Characterization of two different Ly-1+ T cell populations that mediate delayed-type hypersensitivity

This paper describes two functionally different T cell populations that mediate delayed-type hypersensitivity (DTH) reactions in contact-sensitized mice. Both of these T cells are Ly-1+, Qa-2-, and Vicia villosa lectin nonadherent. One of these T cell subpopulations is responsible for the classical 24- to 48-hr component of DTH reactions, is induced 3 to 4 days after immunization, is H-2 restricted, is sensitive to irradiation and to antigen-specific T cell-derived suppressor factors, and is found in nylon wool-nonadherent as well as nylon wool-adherent populations. In contrast, the T cell population that is responsible, via an antigen-specific T cell factor, for a recently described early component of DTH, which is an obligatory initial step for expression of DTH, is induced within 24 hr after immunization, requires much less antigen for immunization, is not H-2 restricted, is not sensitive to irradiation nor to T suppressor factors, and is found exclusively in the nylon wool-nonadherent fraction. These results support a new formulation of DTH. According to this formulation, Ly-1+ T cells produce an antigen-specific, tissue-sensitizing, mast cell-activating factor, and via this factor induce the early component of DTH, which is an obligatory first step in which local antigen challenge induces increased local vascular permeability. This required opening of gaps between endothelial cells is due to T cell factor-dependent release of the vasoactive amine serotonin from cells such as mast cells. This first step allows the second, H-2-restricted, Ly-1+ T cell population to enter the reaction site, and to then be triggered by antigen to release lymphokines that attract the subsequent influx of blood-borne, bone marrow-derived leukocytes to constitute the classical delayed-in-time component of DTH reactions.     

Properties of cloned T cells that mediate delayed-type hypersensitivity against ovalbumin in mice

Cloned T-cell lines that mediate delayed-type hypersensitivity (DTH) against soluble protein antigen, ovalbumin (OA), were established in (C57BL/6 X DBA/2)F1 mice and their properties were examined. They induced antigen-specific delayed-type footpad reactions, characterized histologically by a predominant mononuclear cell infiltration, when transferred intravenously into syngeneic mice. Morphologically, they were medium or large lymphoblasts with granules in the cytoplasm and expressed Lyt 1 cell surface antigens. One of them proliferated antigen specifically under the presence of both C57BL/6 and F1 accessory cells, while others proliferated antigen specifically only under the presence of F1 accessory cells. They also produced macrophage-activating factor (MAF) and substances which mediate a DTH-like footpad inflammatory reaction with a maximum 6 hr after injection into the footpad of normal mice, when incubated in the presence of specific antigen and specific accessory cells in a serum-free medium for 24 hr. These results demonstrate that cloned DTH-effector T cells, established here against soluble protein antigen, are Lyt 1-positive, large lymphoblasts and that they produce MAF and footpad-reactive inflammatory substances antigen specifically under the presence of specific accessory cells.     

The in vitro induction of T cells which mediate delayed-type hypersensitivity toward horse red blood cells.

Humoral and cell-mediated immunity to the antigen horse red blood cells (HRBC) were induced in vitro. The type of immune response induced, however, was dependent on the concentration of antigen present in the culture. Whereas intermediate concentrations of HRBC induced antibody-forming cells, high and low concentrations of HRBC induced T cells which, on transfer, mediated delayed-type hypersensitivity reactions. The inverse relationship between humoral and cell-mediated immunity often observed in vivo is, therefore, also evident when lymphocytes are stimulated with antigen in vitro.     

Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2

The 29-kDa subtilase homologue, Tri r 2, derived from the dermatophyte fungus Trichophyton rubrum, exhibits unique immunologic characteristics in its ability to elicit immediate (IH) and delayed-type (DTH) hypersensitivity skin tests in different individuals. Thus, Tri r 2 provides a model for comparing the T cell repertoire in subjects with distinct immune responses to a single Ag. Recombinant Tri r 2 produced as a GST fusion protein in Escherichia coli stimulated strong in vitro lymphoproliferative responses in 10 IH and 10 DTH responders. Patterns of T cell epitope recognition were compared between skin test groups using 28 overlapping peptides (each in 12 replicate wells) derived from Tri r 2 to stimulate T lymphocyte proliferation in vitro. Peptide 5 (P5; aa 41-60) induced the strongest response in DTH subjects and showed the largest difference between DTH and IH responders in proliferation (mean standardized index, 2.22 and 0.82, respectively; p = 0.0047) and number of positive wells (81 vs 12). Responses to P5 were associated with diverse HLA haplotypes. These results showed that P5 contains an immunodominant epitope specifically associated with DTH and that this peptide is recognized in a permissive manner. Cross-validated linear discriminant analysis using T cell proliferative responses to two regions of Tri r 2 (aa 51-90 and 231-270) gave a 95% predictive accuracy for classification of subjects into IH or DTH groups. We conclude that different immune responses to Trichophyton are mediated by distinct T cell repertoires between individuals with IH and DTH reactions to Tri r 2.     

Regulation of delayed-type hypersensitivity reactions by cyclophosphamide-sensitive T cells.

The onset, intensity, and duration of DTH reactions elicited in mice immunized with either SRBC or products of the major histocompatibility complex can be altered significantly by pretreatment with CY 1 to 2 days before immunization. Such drug pretreatment tends to augment low DTH responses caused by the use of too much antigen and to diminish many responses that are optimal. Thus, pretreatment with CY does not specifically eliminate suppressor cells. Our results are most consistent with the notion that the cellular targets of low doses of CY are positive and negative feedback regulatory cells, which may consist of one population with two effects or, more likely, two distinct cell populations.     

Induction of delayed-type hypersensitivity by the T cell line specific to bacterial peptidoglycans

A T cell line specific for the chemically well-defined peptidoglycan of bacterial cell wall, disaccharide tetrapeptide, was established from Lewis rats immunized with the antigen covalently linked to the autologous rat serum albumin. The antigen specificity was examined with various analogues or derivatives of the peptidoglycan. The cell line was reactive to analogues with the COOH-terminal D-amino acid, but least reactive to those with L-amino acid as COOH terminus. Transferring of the T cell line into X-irradiated normal Lewis rats induced delayed-type hypersensitivity in an antigen specific manner.     

Regulatory action of immune B cells on delayed-type hypersensitivity in mice

Suppressor cells in delayed-type hypersensitivity (DTH) to soluble protein antigens were induced in vitro from BALB/c spleen cells. Transfer of these cells into syngeneic recipients resulted in suppression of the hosts' DTH responses in an antigen-specific manner. These suppressor cells were characterized as B cells by their adherence to nylon-wool columns, resistance to treatment of anti-Thy 1, -Ly 1, and -Ly 2 antibodies plus complement, adherence to anti-mouse immunoglobulin-coated dishes, and nonadherence to uncoated plastic dishes. In addition to being radiation sensitive, these suppressor B cells showed the capability of binding to the primed antigen. Thus, it was demonstrated that our in vitro-induced suppressor cells were antigen-specific B cells. When these suppressor B cells were transferred into the recipients, serum titers of specific antibodies were elevated and effector phase suppressor T cells were induced in the recipients. These results suggest that suppressor B cells exert their suppressor activity through the idiotype-anti-idiotype network.     

B-1 B cell development

CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

Flare-up of delayed-type hypersensitivity initially induced by murine cloned helper T cells

Delayed-type hypersensitivity (DTH) reactions were induced in mice by cloned helper T cells directed against methylated bovine serum albumin (mBSA). The DTH reactions were induced either by local injection of the helper T cells together with the antigen in the hind feet or by intravenous (iv) administration of the cloned T cells and local injection of the antigen. Local or systemic (oral or iv) administration of mBSA after waning of the DTH induced by the cloned helper T cells caused a flare-up reaction. This indicates that functional helper T cells persist at the inflammation site. The inflammations were quantified in a foot swelling assay and were examined histologically. The inflammation measured in the flare-up reaction was generally lower than in the acute reaction. Histologically the acute inflammation showed edema and a large proportion of granulocytes, whereas the flare-up reaction appeared more histiocytic and showed less edema.     

B and T lymphocytes regulated by idiotype anti-idiotype interactions inhibit delayed-type hypersensitivity to BCG in mice

Mice infected subcutaneously with 2 X 10(7) CFU of Mycobacterium bovis strain BCG (BCG) were able to mount a specific DTH response, whereas mice infected intravenously with the same dose of microorganisms were not. The suppression turned out to be mediated by id+ anti-PPD B lymphocytes, which arose very early during the infectious process and induced anti-id B lymphocytes. These cells were found at Day 4 after infection and exerted their effect by activating antigen-specific suppressor T lymphocytes, which affected the efferent phase of the DTH response. These results clearly indicate that the activation of a complex immunosuppressive circuit represents a mechanism by which BCG may interfere with the host's immune response already during the very early phases of infection.     

Inhibition of the delayed-type hypersensitivity response by staphylococcal enterotoxin B-induced suppressor T cells

Staphylococcal enterotoxin B (SEB) is a member of a family of gram-positive bacterial exotoxins which act as superantigens in both mouse and man. The administration of this toxin has been shown to inhibit antibody responses in vivo. We have previously shown that SEB is a potent inducer in vitro of multiple T suppressor cell populations. The present studies show that administration of microgram quantities of this toxin result in a reduced capacity to manifest a delayed-type hypersensitivity (DTH) response. In addition, we find that the failure to generate a normal DTH response appears to be due to the generation of a T suppressor cell population following SEB administration. Adoptive transfer studies show that the suppressor cells bear the CD5+ I-J+ CD4- CD8- Thy 1+ surface phenotype. The relationship of these cells to suppressor T cell populations generated following in vitro activation by SEB is discussed.     

Suppression of delayed-type hypersensitivity to third party "bystander" alloantigens by antigen-specific suppressor T cells

Delayed-type hypersensitivity (DTH) against alloantigens can be induced by sc immunization with allogeneic cells. The induction of DTH can be suppressed by iv preimmunization of the mice with similar allogeneic spleen cells, provided the cells are irradiated before injection. This suppression is mediated by T cells. The suppressor activity can be induced not only by H-2-and non-H-2-coded antigens, but also by H-2 subregion-coded antigens. Suppression induced by K, I, or D subregion-coded antigens is specific for that particular subregion as well as for its haplotype. I-J-coded alloantigens were found to not be necessary for the induction of antigen-specific suppressor T cells. After restimulation of suppressor T cells by the "specific" alloantigens, the DTH to simultaneously administered third-party alloantigens becomes suppressed as well. This nonspecific suppression of DTH to third party "bystander" alloantigens also occurs when the specific and the third-party antigens are presented on separate cells, provided that both cell types are administered together at the same site. The simultaneous presentation of both sets of alloantigens during the induction phase of DTH only is sufficient to prevent the normal development of DTH to the third-party antigens.     

Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis

Contact hypersensitivity (CHS) is a T cell-mediated, Ag-specific skin inflammation induced by skin exposure to haptens in sensitized individuals. Th1/T cytotoxic 1 cells are effector cells of CHS, whereas Th2/T regulatory CD4(+) T cells have down-regulating properties. We have previously shown that CHS to 2,4-dinitrofluorobenzene is mediated by specific CD8(+) effector cells, whose cytolytic activity is mandatory for induction of skin inflammation. In this study, using immunohistochemistry and RT-PCR analysis, we show that CD8(+) T cells are rapidly recruited into the skin at the site of hapten challenge before the onset of clinical and histological signs of skin inflammation. This early CD8(+) T cell recruitment is concomitant with: 1) transient IFN-gamma mRNA expression suggesting local activation of effector cells; and 2) induction of keratinocyte (KC) apoptosis which gradually increased to a maximum at the peak of the CHS response. Alternatively, skin infiltration of CD4(+) T cells occurred later and coincided with the peak of the CHS reaction and the beginning of the resolution of skin inflammation. Mice deficient in CD8(+) T cells did not develop CHS, whereas mice deficient in CD4(+) T cells developed an enhanced inflammatory response with increased numbers of CD8(+) T cells recruited in the skin associated with massive KC apoptosis. These data show that CHS is due to the early and selective recruitment in the skin of CD8(+) T cytotoxic 1 effector cells responsible for KC apoptosis.     

Hapten-Ficoll conjugates induce T-cell-dependent IgM anti-hapten responses and T cells mediating hapten-specific delayed-type hypersensitivity

It was generally believed until recently that the B-cell IgM response induced by type-II "thymus-independent" antigens, that are not mitogenic for B cells, was T cell independent. Neither has it been possible in the past to demonstrate that these antigens activate specific T cells. This has led to the general belief that type-II thymus-independent antigens have the anomalous property of not being able to activate such cells. Recent evidence has shown that the IgM response to hapten-Ficoll conjugates, the prototype of type-II thymus-independent antigens, is T cell dependent in at least some circumstances. Here evidence that these antigens activate T cells mediating hapten-specific delayed-type hypersensitivity (DTH) is presented. Furthermore, the conditions under which they do so are similar to those that allow thymus-dependent antigens to activate DTH-mediating T cells.     

B lymphocytes are required for the generation of T cells that mediate healing of cutaneous leishmaniasis

The role that B lymphocytes and/or antibodies play in the healing of Leishmania major infections in genetically resistant C3H/HeN mice was investigated by monitoring the course of infection in animals that had been B cell depleted by treatment from birth with anti-IgM sera (mu-suppressed). L. major infection of mu-suppressed C3H/HeN mice produced lesions that were significantly larger than those induced in control animals, and failed to heal. Moreover, vaccinated mu-suppressed mice also developed chronic nonhealing infections, although their lesions were initially smaller than those developed by nonvaccinated mu-suppressed controls. The enhanced susceptibility of mu-suppressed mice could be completely overcome by adoptive transfer of T lymphocytes from mice that had spontaneously healed their lesions, and to a lesser extent by T lymphocytes from normal animals. Anti-leishmanial antibody responses were completely absent in mu-suppressed mice, regardless of whether they were lymphocyte reconstituted, whereas delayed type hypersensitivity (DTH) to leishmanial antigens was present in normal and mu-suppressed animals. The ability of immune T cells to protect mu-suppressed mice without restoring humoral responsiveness clearly indicates that antibodies are not necessary for healing leishmanial infections. Instead, the observed effect of mu-suppression argues that B lymphocytes are required for the generation of an effector T cell population, apparently unrelated to DTH, which mediates the healing of cutaneous lesions. These results thus provide the first evidence for the B cell and/or Ig dependency of a T cell population that is critical for the development of immunity against a microbial agent.