Action of dexamethasone in the suppression of delayed-type hypersensitivity in reconstituted SCID mice

OBJECTIVE AND DESIGN: It is not known whether the amelioration of inflammation by corticosteroids is mediated through a direct effect on the T cells or by effects on other inflammatory cells and/or their products. To explore the mechanisms whereby corticosteroids exert their effects on T cell dependent inflammation we have used severe combined immunodeficiency (SCID) mice. These mice are largely devoid of functional T and B lymphocytes, hence being unable to raise delayed type hypersensitivity (DTH), but display intact antigen presenting capacity. The aim of this study was to analyse the target cell population participating in DTH and being affected by corticosteroid treatment. MATERIALS AND METHODS: SCID mice were reconstituted with naive thymocytes from congeneic C.B-17 mice. The day after cell transfer, recipient SCID mice were sensitised by epicutaneous application with oxazolone (OXA). One week later all the mice were challenged with OXA and DTH reaction was registered. Recipient SCID mice were administered a single dose of Dexamethasone (Dex) either one day before cell transfer or one day before the challenge. A group of donor mice was treated with Dex prior to cell preparation for transfer. Also intact C57B1/6 (B6) mice were treated with corticosteroids either before sensitisation or before challenge. RESULTS: In B6 mice, a single injection of Dex significantly decreased DTH when given one day before challenge but had no effect when given one day before sensitisation. In SCID mice treated with various doses of Dex one day before challenge, a profound depression (P < 0.01-0.001) of DTH reactivity was observed, compared to controls. In contrast, administration of Dex to recipient SCID mice before cell transfer resulted in an increased rather than decreased DTH response among SCID recipients. SCID mice receiving Dex exposed thymocytes displayed no significant differences in DTH response, compared to the controls. CONCLUSIONS: Our results indicate that corticosteroids exert suppressive impact on the effector phase of DTH response in mice by anti-inflammatory properties of the hormone or by effects on OXA-sensitised memory T cells.     

Factors influencing the enhancement of delayed-type hypersensitivity to ovalbumin by cyclosporin A in the guinea pig: possible role of suppressor cells

Enhancement of delayed-type hypersensitivity (DTH) reactions to ovalbumin (OVA) was demonstrated in guinea pigs given a single, high dose of cyclosporin A (CsA) intraperitoneally, 2 days before immunization. Courses of oral CsA, commencing at the time of immunization and lasting until day 4 or 13 also resulted in augmented DTH responses at days 14 and 28, respectively. However, the enhancing protocol (CsA; day 0-4) did not significantly affect circulating anti-OVA antibody titres. The capacity to express increased DTH could be adoptively transferred to naive recipients by systemic injection pooled spleen and peritoneal exudate cells. Moreover, the expression of augmented responses was inhibited by transfer of cells from normally immunized donors. Although augmentation of DTH was accompanied by increased lymphocyte proliferative responses to OVA in vitro, there was no similar effect on T cell responses to polyclonal mitogens. The data support the view that augmentation of DTH by CsA is attributable to suppressor cell dysfunction, but that this is unlikely to be a non-specific suppressor cell impairment.     

Role of L3T4+ lymphocytes in protective immunity to systemic Candida albicans infection in mice.

Protective immunity to lethal Candida albicans challenge in vivo and activation of splenic macrophages with highly candidacidal activity in vitro were detected in mice infected with low-virulence agerminative yeast cells of the variant strain PCA-2, at a time when a strong delayed-type hypersensitivity (DTH) reaction to C. albicans occurred in the footpads of PCA-2-treated mice. The DTH reaction was transferable with spleen cell populations from these animals, and enrichment of splenic lymphocytes in L3T4+ cells significantly increased the footpad swelling. The reactivity transferred by L3T4+ cells was a radiosensitive (2,500 rads in vitro) phenomenon that required collaboration with radioresistant, silica-sensitive syngeneic cells in the host and was inhibited by treatment of recipient mice with antibodies to the L3T4 antigen or murine gamma interferon. In vitro, the PCA-2-immune L3T4+ cells produced various lymphokine activities upon incubation with C. albicans, including gamma interferon and granulocyte-macrophage colony-stimulating factor. Anti-L3T4 monoclonal antibody treatment of PCA-2-infected mice significantly impaired their footpad reaction and resistance to C. albicans, as shown by increased recovery of yeast cells from the kidneys of anti-L3T4-treated mice. These results suggested that the mechanisms of anti-Candida resistance induced by PCA-2 may involve specific induction of a DTH response mediated by inflammatory L3T4+ T cells and lymphokine-activated phagocytic effectors. However, the survival rate of the PCA-2-immune mice challenged with C. albicans was not significantly modified by administration of the anti-L3T4 antibody, thus allowing for the conclusion that compensatory mechanisms lead to considerable anti-Candida resistance when the activity of L3T4+ cells is deficient.     

L3T4+ T cells able to mediate parasite-specific delayed-type hypersensitivity play a role in the pathology of experimental Chagas' disease

During the chronic phase of infection with the parasite Trypanosoma cruzi, mice develop inflammatory lesions in the heart and skeletal muscles, as well as in peripheral nerves and the liver. We demonstrated the presence, in the blood of chronically infected mice, of L3T4+ T cells able to transfer a specific T. cruzi delayed-type hypersensitivity (DTH) reaction. Transfer of the chronic inflammatory lesions was obtained by injecting Lyt-2+-depleted lymphocytes from either lymph node or blood of infected mice. T cell lines were established from the chronically infected mice by culturing peripheral blood lymphocytes or lymph node cells with either T. cruzi extracts (TC) or mouse peripheral nerve extracts (PN). Those cell lines that presented an L3T4+ phenotype were also able to specifically transfer a local DTH reaction to naive recipients. Examination of the antigen specificities of these TDTH lines revealed three types: those that mediated a DTH reaction to TC, those that responded to both TC and PN and those that provoked a DTH response when injected with subinflammatory doses of an irrelevant antigen. Some of the lines, when injected into the sciatic nerve of naive recipients, provoked demyelination of the type observed in chronically infected animals. These results suggest that T. cruzi and the host nervous system share common epitopes that can be recognized by TDTH cells.     

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.     

Biological properties of factors secreted by antigen-reactive suppressor cells in mice infected with Mycobacterium lepraemurium.

Antigen-reactive cells were isolated from the spleens of Mycobacterium lepraemurium-infected C57BL/6 mice on petri dishes coated with mycobacterial antigens. When adoptively transferred to syngeneic mice, the mycobacterial antigen-reactive cells were found to depress the induction and expression of the delayed-type hypersensitivity (DTH) reaction to M. lepraemurium antigens. The adoptive transfer of soluble suppressor factors (SF) secreted by these cells inhibited only the expression of DTH. The cells depressing the induction of DTH mainly belonged to the L3T4+ (CD4+) T-lymphocyte subset, whereas those depressing its expression differed from the L3T4+ and Lyt-2+ (CD8+) subsets. Treatment of M. lepraemurium-infected mice with SF reduced their mean survival time and enhanced the multiplication of bacilli at the site of infection and their dissemination to the spleen and liver. In vitro at least, SF appeared to interfere at the level of mycobacterial antigen recognition by T lymphocytes rather than at the levels of antigen processing and presentation by macrophages.     

T-enriched spleen cells in delayed-type hypersensitivity to influenza virus in mice

Restimulation in vitro of T-enriched spleen cells from CBA mice with influenza virus A/Bangkok 1/79/H3N2 or its hemagglutinin (HA) leads to enhancement of delayed-type hypersensitivity (DTH) to virus and HA in recipients of transfer. The enhancement of DTH measured by tail swelling is accompanied by 20-fold increase of binding affinity of transferring cells to HA measured by saturation analysis. DTH induced by HA in vivo is weaker than induced by virus in this system. However, when HA is used in vitro as a restimulating antigen of virus primed in vivo lymphocytes it leads to generation of such lymphocytes population which after transfer mediates DTH response to virus or HA to the same level as virus restimulated cells. The increase of binding affinity of restimulated T lymphocytes to HA accompanying the enhancement of DTH activity is considered in the relation to quantitative and qualitative changes of antigen binding cell populations and their role in antiviral response in this systems.     

Contact sensitivity in the murine oral mucosa. I. An experimental model of delayed-type hypersensitivity reactions at mucosal surfaces.

We have examined in a murine model, the potential of the oral mucosa (OM) to serve as inductive and/or expression site(s) of delayed-type hypersensitivity (DTH) reactions. The expression of DTH reactions in the murine buccal mucosa was studied after topical application of oxazolone or picryl chloride onto the OM of animals previously sensitized with either hapten. Irrespective of the site of priming (skin or buccal mucosa), inflammatory cells appeared in the OM following buccal elicitation with the pertinent hapten. The density of infiltrating cells peaked at 24 h after hapten elicitation. Such inflammatory reactions, which comprised mainly mononuclear cells at 24 h, were preceded by an early inflammatory reaction that developed only in animals previously sensitized at skin sites. This early reaction, comprising mainly PMN neutrophils, peaked at 6-8 h, declined by 8-16 h, and was not observed in mice previously sensitized in the buccal mucosa. The 24 h reactions failed to develop in nude mice similarly treated, in intact unsensitized mice, as well as in animals sensitized with an irrelevant hapten. These reactions could be adoptively transferred to naive animals by LN cells but not by serum from sensitized syngeneic donors. Furthermore, LN cell suspensions depleted of T cells failed to transfer sensitization for subsequent OM DTH. Topical application of contact sensitizing haptens onto OM induced priming for subsequent DTH reactions elicited with recall antigen applied at a distant skin site or at a local buccal site. These results demonstrate that the OM has the capacity to serve both as an inductive and as an expression site for T cell-mediated inflammatory reactions, be these expressed or induced at local mucosal sites or at remote systemic (skin) sites. This animal model should be valuable for studying the regulation of T cell-mediated inflammatory responses at mucosal surfaces.     

Difference in the functional maturation of T cells against Listeria monocytogenes in lymph nodes and spleen.

After subcutaneous immunization of mice with viable Listeria monocytogenes (LM), we evaluated the function of T cells induced in draining lymph nodes (LN) and spleen as determined by the local transfer of delayed-type hypersensitivity (DTH), acquired cellular resistance (ACR) and in vitro lymphokine production. LN cells could transfer specifically DTH but not ACR. In contrast, spleen cells from the same donor mice evoked the DTH response as well as bacterial clearance at the reaction site. Comparison of bacterial counts in spleen and in LN upon subcutaneous inoculation of mice with LM suggested that the lack of bacterial proliferation in LN underlay the failure to induce protective T cells in this lymphoid tissue. Spleen and LN T cells expressed CD4 and CD8 surface antigens equally and DTH response was solely dependent on CD4+ cells. Another major difference between LN and spleen cells was the profile of lymphokines produced in vitro. Upon the in vitro restimulation with killed Listeria, immune spleen cells produced macrophage chemotactic factor (MCF), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In contrast, LN cells could produce all of the measured lymphokines but not IFN-gamma. The results provided strong evidence for the dissociation of DTH and ACR. Listerial growth appeared to be the requirement for full maturation of anti-listerial immunity that may coincide with the generation of IFN-gamma-producing T cells.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. II. Inflammatory helper T cells and effector T cells in mice with delayed-type hypersensitivity and in suppressed mice.

Peritoneal exudate cells were induced in mice 4 days after immunization with SRBC. A low dose of SRBC (10(6) i.v.) caused T lymphocytes to appear in inflammatory exudates. These cells, not only transferred DTH reactions, but also functioned as helper T cells in antibody production after transfer to syngeneic nu/nu recipient mice. After a high dose of SRBC (10(9) i.v.), very few helper T cells and no DTH transferring T cells were found in inflammatory exudates, although they were present in the spleen. It is postulated that T cells mediating DTH reactions and helper T cells behave similarly as far as those dose dependency of appearance in inflammatory exudates is concerned. A high dose of sensitizing antigen causes retention of helper and effector T cells in the spleen, in this way favouring antibody formation; low doses of antigen allow them to leave the spleen, thus favouring mediation of DTH reactions in the periphery.     

Inhibition of T lymphocyte heparanase by heparin prevents T cell migration and T cell-mediated immunity

Previously we reported that activated T lymphocytes express a heparanase enzyme that degrades the heparan sulfate moiety of the proteoglycan of the extracellular matrix (ECM). Expression of the heparanase enzyme was found to be associated with the ability of activated T lymphocytes to penetrate blood vessel walls and accumulate in target organs. We recently found that relatively low doses of heparin administered to mice or rats inhibited T cell-mediated immune reactions. In the present study we investigated the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a delayed-type hypersensitivity (DTH) reaction. We found that heparanase was induced by immunizing mice with antigen in vivo or by activating T lymphocytes with concanavalin A in vitro. Relatively low doses of heparin administered once daily in vivo (5 micrograms) or present in vitro (0.1 microgram/ml) inhibited the expression of heparanase induced by immunization or by concanavalin A incubation. Higher or lower doses of heparin did not have these effects. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of the lymph node cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings suggest that modulation of cell-mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.     

Genetic regulation of delayed-type hypersensitivity responses to poly (Tyr,Glu)-poly(DLAla)--poly(Lys): expression of the genetic defect in the induction and manifestation phases in H-2s and H-2f mice.

The genetic defect of H-2s and H-2s non-responder mouse strains in both the induction and manifestation phases of delayed-type hypersensitivity (DTH) responses to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L] was analysed. Utilizing an in vitro system to activate DTH effector T cells, we observed that non-adherent T cells of (H-2f X H-2b) F1 or (H-2s X H-2b)F1 responder mice, could not be activated on antigen bearing adherent cells of H-2f or H-2s haplotypes. On the other hand, these T cells were effectively sensitized on adherent cells derived from either F1 or parental (H-2b) responder mice. These results indicate that in these mouse strains the genetic defect, in the induction phase of DTH, is expressed at the level of the antigen presenting cell. In subsequent experiments, we were able to "correct' the non-responsiveness of H-2s recipients by transfer of educated and irradiated (H-2s X H-2b)F1 T cells together with normal F1 adherent cells. Normal non-adherent and nylon wool enriched T cells failed to restore these responses. Similarly, antigen-pulsed F1 irradiated peritoneal exudate cells could stimulate DTH responses in SJL recipients of (SJL X C57BL/6)F1 (T,G)-A--L educated cells. The genetic defect of H-2s mice in the manifestation phase of the DTH reaction is thus also expressed on the antigen presenting cell.     

Cyclosporin A prevents suppression of delayed-type hypersensitivity in mice immunized with high-dose sheep erythrocytes.

When administered intraperitoneally to mice 2 days before immunization with a tolerogenic dose (10(9)) of sheep red blood cells (SRBC), cyclosporin A (CsA; 200 mg/kg) strikingly augmented 4-day delayed-type hypersensitivity (DTH) footpad reactions. These enhanced responses were similar in magnitude to those seen in animals sensitized with an immunogenic, low-dose (10(6)) SRBC. The stimulatory effect of CsA was observed over the dose range of 5-200 mg/kg and was obtained in animals given the drug in one injection, up to 7 days before sensitization. The augmentation of DTH was characterized by footpad swelling, intense mononuclear cell infiltration and increased deposition of 125I-fibrinogen within the challenge site. In addition, increased expression of procoagulant activity by spleen cells in response to antigen was observed. Cell transfer experiments showed that the CsA-enhanced DTH could be adoptively transferred to naive recipients. Additional transfers conducted at the time of antigen challenge suggested that, under the conditions described, CsA inhibited the action of a population of suppressor cells normally effective during DTH reactions.     

Characterisation of cyclosporine-induced suppressor cells in murine delayed-type hypersensitivity responses

The effect of Cyclosporine A (CsA) on T cell-mediated delayed-type hypersensitivity (DTH) reactions to murine alloantigens was analysed by transferring spleen cells from sensitised and suppressed mice into irradiated naive recipients. Selective removal of Thy1+, Ly1+ or Ly2+ cells prior to intravenous transfer of cells from alloantigen-sensitised mice showed that Ly1+ Thy1+ cells transferred sensitivity. Concomitant treatment of mice with alloantigen and CsA suppressed the DTH response to alloantigens, and spleen cells transferred from these suppressed mice abrogated the response of sensitised cells transferred at the same time. The suppressor cells were strain-restricted and antigen-specific with the surface phenotype Thy1+, Ly1-Ly2+.     

Augmentation of delayed-type hypersensitivity to high dose sheep erythrocytes by cyclosporin A in the mouse: influence of drug dosage and route of administration and analysis of spleen cell populations.

When administered by various routes 48 h before a high systemic dose (10 degrees) of sheep red blood cells (SRBC), Cyclosporin A (CsA) prevented the suppression of delayed-type hypersensitivity (DTH) reactions elicited 4 days later. Augmentation of DTH was observed over a wide range (5-200 mg/kg) and with circulating CsA levels ranging below 45 ng/ml at the time of immunization or antigen challenge. Splenic lymphocytes from vehicle- and CsA-treated mice exhibited good proliferative responses to mitogen in vitro, but only those from CsA-treated animals responded to antigen. Expression of DTH was associated with a progressive, 2-fold increase in the absolute numbers of splenic L3T4+ cells, whereas no significant alteration in the number of Lyt-2+ lymphocytes was recorded. B cell and macrophage numbers in the spleen were unaffected by CsA. In contrast to its potentiating effects on cell-mediated immunity, CsA caused profound (up to 100%) suppression of the concomitant production of splenic anti-SRBC IgM-secreting plasma cells. Circulating anti-SRBC antibody levels were also markedly reduced. These data show that CsA can permit induction of TDTH, whilst suppressing T-dependent humoral immunity and without significant change in absolute numbers of Lyt-2+ cells.     

Increased infiltration by monocytes in delayed type hypersensitivity site following cyclophosphamide treatment.

Sensitized lymphocytes transferred locally with antigens into footpads of unprinted mice can elicit a delayed-type-hypersensitivity (DTH) reaction. Treatment of recipients with cyclophosphamide (CY) increased the infiltration observed at the DTH site. The enlargement of footpads was detected with low doses of DTH mediating cells as with large ones. An enumeration of circulating monocytes performed on the test days and the preceding days has shown a three to ten fold increase of number of monocytes. During the recovery following CY treatment (20 mg/kg and 200 mg/kg) a rebound effect at level of precursors of monocytes was observed. Thus a dual mechanism is proposed to explain the effect of CY on DTH reaction: a liberation of DTH cells by inhibition of B response as previously described; an increased number of monocytes which can be recruited by DTH-cells in site of antigen injection.     

In Vivo Collaboration between Precursor T Cells and Helper T Cells in the Development of Delayed-Type Hypersensitivity Reaction to Influenza Virus in Mice

Nude, athymic mice do not mount a delayed-type hypersensitivity (DTH) response to influenza A virus. A single injection of T helper cells (gamma-irradiated, 2-day immune spleen cells) or three injections over 3 days of a concanavalin-A-activated spleen cell supernatant to virus-sensitized nude mice resulted in a 'normal' DTH response when the mice were challenged with the virus. It was previously shown that the cells responsible for the reaction were T cells and required I-region compatibility. Injection of T helper cells into normal mice did not affect the level of the subsequent DTH response. However, injection of such cells into mice pretreated with anti-thymocyte serum (ATS) restored the ability of the mice to mount a DTH response. The results show that (1) nude mice contain precursor T cells for influenza virus antigen; (2) an I region-restricted response can be generated in the absence of a thymus; and (3) in vivo collaboration between DTH T-cell precursors and helper T cells can be shown to occur in congenitally nude mice and ATS-treated mice.     

Use of a skin test assay to determine tumor-specific CD8+ T cell reactivity

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum^?) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum^? peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8⁺ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2–6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8⁺ anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.     

Administration of anti-type II collagen antibody sustains footpad swelling of mice caused by a delayed-type hypersensitivity reaction and induces severe arthritis

Delayed-type hypersensitivity (DTH) is an immune reaction induced by antigen. In the mice footpads at which DTH is elicited, transient swellings which usually peaks at 24-48 h after the antigen challenge are observed. We found that the footpad swellings of mice are sustained for at least 7 days after the antigen challenge if the mice were injected with anti-type II collagen monoclonal antibody (anti-CII MoAb) before the antigen challenge. A histological section of the swelled hindpaw revealed that severe joint inflammation and bone destruction was induced. These features were not observed in the footpads of the DTH-induced mice. Analysis of the inflammatory reaction induced by both the DTH and the anti-CII MoAb injection, here named as DTH arthritis, revealed the following: (1) DTH arthritis is elicited in an antigen-specific manner; and (2) the development of DTH arthritis is mediated by antigen-specific T cells, especially CD4+ T cells.     

Induction of delayed-type hypersensitivity to Leishmania major and the concomitant acceleration of disease development in progressive murine cutaneous leishmaniasis.

BALB/c mice injected intradermally with 10(5) or higher doses of formaldehyde-fixed promastigotes (FFP) of Leishmania major developed strong delayed-type hypersensitivity (DTH) to leishmanial antigens injected into the hind footpad 3 to 10 days later. The DTH peaked 15 to 18 h after footpad injection and disappeared by 48 h. This specific DTH correlated with the homing of 51Cr-labeled syngeneic bone marrow cells and the infiltration of proliferating cells to the site of antigen administration. Spleen cells from FFP-sensitized mice also gave significant proliferative response to FFP in vitro. The DTH was adoptively transferable by Lyt-1+2-L3T4+ T cells and was H-2 restricted. DTH could be substantially enhanced by pretreatment with cyclophosphamide or pertussigen. Such DTH enhancement was accompanied by concomitant exacerbation of disease progression after L. major infection. Mice injected intravenously with FFP developed substantial immunity to cutaneous leishmaniasis but specifically suppressed DTH reactivity. Treatment of mice with pertussigen before intravenous immunization, however, abolished the protection and reversed the suppression of DTH. These results therefore demonstrate that the early-appearing type of DTH is not involved in host protection but that it actually facilitates disease progression in cutaneous leishmaniasis. Further evidence, which also shows the nonspecific nature of this disease exacerbation, is provided by local cell transfer experiments. Splenic T cells from mice sensitized to keyhole limpet hemocyanin or FFP induced significantly larger lesions compared with normal T cells when they were transferred into the footpad together with specific antigen and L. major promastigotes.