeloid and lymphoid DC, and activated CD4(+) T cells in DLN and (ii) diminishes the total numbers of cells, the numbers of activated CD4(+) T cells, and amount of gamma interferon at the DTH reaction site. Although TNF-alpha neutralization during induction of the nonprotective CMI response had little effect on cellular and cytokine parameters measured, it did cause a reduction in footpad swelling when mice received challenge in the footpad. Our findings show that TNF-alpha functions during induction of the protective CMI response by influencing the accumulation of all three DC subsets into DLN. Without antigen stimulated DC in DLN, activated CD4(+) T cells are not induced and thus not available for the expression phase of the CMI response.

Differences in components at delayed-type hypersensitivity reaction sites in mice immunized with either a protective or a nonprotective immunogen of Cryptococcus neoformans.

Cell-mediated immunity is the major protective mechanism against Cryptococcus neoformans. Delayed swelling reactions, i.e., delayed-type hypersensitivity (DTH), in response to an intradermal injection of specific antigen are used as a means of detecting a cell-mediated immune (CMI) response to the antigen. We have found previously that the presence of an anticryptococcal DTH response in mice is not always indicative of protection against a cryptococcal infection. Using one immunogen that induces a protective anticryptococcal CMI response and one that induces a nonprotective response, we have shown that mice immunized with the protective immunogen undergo a classical DTH response characterized by mononuclear cell and neutrophil infiltrates and the presence of gamma interferon and NO. In contrast, immunization with the nonprotective immunogen results in an influx of primarily neutrophils and production of tumor necrosis factor alpha (TNF-alpha) at the DTH reaction site. Even when the anticryptococcal DTH response was augmented by blocking the down-regulator, CTLA-4 (CD152), on T cells in the mice given the nonprotective immunogen, the main leukocyte population infiltrating the DTH reaction site is the neutrophil. Although TNF-alpha is increased at the DTH reaction site in mice immunized with the nonprotective immunogen, it is unlikely that TNF-alpha activates the neutrophils, because the density of TNF receptors on the neutrophils is reduced below control levels. Uncoupling of DTH reactivity and protection has been demonstrated in other infectious-disease models; however, the mechanisms differ from our model. These findings stress the importance of defining the cascade of events occurring in response to various immunogens and establishing the relationships between protection and DTH reactions.

Dendritic cells in the induction of protective and nonprotective anticryptococcal cell-mediated immune responses.

Dendritic cells (DC) can be divided into three subsets, Langerhans cells, myeloid DC (MDC), and lymphoid DC (LDC), based upon phenotypic and functional differences. We hypothesized that different DC subsets are associated with the development of protective vs nonprotective cell-mediated immune (CMI) responses against the fungal pathogen, Cryptococcus neoformans. To test this, mice were immunized with protective and/or nonprotective immunogens, and DC subsets in draining lymph nodes were assessed by flow cytometry. The protective immunogen (cryptococcal culture filtrate Ag-CFA), in contrast to the nonprotective immunogen (heat-killed cryptococci-CFA), the nonprotective immunogen mixed with the protective immunogen (cryptococcal culture filtrate Ag + heat-killed cryptococci-CFA), or controls, stimulated significant increases in total leukocytes, Langerhans cells, MDC, LDC, and activated CD4+ T cells in draining lymph nodes. The protective immune response resulted in significantly increased levels of anticryptococcal delayed-type hypersensitivity reactivity and activated CD4+ T cells at the delayed-type hypersensitivity reaction site. Draining lymph node LDC:MDC ratios induced by the protective immunogen were significantly lower than the ratios induced by either immunization in which the nonprotective immunogen was present. In contrast, mice given the nonprotective immunogen had LDC:MDC ratios similar to those of naive mice. Our data indicate that lymph node Langerhans cells and MDC are APC needed for induction of the protective response. The predominance of LDC in mice undergoing nonprotective responses suggests that lymph node LDC, like splenic LDC, are negative regulators of CMI responses. In addition to showing DC subsets associated with functional differences, our data suggest that the LDC:MDC balance, which can be modulated by the Ag, determines whether protective or nonprotective anticryptococcal CMI responses develop.

Role of the C-C Chemokine, TCA3, in the Protective Anticryptococcal Cell-Mediated Immune Response

Activated T lymphocytes play a crucial role in orchestrating cellular infiltration during a cell-mediated immune (CMI) reaction. TCA3, a C-C chemokine, is produced by Ag-activated T cells and is chemotactic for neutrophils and macrophages, two cell types in a murine CMI reaction. Using a gelatin sponge model for delayed-type hypersensitivity (DTH), we show that TCA3 is a component of the expression phase of an anticryptococcal CMI response in mice. TCA3 mRNA levels are augmented in anticryptococcal DTH reactions at the same time peak influxes of neutrophils and lymphocytes are observed. Neutralization of TCA3 in immunized mice results in reduced numbers of neutrophils and lymphocytes at DTH reaction sites. However, when rTCA3 is injected into sponges in naive mice, only neutrophils are attracted into the sponges, indicating TCA3 is chemotactic for neutrophils, but not lymphocytes. We show that TCA3 is indirectly attracting lymphocytes into DTH-reactive sponges by affecting at least one other chemokine that is chemotactic for lymphocytes. Of the two lymphocyte-attracting chemokines assessed, monocyte-chemotactic protein-1 and macrophage-inflammatory protein-1alpha (MIP-1alpha), only MIP-1alpha was reduced when TCA3 was neutralized, indicating that TCA3 affects the levels of MIP-1alpha, which attracts lymphocytes into the sponges. TCA3 also plays a role in protection against Cryptococcus neoformans in the lungs and brains of infected mice, as evidenced by the fact that neutralization of TCA3 results in increased C. neoformans CFU in those two organs.

T‐cell receptor CDR3 size distribution analysis to evaluate specific T‐cell response to cancer vaccines

To evaluate immunization procedures in cancer patients, it is important to define which biological parameters reflecting a specific immune response to the vaccine should be followed. One of these may be the recruitment or expansion of clonally amplified T-cell subpopulations previously primed by tumor-specific antigen that could be detected in tiny samples by CDR3 length analysis of T-cell receptor Vβ transcripts. To evaluate this procedure, we studied one patient with metastatic colorectal adenocarcinoma who had received 4 intradermal injections of irradiated autologous tumor cells plus IL-2 on days 1, 8, 15 and 36. Skin tests for delayed type hypersensitivity (DTH) reaction to irradiated autologous tumor cells were performed on days 1 and 43. Although no change was observed in day 43 PBMCs, some recurrent transcripts were detected with similar CDR3 size patterns in both vaccine and DTH sites. Fine analysis of these Vβ-Cβ PCR products with Jβ primers confirmed that transcripts with similar length were recruited in both vaccine and DTH sites. Induction of an inflammatory response in both DTH and vaccine sites may therefore be associated with the recruitment of few T-cell clonotypes. In addition, a Vβ15-Jβ2.5 transcript similar to those detected in vaccine and DTH sites was also identified in enzymatically dissociated tumor cells and in a tumor fragment. Sequencing confirmed that an identical junctional sequence was shared by Vβ15-Jβ2.5 transcripts from both DTH and tumor samples. Our results indicate that a T-cell clone similar to the one amplified in the tumor was recruited at the vaccine and DTH reaction sites. We therefore suggest that such an approach would be useful in assessing specific expansion of T-cell clones induced by cancer vaccines. Int. J. Cancer 71: 972-977, 1997. © 1997 Wiley-Liss Inc.

MIP-1 alpha contributes to the anticryptococcal delayed-type hypersensitivity reaction and protection against Cryptococcus neoformans.

Leukocyte infiltration into infected tissues is essential for the clearance of microorganisms. In animals with a cell-mediated immune (CMI) response to the infectious agent, as opposed to naive animals, leukocyte migration is greatly enhanced into sites of the organism or antigen. The role of the,chemotactic cytokine or chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), in the expression phase of the CMI response and in protection against Cryptococcus neoformans was assessed. With the use of a gelatin sponge model in mice as a means of detecting an anti-cryptococcal delayed-type hypersensitivity (DTH) reaction, we found that MIP-1 alpha levels in fluids from cryptococcal antigen (CneF)-injected sponges in immunized mice (DTH-reactive sponges) were significantly increased over levels of MIP-1 alpha in fluids from saline-injected control sponges at 12 and 24-30 h after injection. MIP-1 alpha levels peaked before increases in neutrophils and lymphocytes in the DTH-reactive sponges, suggesting that MIP-1 alpha was responsible, at least in part, for attracting these leukocyte types. Immunized mice treated with neutralizing antibody to MIP-1 alpha before sponge injection with CneF had reduced numbers of neutrophils and lymphocytes in the DTH-reactive sponges and showed reduced clearance of C. neoformans from the lungs, spleens, livers, and brains when compared with controls. Furthermore, injection of rmMIP-1 alpha into sponges in naive mice resulted in an increase in the influx of neutrophils and lymphocytes into the sponges compared with saline-injected sponges. Together our findings provide solid evidence that MIP-1 alpha is a component of the anticryptococcal DTH reaction. In addition, MIP-1 alpha influences neutrophil influx and attracts lymphocytes into the DTH reaction site. Finally, we showed that MIP-1 alpha plays a role in protection against C. neoformans.

Role of Macrophage Tissue Factor in the Development of the Delayed Hypersensitivity Reaction in Monkey Skin

Delayed-type hypersensitivity (DTH) reaction is a cell-mediated immune response, characterized by fibrin deposition. To determine whether macrophage tissue factor (TF), an initiator of blood coagulation, was associated with the DTH reaction, TF expression on macrophages and resulting fibrin deposition at the site of DTH, induced in Bacille-Calmette-Guerin (BCG)-sensitized Japanese monkeys with intradermal administration of pure protein derivative of tuberculin (PPD), were examined immunohistochemically using monoclonal antibodies (MoAbs) against TF and fibrin. Most mononuclear cells, but not polymorphonuclear cells, in DTH reaction sites were TF-positive and were identified as macrophages by double-immunostaining using anti-TF and anti-macrophage MoAbs. Fibrin deposition was only observed around TF-positive macrophages in the stroma of DTH reaction sites and the deposition was not seen in the periphery of other TF-positive tissues (epidermis, trichoepithelium, and blood vessels). The development of induration and the extent of fibrin deposition paralleled an increase of TF-positive macrophages in the DTH reaction sites. In PPD-injected skin sites of nonsensitized monkeys, neither TF-positive macrophages nor fibrin were seen but epidermis, trichoepithelium, and blood vessels were TF-positive. Moreover, the development of induration was inhibited by simultaneous administration of anti-TF MoAb and PPD into BCG-sensitized monkeys. These results suggest that TF expression on macrophages in the reaction site is a key factor for the DTH reaction, through the activation of blood coagulation, resulting in fibrin deposition.

Evaluation of the antigen specificity of the cellular infiltrate at the delayed type hypersensitivity reaction site with the use of an antigen-specific T cell clone

Antigen specific T lymphocytes in delayed type hypersensitivity (DTH) test sites were analyzed in cell-transfer experiments. T lymphocytes from B6 mice were transferred into B6PL mice which differ from B6 at the Thyl allele. When peritoneal exudate cells (PEC) from ovalbumin (OA)-immunized B6 mice were transferred into B6PL mice intravenously, DTH reaction was observed on footpads. A greater number of B6 T cells was identified at the DTH site. OA-reacting B6 T cell clones were then transferred with antigens into the footpads of B6PL mice. DTH reaction was induced at the footpads. More T cell clones were identified at the DTH test sites than in the control.

Effects of pertussis toxin (PT) on T-cell populations sensitized for delayed-type hypersensitivity in mice

Effects of pertussis toxin (PT) on sensitized T-cell populations for delayed-type hypersensitivity (DTH) were examined in mice. DTH was induced by sensitizing mice with ovalbumin (OA) and elicited by injecting OA into the footpad. DTH could be conferred on naive recipient mice by injecting sensitized spleen cells either intravenously into mice or locally into the footpad. When the sensitized mice were given PT at the time of DTH-elicitation, they did not express a high DTH reaction, with the maximum reaction 24 hr after elicitation. When the recipient mice were given PT just before intravenous injection of sensitized spleen cells, DTH was not conferred. In addition, when the sensitized spleen cells were treated with PT in vitro and then transferred intravenously, DTH was not conferred in recipient mice. However, DTH was conferred by local transfer of the sensitized spleen cells even after treatment with PT in vitro. Migration experiments using 51Cr-labeled, sensitized splenic T cells demonstrated that PT treatment of the T-cell population inhibited its accumulation in the DTH reaction site 24 hr after intravenous transfer. On the other hand, experiments on in vitro lymphokine production by the sensitized splenic T cells demonstrated that the PT treatment did not inhibit antigen-dependent production of a macrophage activating factor (MAF). These results suggest that PT suppresses the migration of the sensitized T-cell population from the circulation to the DTH reaction site but not their MAF production. Based on these findings, possible mechanisms by which PT affects DTH are discussed.

A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity

Many drugs and other chemicals can alter cell-mediated immunity (CMI), a response that often correlates with delayed-type hypersensitivity (DTH). Several DTH assays were evaluated to determine a method best suited for assessing chemically induced modulation of CMI in rats. The effects of various antigens, adjuvants, doses, routes, and immunosuppressants were investigated. The DTH method which produced optimum results in rats uses a footpad swelling reaction elicited by specific preparations of bovine serum albumin (BSA) and Freund's complete adjuvant (FCA). The rats were sensitized with 100 μg BSA in FCA injected subcutaneously at the base of the tail, and were challenged 7 days later with 75 μl of 2% heat-aggregated BSA suspension injected into the left rear footpad. Footpad swelling was measured with pressure calipers 24 h later and compared to the contralateral footpad which was sham-injected with 75 μl of physiological saline. Antigen-injected footpads were nearly double the thickness (7–8 mm) of the control footpads (3–4 mm), and variation between animals was small (CV = 5%). Dexamethasone and cyclophosphamide significantly suppressed the DTH reaction. Histopathological examination of the DTH reaction sites revealed a mononuclear cell infiltrate which is characteristic of type IV hypersensitivity. In addition to being highly quantitative and sensitive, this method provides a simple and reproducible assessment of CMI responses in the rat.

Role of complement in the expression of delayed-type hypersensitivity in rats: studies with cobra venom factor

The hypothesis was tested that delayed-type hypersensitivity (DTH) to the complement-activating bacterium Listeria monocytogenes is initiated by complement-derived mediators that attract sensitized lymphocytes to reaction sites. To this end DTH and acquired resistance to L. monocytogenes were measured in rats injected with cobra venom factor, a potent inactivator of C3. Treatment with cobra venom factor reduced the hemolytic power of serum to less than 0.5% of the control value. Such decomplemented animals expressed both DTH and antimicrobial resistance, although expression of DTH was reduced (ca. 50%) when compared with complement-sufficient controls. The observed depression of DTH in cobra venom factor-treated rats was associated with a reduction in the number of recently activated lymphocytes (lymphoblasts) and macrophages that accumulated in DTH reaction sites. The above findings are explained, in part, by inhibition of inflammation during the early postinduction period. Supporting evidence came from measurements of labeled lymphoblast sequestration in saline injection sites and the slower accumulation of macrophages in nitrocellulose filters that were implanted subcutaneously in complement-depleted rats. The ability of cobra venom factor-treated rats to express DTH and protect themselves against a Listeria challenge seems to exclude C3-dependent factors as essential mediators in the attraction of antigen-reactive lymphocytes to reaction sites.

Requirement for vasoactive amines for production of delayed-type hypersensitvity skin reactions.

The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5-hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice. We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell. Spleen cells of immune, reserpine-treated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice. We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines. If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate. The secondarily arriving vasoactive amine-dependent cells are responsible for the micro- and macroscopic lesions of DTH reactions. Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called.