The journal of Dermatology Vol. 19: 722-725, 1992
WS In-2
The Interaction between Langerhans Cells and CD4+ T Cells Conrad Hauser Abstract
The human skin is increasingly exposed to haptens and environmental protein antigens. Because Langerhans cells represent the outermost network of MHC class 11+ antigen presenting cells in mammalians, we investigated their interaction with CD4+ T cells. Hapten-modified Langerhans cells induced proliferation and IL-2 production in naive resting CD4+ T cells. T cells activated in this manner and subsequently cultured with IL-2 mediated contact sensitivity in vivo and produced IL-2 but no IL-4 upon restimulation in vitro. Thus they corresponded to Th1 cells. Repeated stimulation with. Langerhans cells induced a modulation of the lymphokine pattern: IL-2- and IL-4-producing ThO-like cells were identified after 3 to 4 rounds of restimulation; after >5 rounds, Thz-like cells with an IL-4+IL-2- pattern and the capacity for inducing IgE synthesis in B cells was identified. Th2 cells were also recently fOHpd to mediate inflammatory tissue lesions containing a cellular infiltrate. This demonstrates that Langerhans cells may activate resting CD4+ T cells, Th1-, ThO- and Th2-like cells. It further shows that Langerhans cells may promote the differentiation of postthymic CD4+ T cells into subsets with distinct immune functions: Th1 cells which have the potential to mediate inflammatory reactions such as allergic contact sensitivity and Th2 cells which may be responsible for abnormalities associated with atopic dennatis, such as elevated IgE and inflammatory skin lesions containing a cellular infiltrate. Introduction
Epidermal Langerhans cells (see ref 1, 2 for a review) are bone marrow derived cells that are typically located within the suprabasal layer of the epidermis and form, with the aid of their dendrites, a contiguous horizontal network. Langerhans cells bear a number of immunologically relevant molecules on their surface. Among these, the CD1a antigen is commonly used for the identification of Langerhans cells within the human epidennis.· Langerhans cells in all species so far investigated constitutively express major histocompatibility class II molecules. The presence of this molecule on epidermal Langerhans cells led Stingl and coworkers in 1978 to the discovery of their antigen presenting function (3). The fate of an antigen within an antigen presenting cell has partly been elucidated in recent years (1, 2). After uptake by fluid phase endocytosis or receptor mediated endocytosis, antigen is proteolytically cleaved within an acidic endosomal compartment. This compartment Department of Dermatology, Hopital Cantonal Universitaire, 24, rue Micheli-Du-Crest, 1211 Geneva 4, Switzerland.
is also the export pathway for the major histocompatibility class II molecules. There is evidence that the peptide binding groove of the class II molecules is loaded by antigen-derived peptides at this crossover of endocytosis and export. The peptide laden class II molecules are transported to the cell surface and displayed. for binding to their natural counter receptor. If one of the clonally distributed receptors for antigen plus class II expressed on T lymphocytes has a certain threshold affinity for class II plus peptide, class II restricted and antigen specific interaction can occur. This interaction is strengthened by the binding of CD4 to nonpolymorphic determinants on the class II molecule. CD4 bearing T cells, often referred to as T helper cells, are thus molecularly determined interaction partners of class II bearing Langerhans cells. However, additional molecular interactions between Langerhans cells and CD4+ T cells must occur before the T cells respond by activation. These additional signals derived from antigen presenting cells are collectively called second signals. They may depend on the activation and differentiation state of the T cell and the T cell response to be measured (proliferation, lymphokine production). When cultured for 2-4 days, epidermal cell sus-
Langerhans Cells and T Helper Cells
723
FITC- AND TNP-SPECIFIC CONTACT SENSITIVITY EFFECTOR T CELLS CAN BE GENERATED IN VITRO WITH cLC HAPTEN ON cLC
HAPTENFOR CHALLENGE
EAR SWELLING(mm x 102) 10
TNCB
TNCB
FITC
TNCB
FITC
FITC
TNCB
FITC
15
20
Fig. 1. CD4+ T cells from naive mice sensitized in vitro with FITC- or TNP-modified cultured Langerhans cells (cLC) were injected into naive recipients. Ear swelling in response to challenge with the relevant and the irrelevant hapten were recorded (for methods see ref. 8).
pensions are a source of potent antigen presenting cells. It has been established by Schuler and colleagues that Langerhans cells mature under the influence of the cytokines GM-CSF and IL-I, both secreted by keratinocytes in culture, into powerful antigen presenting cells (4, 5). Since cultured Langerhans cells have been shown to be capable of activating both naive and previously activated (memory) T cells, we selected them to study their effects on CD4+ T cells. For the work presented here, we primarily used haptens coupled to cultured Langerhans cells because they are thought to present haptens to T cells in vivo and because hapten-specific T cells have a high precursor frequency within nonsensitized T lymphocyte populations. Thus, a T cell response may readily be measured.
The interaction ofLangerhans ceUsand naive CD4+ T cells results in generation ofThI effector'ceUs When hapten-modified Langerhans cells were cultured with naive syngeneic CD4+ T cells, marked proliferation was observed after 5 days (6). Haptendependent proliferation was blocked with antibodies to class II molecules, indicating that hapten was recognized in the context of class II (6). The response was also blocked with antibodies to LFA-I (6), suggesting that the LFA-l/lCAM-I pair of adhesion receptors were involved as one of the secondary signals for hapten-dependent primary CD4+ T cell proliferation. Restimulation experiments showed that the activated CD4+ T cells were specific for the hapten used for primary T cell activation (6). Primary stimulation, as well as restimulation with the relevant hapten bearing antigen presenting cells, led to the release of IL-2 by CD4+ T cells (7). After expansion with recombinant
Fig. 2. Synopsis of the interaction between Langerhans cells and CD4+ T cells.
IL-2, hapten-specific CD4+ T cells also released IL-2 upon restimulation in vitro (8). In addition, these cells were capable of transferring contact sensitivity in vivo when injected into naive syngeneic mice (8, Fig. I). These results demonstrated that Langerhans cells can induce Thl-like effector cells after a single round of interaction with naive CD4+ T cells.
Repeated interaction of activated CD4+ T cells with Langerhans ceUs results in Th2 effector ceUs that stimulate IgE synthesis and mediate tissue intJam· mation When activated hapten-specific CD4+ T cells were stimulated for several cycles with hapten-modified Langerhans cells, the lymphokine secreted by these T cell lines in response to hapten was identified as IL-4. No IL-2 production was observed (7). Therefore, hapten-specific CD4+ T cells generated in this manner resembled Th2 cells. IL-4 was previously reported to induce the secretion of IgE in activated B cells (9). Recognition of hapten on B cells should lead to activation of the B cell. TNP modification of B cells should provide all B cells with hapten recognizable by hapten-specific T cells. Thus, recognition of hapten modified B cells should potentially activate all B cells regardless of their specificity. As a consequence, all activated B cells are expected to respond to IL-4. Using this polyclonal but hapten-specific readout system, we were able to show that IL-4 secreting hapten-specific
724
Hauser
Table 1. Clonal allogeneic CD4+ T cell cultures switch from IL-2 to IL-4 production
Clone
Restimulation cycle
% Inhibition of response by anti-IL-2 anti-IL-4
2Fll
1 4
100 4.1
10.8 90.9
2D4
1 2 4
100 31.4 1.5
18.6 81.8 100
IH4
1 4
9004 18.6
13.1 92.2
IGll
1 4
8304 6.7
18.8 95.2
Replicate cultures containing 7 x 1(}l irradiated cultured Langerhans cells from Balb/c mice and 20 or 60 CD4+ T cells from nonimmunized C3H mice were set up. Cultures were fed with cultured Langerhans cells and fresh media once a week. Culture supernatants were harvested 2 days later and incubated with CTLL cells that respond to both IL-2 and IL-4 and S4B6 (anti-IL-2) or llBll (anti-IL-4) or no antibody. Percent inhibition was calculated from 100% x cpm with antibody/cpm without antibody. The frequency of responding wells was
WS In-2
The Interaction between Langerhans Cells and CD4+ T Cells Conrad Hauser Abstract
The human skin is increasingly exposed to haptens and environmental protein antigens. Because Langerhans cells represent the outermost network of MHC class 11+ antigen presenting cells in mammalians, we investigated their interaction with CD4+ T cells. Hapten-modified Langerhans cells induced proliferation and IL-2 production in naive resting CD4+ T cells. T cells activated in this manner and subsequently cultured with IL-2 mediated contact sensitivity in vivo and produced IL-2 but no IL-4 upon restimulation in vitro. Thus they corresponded to Th1 cells. Repeated stimulation with. Langerhans cells induced a modulation of the lymphokine pattern: IL-2- and IL-4-producing ThO-like cells were identified after 3 to 4 rounds of restimulation; after >5 rounds, Thz-like cells with an IL-4+IL-2- pattern and the capacity for inducing IgE synthesis in B cells was identified. Th2 cells were also recently fOHpd to mediate inflammatory tissue lesions containing a cellular infiltrate. This demonstrates that Langerhans cells may activate resting CD4+ T cells, Th1-, ThO- and Th2-like cells. It further shows that Langerhans cells may promote the differentiation of postthymic CD4+ T cells into subsets with distinct immune functions: Th1 cells which have the potential to mediate inflammatory reactions such as allergic contact sensitivity and Th2 cells which may be responsible for abnormalities associated with atopic dennatis, such as elevated IgE and inflammatory skin lesions containing a cellular infiltrate. Introduction
Epidermal Langerhans cells (see ref 1, 2 for a review) are bone marrow derived cells that are typically located within the suprabasal layer of the epidermis and form, with the aid of their dendrites, a contiguous horizontal network. Langerhans cells bear a number of immunologically relevant molecules on their surface. Among these, the CD1a antigen is commonly used for the identification of Langerhans cells within the human epidennis.· Langerhans cells in all species so far investigated constitutively express major histocompatibility class II molecules. The presence of this molecule on epidermal Langerhans cells led Stingl and coworkers in 1978 to the discovery of their antigen presenting function (3). The fate of an antigen within an antigen presenting cell has partly been elucidated in recent years (1, 2). After uptake by fluid phase endocytosis or receptor mediated endocytosis, antigen is proteolytically cleaved within an acidic endosomal compartment. This compartment Department of Dermatology, Hopital Cantonal Universitaire, 24, rue Micheli-Du-Crest, 1211 Geneva 4, Switzerland.
is also the export pathway for the major histocompatibility class II molecules. There is evidence that the peptide binding groove of the class II molecules is loaded by antigen-derived peptides at this crossover of endocytosis and export. The peptide laden class II molecules are transported to the cell surface and displayed. for binding to their natural counter receptor. If one of the clonally distributed receptors for antigen plus class II expressed on T lymphocytes has a certain threshold affinity for class II plus peptide, class II restricted and antigen specific interaction can occur. This interaction is strengthened by the binding of CD4 to nonpolymorphic determinants on the class II molecule. CD4 bearing T cells, often referred to as T helper cells, are thus molecularly determined interaction partners of class II bearing Langerhans cells. However, additional molecular interactions between Langerhans cells and CD4+ T cells must occur before the T cells respond by activation. These additional signals derived from antigen presenting cells are collectively called second signals. They may depend on the activation and differentiation state of the T cell and the T cell response to be measured (proliferation, lymphokine production). When cultured for 2-4 days, epidermal cell sus-
Langerhans Cells and T Helper Cells
723
FITC- AND TNP-SPECIFIC CONTACT SENSITIVITY EFFECTOR T CELLS CAN BE GENERATED IN VITRO WITH cLC HAPTEN ON cLC
HAPTENFOR CHALLENGE
EAR SWELLING(mm x 102) 10
TNCB
TNCB
FITC
TNCB
FITC
FITC
TNCB
FITC
15
20
Fig. 1. CD4+ T cells from naive mice sensitized in vitro with FITC- or TNP-modified cultured Langerhans cells (cLC) were injected into naive recipients. Ear swelling in response to challenge with the relevant and the irrelevant hapten were recorded (for methods see ref. 8).
pensions are a source of potent antigen presenting cells. It has been established by Schuler and colleagues that Langerhans cells mature under the influence of the cytokines GM-CSF and IL-I, both secreted by keratinocytes in culture, into powerful antigen presenting cells (4, 5). Since cultured Langerhans cells have been shown to be capable of activating both naive and previously activated (memory) T cells, we selected them to study their effects on CD4+ T cells. For the work presented here, we primarily used haptens coupled to cultured Langerhans cells because they are thought to present haptens to T cells in vivo and because hapten-specific T cells have a high precursor frequency within nonsensitized T lymphocyte populations. Thus, a T cell response may readily be measured.
The interaction ofLangerhans ceUsand naive CD4+ T cells results in generation ofThI effector'ceUs When hapten-modified Langerhans cells were cultured with naive syngeneic CD4+ T cells, marked proliferation was observed after 5 days (6). Haptendependent proliferation was blocked with antibodies to class II molecules, indicating that hapten was recognized in the context of class II (6). The response was also blocked with antibodies to LFA-I (6), suggesting that the LFA-l/lCAM-I pair of adhesion receptors were involved as one of the secondary signals for hapten-dependent primary CD4+ T cell proliferation. Restimulation experiments showed that the activated CD4+ T cells were specific for the hapten used for primary T cell activation (6). Primary stimulation, as well as restimulation with the relevant hapten bearing antigen presenting cells, led to the release of IL-2 by CD4+ T cells (7). After expansion with recombinant
Fig. 2. Synopsis of the interaction between Langerhans cells and CD4+ T cells.
IL-2, hapten-specific CD4+ T cells also released IL-2 upon restimulation in vitro (8). In addition, these cells were capable of transferring contact sensitivity in vivo when injected into naive syngeneic mice (8, Fig. I). These results demonstrated that Langerhans cells can induce Thl-like effector cells after a single round of interaction with naive CD4+ T cells.
Repeated interaction of activated CD4+ T cells with Langerhans ceUs results in Th2 effector ceUs that stimulate IgE synthesis and mediate tissue intJam· mation When activated hapten-specific CD4+ T cells were stimulated for several cycles with hapten-modified Langerhans cells, the lymphokine secreted by these T cell lines in response to hapten was identified as IL-4. No IL-2 production was observed (7). Therefore, hapten-specific CD4+ T cells generated in this manner resembled Th2 cells. IL-4 was previously reported to induce the secretion of IgE in activated B cells (9). Recognition of hapten on B cells should lead to activation of the B cell. TNP modification of B cells should provide all B cells with hapten recognizable by hapten-specific T cells. Thus, recognition of hapten modified B cells should potentially activate all B cells regardless of their specificity. As a consequence, all activated B cells are expected to respond to IL-4. Using this polyclonal but hapten-specific readout system, we were able to show that IL-4 secreting hapten-specific
724
Hauser
Table 1. Clonal allogeneic CD4+ T cell cultures switch from IL-2 to IL-4 production
Clone
Restimulation cycle
% Inhibition of response by anti-IL-2 anti-IL-4
2Fll
1 4
100 4.1
10.8 90.9
2D4
1 2 4
100 31.4 1.5
18.6 81.8 100
IH4
1 4
9004 18.6
13.1 92.2
IGll
1 4
8304 6.7
18.8 95.2
Replicate cultures containing 7 x 1(}l irradiated cultured Langerhans cells from Balb/c mice and 20 or 60 CD4+ T cells from nonimmunized C3H mice were set up. Cultures were fed with cultured Langerhans cells and fresh media once a week. Culture supernatants were harvested 2 days later and incubated with CTLL cells that respond to both IL-2 and IL-4 and S4B6 (anti-IL-2) or llBll (anti-IL-4) or no antibody. Percent inhibition was calculated from 100% x cpm with antibody/cpm without antibody. The frequency of responding wells was
