How To Understand The Cause Of Psoriasis

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1 CONTINUING MEDICAL EDUCATION The immunologic basis for the treatment of psoriasis with new biologic agents James G. Krueger, MD, PhD New York, New York Psoriasis vulgaris is the most prevalent T-cell mediated inflammatory disease in humans. The pathogenesis of psoriasis is linked to activation of several types of leukocytes that control cellular immunity and to a T- cell dependent inflammatory process in skin that accelerates the growth of epidermal and vascular cells in psoriasis lesions. Critical steps in immunologic activation include Langerhans cell maturation (activation), T-cell activation, differentiation and expansion of type 1 T cells, selective trafficking of activated T cells to skin, and induction of an inflammatory cytokine and chemokine cascade in skin lesions. In turn, each of these steps offers an opportunity for intervention with engineered biologic therapeutics. (J Am Acad Dermatol 2002;46:1-23.) Learning objective: By reading this article, the participant will be able to understand molecules involved in regulating immune responses and the ways in which rationally designed biologic agents may be used to intercept pathologic immune activation. Psoriasis is a common skin disease affecting an estimated 2.6% of the US population. 1 Over the past decade, the view of the pathogenesis of psoriasis has dramatically changed. Previously it was assumed that keratinocyte hyperproliferation associated with abnormal epidermal differentiation was the primary cause of psoriasis. However, it is now recognized that epidermal hyperplasia is a reaction to the activation of the immune system in focal skin regions, which, in turn, is mediated by CD8 + From the Laboratory for Investigative Dermatology, The Rockefeller University. Funding sources: National Institutes of Health grants RR00102, AI39214, AI49572, and AI49832 (to author). Manuscript prepration and publication costs provided by an educational grant from Protein Design Labs, Inc. Conflicts of interest: The author has been a consultant or has had laboratory support from several companies that are developing or testing biologic immune modifiers: Amgen, Biogen, Boehringer, Centecor, Genentech, Genetics Institute, Immune Response Corporation, Johnson & Johnson, Ligand, Med- Immune, Novartis, Protein Design Labs, Roche, Seragen, Squibb, and Xoma.The author has received no compensation for writing this article. Reprint requests: James G. Krueger, MD, PhD, Associate Professor and Physician, Head, Laboratory for Investigative Dermatology, The Rockefeller University, 1230 York Ave, New York, NY kruegej@rockvax.rockefeller.edu. Copyright 2002 by the American Academy of Dermatology, Inc /2002/$ /2/ doi: /mjd and CD4 + T lymphocytes that accumulate in diseased skin. Indeed, psoriasis is now recognized as the most prevalent T-cell mediated inflammatory disease in humans. Our understanding of the immunologic basis of psoriasis has now opened the door to the rational design and testing of new therapeutic agents. This article is focused on explaining molecular control points in cutaneous immune reactions and the ways in which specifically engineered biologic agents can be used to block these events. Indeed, the application of new biologic therapeutics to psoriasis is emerging as one of the most exciting areas in all of molecular medicine. This stands in sharp contrast to the development of prior therapeutics for this disease, in which serendipity played the major role in drug discovery. The need for new therapies to treat psoriasis is underscored by the need to develop long-term treatment strategies, given the fact that treatment for 40 to 50 years is not uncommon, and by toxicities that often appear with long-term use of standard treatments. The major alternative treatments, including retinoids, photochemotherapy with psoralens and ultraviolet A (PUVA), phototherapy with ultraviolet B (UVB), methotrexate, and cyclosporine, all have well-known and characteristic side effects. 2 Consequently, it is recommended that patients receive each therapeutic modality for a maximum of 1 to 2 years and then switch to another 1

2 2 Krueger J AM ACAD DERMATOL JANUARY 2002 Abbreviations used: APCs: antigen-presenting cells CLA: cutaneous lymphocyte-associated antigen CTLs: cytotoxic T lymphocytes DC-LAMP: dendritic cell lysosome-associated membrane protein IFN-γ interferon gamma GM-CSF: granulocyte-macrophage colony stimulating factor HEV: high endothelial venules HLA: human leukocyte antigen ICAM: intercellular adhesion molecule IL: interleukin IL-12R: IL-12 receptor IL-2R: IL-2 receptor LFA: lymphocyte function associated antigen MHC: major histocompatibility complex NK: natural killer NK-T: natural killer (T)-cell PNAd: peripheral lymph node addressin PUVA: psoralens and ultraviolet A RANTES: regulated upon expression, normal T-cell expressed and secreted Tc1: type 1 cytotoxic T cell Tc2: type 2 cytotoxic T cell TCR: T-cell receptor T H 1: type 1 helper T cell T H 2: type 2 helper T cell TNF-α tumor necrosis factor α TRANCE: tumor necrosis factor related activation-induced cytokine UVB: ultraviolet B VCAM: vascular cell adhesion molecule VEGF: vascular endothelial growth factor VLA-4: very late antigen 4 form. By using rotational treatment, cumulative toxicity associated with each individual treatment is theoretically minimized, and it is hoped that patients can tolerate long-term therapy. 3,4 However, new biologic treatments offer the potential to deliver highly selective therapeutics that can minimize side effects on cells outside the immune system. Consequently, it may be possible to improve the therapeutic index for long-term treatment of patients with psoriasis. One example of the potential for biologic therapies is illustrated in Fig 1. 5 This figure shows a patient with psoriasis vulgaris who was given humanized antibodies to a T-cell surface protein (the T-cell growth factor receptor or interleukin 2 [IL-2] receptor) as recently reported. 5 This dramatic improvement in the skin lesions occurred during a period of several weeks in which IL-2 receptors (IL-2R) were blocked by daclizumab, a humanized antibody to the IL-2R. The rationale for the use in psoriasis of immune antagonists of this type is explained in more detail in the sections that follow. PSORIASIS AS A LYMPHOCYTE- MEDIATED INFLAMMATORY DISEASE In the sections that follow, the evidence that psoriasis is a T-cell mediated inflammatory disease is reviewed briefly. Then, molecular and cellular control points in cutaneous immune activation are presented with the goal of explaining mechanistic overlaps between normal immunity and pathogenic immune activation in psoriasis. In a final section, the ability to manipulate cellular and molecular immune control points with biologic therapeutics is discussed. A HISTORICAL PERSPECTIVE: IDENTITY, CLASSIFICATION, AND ACTIVITY OF T CELLS IN PSORIASIS Because the clinical appearance of psoriasis is largely caused by epidermal changes, the disease has traditionally been considered one of excessive keratinocyte proliferation and abnormal differentiation. 6 Within psoriatic lesions, the keratinocyte cell cycle time is reduced approximately 8-fold (36 vs 311 hours in normal skin) and the number of dividing cells is doubled, resulting in a hyperplastic epithelium. 7 More recently, infiltration of T lymphocytes in skin lesions has been recognized to be an integral feature of psoriasis, and current evidence suggests that epidermal changes in psoriasis are caused by actions of T lymphocytes in skin lesions. 8 T cells in psoriasis vulgaris Before antibodies became available to phenotype different types of leukocytes, it was clear that the dermis of psoriasis vulgaris lesions contained numerous mononuclear inflammatory cells (leukocytes) and that these cells appeared in early lesions before obvious epidermal changes were apparent. 9 In 1978, T lymphocytes and macrophages were identified as the chief cell types in these dermal infiltrates through cell-binding assays. 10,11 By 1983, monoclonal antibodies were available to more precisely phenotype infiltrating leukocytes in psoriasis lesions. Studies at that time showed accumulations of both CD4 + and CD8 + T lymphocytes, increased dermal Langerhans cells, and scattered dermal monocytes (macrophages). 12 Accordingly, it was suggested that psoriasis might be best considered as a chronic inflammatory condition as a result of persistent stimulation of T-cells by immunogen(s) of epidermal origin. 12 This view was further supported by the finding that epidermal keratinocytes in psoriatic lesions

3 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 3 synthesize human leukocyte antigen (HLA)-DR molecules, 13 since a specific product of activated T cells, interferon gamma (IFN-γ), will induce synthesis of HLA-DR in keratinocytes. Fig 2 illustrates the striking increase in T lymphocytes in the epidermis and dermis of psoriasis vulgaris skin lesions as compared with only a few T cells in the dermis of uninvolved skin of patients with psoriasis. The routine availability of monoclonal antibody probes to leukocytes has now solidified the view that T lymphocytes are always increased in skin lesions of psoriasis vulgaris. Furthermore, monoclonal antibodies have permitted extensive characterization of T-cell subsets and other leukocytes found in psoriasis lesions. For example, CD8 + T cells (mainly cytotoxic or killer lymphocytes) are highly concentrated in psoriatic epidermis 14,15 (illustrated in Fig 2). Subsequent studies showed that both CD4 + and CD8 + T cells have markers of persistent activation such as increased IL- 2R and HLA-DR surface molecules, 16 whereas the differentiation of both cell types is strongly polarized in the type 1 pathway. Infiltrating T cells in psoriasis lesions are thus mainly activated type 1 helper T cells (T H 1) (CD4 + ) and type 1 cytotoxic T cells (Tc1) (CD8 + ). 17 T H 1 and Tc1 lymphocytes have the ability to manufacture the inflammatory cytokines IFN-γ and tumor necrosis factor α (TNF-α) upon activation, and both of these cell types are considered to be effector cell populations (as opposed to regulatory T cells, which are chiefly type 2 cells). Clonal expansion of T cells in psoriasis vulgaris lesions has been observed only in CD8 + T cells, which suggests that the Tc1 subset is the major antigen-reactive population in this type of psoriasis. 18 T cells in guttate psoriasis The identification of T cells in skin lesions of guttate psoriasis led to the suggestion that T cells might be critical pathogenic regulators. However, a pathogenic role has been suggested only for CD4 + T cells, whereas CD8 + T cells have been proposed as suppressor lymphocytes. 19 A precise characterization of CD4 + versus CD8 + T-cell differentiation needs to be established by modern techniques in guttate psoriasis. It has been suggested that T cells are initially reactive with streptococcal antigens in guttate psoriasis, 20 but there is much less evidence for this trigger in psoriasis vulgaris. Fig 1. Marked reduction of psoriatic skin lesions after in vivo blockade of CD25 (high-affinity IL-2 receptor) (top) before treatment and (bottom) after 6 weeks of treatment with daclizumab, a humanized antibody. 5 Therapeutic modulation of cellular immune infiltrates Although psoriasis could tentatively be classified as an inflammatory disease based on the selective accumulation of T lymphocytes in skin lesions, direct evidence that T lymphocytes induce/sustain the disease process is relatively recent. However, several observations influenced thought about the role of activated cellular immunity in pathogenesis. After T cells were characterized in psoriasis vulgaris lesions, it was found that PUVA therapy depleted lymphocytes in concert with disease improvements. 21,22 Although these data were consistent with a role for T cells in pathogenesis, there was no evidence for selective actions of PUVA on T cells. Cyclosporine was found to have dramatic effects on disease activity. 23 Since cyclosporine has a major inhibitory effect on T-cell activation, arguments began to be made that psoriasis was fundamentally an inflammatory disease. 24 However, cyclosporine also has strong antiproliferative effects on human epidermal keratinocytes at therapeutically relevant concentrations. 25 Accordingly, the role of T lymphocytes in disease pathogenesis was not definitely known until a specific T-cell antagonist became available. Even so, more indirect evidence was accumulating for a role of cellular immunity in psoriasis. For example, perturbation of the immune system by infection (eg, streptococcal infection and HIV infection) 26,27 or treatment with a variety of interferons 28 caused a flare of symptoms. Moreover, the injection of IFN-γ

4 4 Krueger J AM ACAD DERMATOL JANUARY 2002 A B C Fig 2. Histologic sections of nonlesional skin (A) and psoriatic plaque (B and C). T cells present were detected by immunostaining with monoclonal antibodies to CD3 (A and B) or CD8 (C). into nonlesional skin of a patient with psoriasis induced new lesions. 29 In addition, FK506 (a T-cell selective immunosuppressant, like cyclosporine), was found to have impressive therapeutic activity. 30 The most direct pathogenic link between T lymphocytes and overall disease activity was made in a clinical experiment using the immunotoxin DAB 389 IL-2, a fusion protein that carries a potent cellular toxin only to cells expressing functional IL-2R. 31 Hence keratinocytes were not directly affected by this targeted toxin. When DAB 389 IL-2 was administered in vivo to patients with psoriasis, some experienced clinical resolution that was associated with histopathologic reversal of epidermal hyperplasia (regenerative epidermal growth was reversed), angiogenesis, and neutrophil influx. All cellular aberrations that defined psoriasis histopathology were effectively eliminated by specific targeting of T lymphocytes in skin lesions. In particular, disease improvements correlated best with elimination of intraepidermal T cells, which were mainly CD8 + or Tc1 lymphocytes. This observation further supported the pathogenic potential of the Tc1 subset in psoriasis vulgaris. The use of DAB 389 IL-2, a rationally engineered biologic immune antagonist, opened the door to treatment of psoriasis with other biologic agents that modify cellular immune reactions. Trials with new biologic agents, as discussed later, have continued to solidify the view that T cells orchestrate pathogenic inflammation in psoriasis vulgaris. Finally, there is strong support from one set of laboratory experiments that T lymphocytes initiate pathologic reactions in psoriatic skin. 32 In these experiments, otherwise normal-appearing skin from a patient with psoriasis was converted to a psoriasis lesion by intradermal injection of peripheral blood mononuclear cells (primarily T lymphocytes) after they were activated with bacterial superantigens. In contrast, injection of defined cytokines induced only modest increases in epidermal growth. Neutrophils were absent from induced psoriasis lesions, suggesting that their role is secondary in initiation of psoriasis skin lesions. Through use of this model system, one other T-cell subset has been identified with pathogenic potential. This cell type, a natural killer (NK) T cell, or a T cell that bears a number of NK cell markers, differs from classic T cells in that it can recognize some antigens independent of the T-cell receptor (TCR). The potential pathogenic actions of NK T cells in psoriasis has been recently reviewed 33 and continued study of this group of T cells in psoriasis should prove interesting. Finally, it should be stated that understanding the central role of T cells in the pathogenesis of psoriasis has led to a re-examination of the therapeutic mechanism of many standard agents. It can now be argued that the most effective therapies for psoriasis, including UVB, 34 PUVA, 35 methotrexate, 36 thioguanine, 37 and cyclosporine, have strong suppressive actions on T lymphocytes, either by preventing elaboration of inflammatory cytokines or by selective cytotoxic actions. Even calcitriol, which has been considered mainly as a modifier of epidermal differentiation, has suppressive effects on IFN-γ elaboration from activated T lymphocytes. 38 Accordingly, through serendipity we may be discovering highly effective immune-modifying therapeutics for psoriasis, but toxic effects of these agents on cells outside the immune system preclude their long-term use. With new biologic immune modifiers, our understanding of cutaneous immunology should allow for construction of highly specific immune-targeted therapeutics that will have a better long-term safety profile than currently available agents.

5 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 5 Fig 3. Generation of a cutaneous T-cell immune response de novo. This figure diagrams the sequence of cellular immune activation and trafficking pathways of Langerhans cells and T lymphocytes during an immune response. Stages in this response are (1) antigen capture by immature Langerhans cells in the epidermis, which then activates maturation and migration of cells to lymph nodes draining skin sites; (2) molecular interactions between a mature Langerhans cell and a naive T cell in a lymph node that activates the T lymphocyte; (3) activated lymphocytes acquire the skin-homing receptor CLA and differentiate into type 1 or type 2 effector lymphocytes; (4) CLA + memory T cells enter the circulation and exit cutaneous blood vessels at sites of inflammation; and (5) T lymphocytes in the dermis or epidermis become activated to release cytokines (or exert other effector actions) upon encountering the initiating antigen. Psoriasis is a disease in which type 1 T lymphocytes are expanded and effector actions in skin involve the release of IFN-γ, as diagrammed. In normal immune responses, antigens are eliminated by T-cell stimulated pathways in the skin and then the immune response ceases. In psoriasis, T-cell infiltration and effector responses persist chronically. For expansion of abbreviations, see the list at the beginning of the article. CELLULAR AND MOLECULAR EVENTS IN ACTIVATION OF THE CUTANEOUS IMMUNE SYSTEM: OVERLAPS BETWEEN NORMAL AND PATHOLOGIC IMMUNITY Fig 3 outlines the cellular activation sequence that must occur to establish a T-cell mediated immune reaction in the skin de novo. Five steps needed to produce a cutaneous immune response are numbered on the figure and described in the figure legend. These steps are the organizing focus for our discussion of molecular control points in generation of a cellular immune response and for discussing pathologic immune activation in psoriasis vulgaris. T-cell trafficking and cutaneous immunity Naive T cells (cells that have not previously been activated by antigen in an immune response) circulate between the blood system and the lymph nodes. 39,40 Activation of a cutaneous immune response to a new antigen requires (1) a mechanism to stimulate the growth of antigen-reactive naive T cells in lymph nodes (whereby they become memory T cells) and (2) a mechanism to alter T-cell circulation such that effector T cells are delivered to skin regions that harbor the activating antigen. The initial step in this process is antigen-driven activation of epidermal Langerhans cells (Fig 3, step 1), which is coupled with migration of antigen-bearing Langerhans cells to skin-draining lymph nodes. In the lymph node, T-cell activation (Fig 3, step 2) and differentiation (Fig 3, step 3) ensue, such that T-cell clones expand according to the type of immune response that needs to be generated. In this process, T cells acquire new adhesion molecules that direct

6 6 Krueger J AM ACAD DERMATOL JANUARY 2002 T-cell activation The activation of T cells in conjunction with mature APCs is a multiple-step process that requires both stimulation of the TCR and several accessory signals delivered through other cell surface receptors. Fig 4 illustrates molecules associated with T-cell activation in more detail. The sequence of activation events can be termed primary stimulation, costimulathem back to sites of cutaneous inflammation (Fig 3, step 4). Before discussing how T-cell activation circuits might be altered in chronic psoriasis vulgaris lesions, let us consider the generation of a normal immune response in more detail. T-cell trafficking between the peripheral circulation and lymph nodes is maintained by a unique set of adhesion molecules. Naive T cells express high levels of L-selectin, allowing them to attach, or tether, and then roll on the surface of the high endothelial venules (HEVs) within lymph nodes. 41 After stimulation by an adhesion-triggering chemokine (6-Ckine) expressed on the luminal surface of the HEV, 42 the rolling T cells arrest and bind tightly through an integrin, lymphocyte functional antigen (LFA) The T cells are now able to extravasate through the HEV into the lymph node. Naive T cells are kept in this circulatory pattern because they lack the specific combinations of adhesion molecules and chemokine receptors necessary to enter extranodal tissues. At the heart of the antigenic activation of naive T lymphocytes is the recognition of antigens bound to class I or II major histocompatibility complex (MHC) molecules on dendritic antigen-presenting cells (APCs) that have migrated from the skin via the afferent lymphatics to skin-draining lymph nodes (Fig 3). In this sense, the T cells are educated by the migrating dendritic APCs. Skin contains large numbers of dendritic APCs in both the epidermis (where they are termed Langerhans cells) and the dermis (where they are termed dermal dendritic cells, unless they possess lineage markers of Langerhans cells [eg, CD1a, langerin, or dendritic cell lysosomeassociated membrane protein (DC-LAMP)]). 44 Langerhans cells typically exist in an immature state and are unable to activate T cells. However, they are in the optimal form for antigen capture and processing. It is proposed that macromolecules in the skin are internalized by Langerhans cells, enzymatically processed, and the antigens subsequently presented on the cell surface in preparation for presentation to naive T cells. 44 After antigen capture, a maturation process occurs (Fig 3, step 1) that is characterized by an increased ability to stimulate T-cell activation. Maturation is associated with an increased synthesis of cell-surface counterreceptors (Fig 4), including CD80 (B7-1), CD86 (B7-2), CD40, and intercellular adhesion molecule (ICAM)-1. Expression of CD1a and langerin (both are lineage markers for immature Langerhans cells) decrease during maturation. Conversely, the proteins DC-LAMP and CD83 are induced during maturation and are expressed only by fully mature cells. These activated dendritic APCs then migrate via the afferent lymphatics and collect in the skin-draining lymph nodes, where the naive T cells are concentrated (Fig 3) and then T-cell activation occurs. T-lymphocyte activation in skin-draining lymph nodes induces expression of cutaneous lymphocyte-associated antigen (CLA), a skin homing receptor, which targets newly activated memory T cells back into the skin (Fig 3). In the course of a normal cutaneous immune reaction, antigen-reactive T cells would enter skin at sites that harbor residual antigen. Subsequently, that antigen would be eliminated by an immune mechanism appropriate to the inciting antigen (Fig 3, step 5). For example, bacterial antigens might be eliminated by macrophages that are activated by cytokines released from antigen-stimulated T H 1 cells. Dermal antigens could be eliminated by antibodies stimulated, in part, by cytokines produced by T H 2 cells, and viral antigens might be eliminated by killing of infected cells by cytotoxic T lymphocytes (CTLs) and/or interferons produced by activated T cells (Tc1 cells). In addition, IL-2, also produced by activated T cells, can activate NK cells to become cytotoxic for some cellular targets. With antigen elimination, the cutaneous immune response would cease. Some memory T cells for that antigen will remain in circulation, but the bulk of antigen-reactive effector lymphocytes will be eliminated through apoptotic death. It is assumed that initial T-cell activation in psoriasis must follow the pathways discussed above, but with chronic plaque psoriasis the immune response is sustained in focal skin regions. We now recognize that fully mature Langerhans cells are found in abundance in the epidermis and dermis of plaque lesions. 45,46 Hence, chronic T-cell stimulation in psoriasis vulgaris could be attributable to ongoing stimuli delivered to skin-homing T cells from mature Langerhans cells located in skin lesions. The psoriatic process may be sustained by a continuous immune response in a peripheral lymphoid tissue (T cells, dendritic cells, and vessels arranged like a T- dependent zone in lymph nodes) that forms in lesional skin, as discussed later. Whether T-cell activation occurs in lymph nodes or in chronic skin lesions, a variety of specific molecular interactions are necessary to stimulate proliferation of antigenreactive T cells clones as described below.

7 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 7 Fig 4. A T cell interacts with a mature Langerhans cell to become activated. The T cell receives 3 sets of signals (as explained in the text) from counter-receptors expressed by the Langerhans cells or by soluble cytokines released during cellular activation. Psoriasis lesions contain mature Langerhans cells that express activation-related molecules diagrammed in this figure. For expansion of abbreviations, see list at the beginning of the article. tion, and mitotic stimulation (diagramed as steps 1, 2, and 3 in Fig 4). The initial interaction that triggers T- cell activation is recognition of an antigenic peptide that is bound to either MHC-I (typical for intracellular antigens) or MHC-II (typical for extracellular antigens) on APCs. The process of APC maturation would have led to uptake and processing of antigenic peptides such that they associate with extracellular MHC molecules on activated cells. Antigens are recognized, in turn, by the TCR complex which is present on the surface of all T cells (Fig 4). Peptide antigens presented on MHC-I are recognized by a TCR complex that contains α/β chains of the TCR protein, 5 protein subunits of CD3 (γ, δ, ε, ξ, η chains), and α/β chains of the CD8 molecule, whereas antigens presented on MHC-II are recognized by a TCR/CD3/CD4 complex. 47 During sampling of peptides presented on the surface of dendritic cells, the surface counterreceptors LFA-1 (an integrin composed of CD11a and CD18 subunits) and ICAM-1 maintain adhesion between a T cell and an APC (Fig 4). If a match occurs between a peptide and a particular TCR variant (TCR genes are rearranged like antibody genes to generate a diversity of binding sites to various antigens), a complex set of biochemical signals ensues (sequential protein phosphorylation, calcium entry, calcineurin activation, and transcription factor activation) that increase synthesis of messenger (m)rnas for activation-associated genes such as IL-2 and the α-subunit of the IL-2R (CD25 molecule). Accessory or costimulatory signals (Fig 4, step 2) are also critical for optimal T-cell activation. Historically, one of the most important costimulatory signals is transduced through a glycoprotein on the surface of the T-cell termed CD28 (Figs 5 and 6). 48 CD28 binds to 2 distinct B7 molecules, CD80 and CD86, which are up-regulated on the surface of dendritic cells during antigen-triggered maturation. The coordinated stimulation of TCR and CD28 pathways regulates the transcription of several cytokines involved in T-cell activation, including IL-2, TNF-α, IFN-γ, and granulocyte-macrophage colony stimulating factor (GM-CSF). 49,50 In contrast, the absence of CD28 costimulation produces only a partial TCR-

8 8 Krueger J AM ACAD DERMATOL JANUARY 2002 Fig 5. The migration pathway for skin-homing T cells in psoriasis. CLA + T cells roll after tethering to selectins (step 1), where chemokines activate cells via specific cell-surface receptors (step 2). Chemokine activation enables integrins to bind to intercellular adhesion molecules and vascular cell adhesion molecules, which mediate firm adhesion to the endothelium (step 3). T cells migrate across endothelium into the dermis (step 4) in response to chemokines that are synthesized in psoriatic lesions. Chemokines and receptors that mediate trafficking of type 1 T cells are indicated. Tc1 lymphocytes then migrate into epidermis (step 5) according to chemokine gradients and adhesion molecules expressed on this T-cell subset. Chemokines and binding pathways are drawn in red, while cell migration pathways in response are drawn in blue. RANTES, Regulated upon expression, normal T-cell expressed and secreted; for other abbreviations, see list at the beginning of the article. generated intracellular signal and, consequently, decreased T-cell responsiveness. However, as illustrated in Fig 5, T-cell activation is more complex than primary stimulation and costimulation through CD28. Other stimuli delivered to the T cell result from interactions between B7 molecules and CTLA4 on T cells. Whereas B7-CD28 interactions deliver positive activation signals to T cells, the interaction of B7 proteins with CTLA4 tends to deliver a growth suppressive signal In addition, there are a number of other accessory signals mediated by the interactions of other proteins on dendritic APCs and T cells that increase T-cell activation. Other accessory or costimulatory counterreceptors include ICAM- 1/LFA-1, LFA-3/CD2, and CD40/CD40L (Fig 4). 54 The third set of signals delivered to the T cell are from the cytokines IL-2 (made by activated T cells) and IL- 12 (made by mature Langerhans cells). Binding of these cytokines to surface receptors expressed on activated T cells regulates mitotic activation and differentiation of T cells into type 1 effectors, respectively (Fig 4, step 3). As discussed earlier, dendritic APCs differentiate or mature during the activation process so that T-cell activation is not only initiated but is actually driven to be more efficient. Consequently, the interactions outlined in Figs 3 and 4 do not all occur at the same time but, rather, are fluid in nature. The early steps in APC maturation appear to be controlled by antigen capture and by cytokines such as GM-CSF, IL-4, and TNFα. 55,56 In contrast, the later stages of differentiation appear to be regulated through contact with T cells. For example, the interaction of CD40 with CD40L on the activated T-cell up-regulates expression of CD40

9 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 9 Fig 6. T-cell mediated inflammatory pathways that stimulate pathogenic pathways in psoriasis vulgaris lesions. Dermal type 1 helper T (T H 1) cells (1) or epidermal type 1 cytotoxic T (Tc1) cells (2) release IFN-γ and other cytokines that cause increased expression of inflammatory proteins on keratinocytes. Tc1 cells produce epidermal damage by mechanisms shown in Fig 8. Cytokine-activated keratinocytes produce chemokines and various other growth factors (3) that then stimulate neutrophil influx, vascular alterations, and keratinocyte hyperplasia, as detailed in the text. PNAd, Peripheral lymph node addressin; for other abbreviations, see list at the beginning of the article. on the dendritic cell. CD40-CD40L interaction also appears to stimulate synthesis of B7 on the APCs. This leads to an enhancement of ongoing stimulation involving a switch to a high-affinity form of CD28 and an increased synthesis of CTLA4 on T cells. 44 In addition, after ligation of CD40, mature dendritic cells synthesize high levels of IL-12 that enhance T-cell activation and differentiation. 57,58 Furthermore, the activation of dendritic APCs by CD40L (made only by antigen-activated CD4 + T cells) functionally activates these cells by an unknown mechanism so that they become capable of stimulating differentiation of cytotoxic CD8 + cells in a much more efficient fashion. Dendritic cell survival and activity are further stimulated by the interaction of tumor necrosis factor related activation-induced cytokine (TRANCE) and the TRANCE receptor. 59,60 A member of the TNF family, TRANCE is synthesized by T cells after engagement of the TCR and binds to its receptor (TRANCE receptor) on dendritic APCs. This results in an inhibition of dendritic cell apoptosis. Clearly, the communication between dendritic APCs and T cells should be considered as an ongoing dialogue rather than as a brief monologue. T-cell proliferation and differentiation After activation, T cells clonally proliferate and differentiate into one of several possible effector cells (Fig 4, step 3). 61,62 T H 1 or Tc1 effector cells are CD4 + or CD8 + T cells that produce type 1 cytokines such as IFN-γ, IL-2, and TNF-α, but not IL-4. This maturation pathway is stimulated by IL-12 released from activated dendritic cells, as described earlier, and by exposure of T cells to type 1 cytokines during maturation. A second pathway of T-cell maturation generates T H 2 or Tc2 effectors that are CD4 + or CD8 + T cells that release type 2 cytokines including IL-4, IL-6, and IL-10, but not IFN-γ. These cells have a regulatory role, as

10 10 Krueger J AM ACAD DERMATOL JANUARY 2002 high concentrations of type 2 cytokines suppress the actions of type 1 effectors, while increasing immunoglobulin production from B cells. 63,64 Finally, T cells can also differentiate into NK T cells, which are cells that can react to some nonprotein antigens. 33 Psoriasis is considered a type 1 disease (T H 1 and Tc1), characterized by type 1 cytokines and a predominance of CD8 + T cells in the epidermis and CD4 + T cells in the dermis (Figs 2 and 3). 65 Both CD4 + and CD8 + T cells produce mainly type 1 cytokines. 15 The precise mechanisms that control the type 1/type 2 balance are not known. Although Langerhans cells can present antigens to both T H 1 and Th2 cells, the APC may play a role in determining down which pathway a T cell matures. When irradiated with UV light, Langerhans cells can only present antigens to T H 2 cells. 66 However, the local concentrations of cytokines probably play the major role in the type 1/type 2 balance. For example, high concentrations of IL-12 favor type 1 differentiation, whereas IL- 4 tends to stimulate type 2 differentiation. Such patterns of cytokines may be specific to anatomic location, as demonstrated by the production of IL-2 by T cells in lymphoid organs draining nonmucosal tissue sites, whereas those draining mucosal sites produce IL Hence increased expression of IL-12 in psoriatic skin lesions, 68,69 together with expression of IL-12R by lesional T-cells 69 could drive the type 1 T-cell differentiation bias that is seen in both skin lesions and circulating lymphocytes of patients with psoriasis. Persistence of mature Langerhans cells, which produce abundant IL-12 (Fig 5), provides the appropriate cellular and cytokine background for direct stimulation of T H 1 and Tc1 T-cell subsets in lesional psoriatic skin. Highly effective immune responses, such as those that eliminate mycobacteria and other pathogenic bacteria, are usually associated with a strong type 1 immune response that generates T H 1 and Tc1 T cells. Most other autoimmune diseases in humans (eg, inflammatory bowel disease, juvenile onset diabetes, or rheumatoid arthritis) are typified by type 1 T-cell infiltrates in target tissues. In contrast, type 2 immune responses often generate ineffective immunity, that is, effector immune responses regulated by type 2 cells cannot eliminate a number of bacterial pathogens. Allergic and antibody-mediated autoimmune diseases tend to be characterized by immune responses biased to type 2 T cells. The effector immune response: Trafficking of CLA + T cells into inflamed skin During maturation, T cells express new cell surface proteins that enable them to exit from the blood vessels and migrate into the skin (see Figs 3 and 5). 70 Perhaps the most important trafficking protein on the memory T cell in psoriasis is a glycoprotein termed CLA, 39 synthesized by the glycosylation of an existing protein (P-selectin glycoprotein ligand 1). 71 Indeed, although memory T cells in psoriasis typically express CLA on their surface, T cells in inflammatory disease involving tissues other than skin are predominantly CLA negative. 39 However, CLA is more than just a marker of skin specificity. It is an adhesion molecule that mediates the initial tethering of T cells to the endothelium in cutaneous postcapillary venules, 70,71 which is necessary for the subsequent slowing, arrest, and extravasation into the skin (Fig 5). 72 The CLA glycoprotein interacts with E-selectin and P-selectin expressed constitutively at low levels on cutaneous microvessels, but is strongly overexpressed during cutaneous inflammation. 73 However, although the CLA-selectin interaction is important during the initial stages of extravasation into the skin, chemokines and interactions between integrins and cell adhesion molecules are also involved (Fig 5). 72 After tethering involving CLA-selectin interactions, the T cells roll slowly on the endothelial surface, allowing the T cells to be exposed to chemokines that were produced by endothelial cells, keratinocytes, and other cell types resident in skin tissue. These chemokines induce modifications in integrins on the T cell, including LFA-1 and very late antigen-4 (VLA-4), so that they can bind to ICAM-1 and vascular cell adhesion molecule (VCAM)-1, respectively, on the venules. This results in arrest and flattening of the CLA + T cells in preparation for extravasation through the endothelial layer, where they respond to chemotactic gradients and move toward the epidermis (Fig 5). 72 In this way, the expression of unique receptors, including CLA, by T cells, and the preferential expression of ligands for chemokines produced by skin cells, all increase the specificity of these T cells for skin. 74 Fig 5 diagrams some of the chemokines that are thought to enhance trafficking of T-cells into psoriasis vulgaris lesions. Although 40 or more different chemokines have been found to have chemotactic effects on different types of leukocytes, 75,76 this discussion is limited to (1) chemokines or receptors that are frequently expressed on CLA + T cells, (2) chemokines that have elevated expression in psoriatic lesions, and (3) chemokine receptors that are expressed at higher levels on T cells in psoriatic lesions. In general, the production of chemokines by various cell types diagrammed in Fig 5 (endothelial cells, keratinocytes, monocytes, and Langerhans cells) is stimulated by IFN-γ and TNF-α released from antigen-activated T cells (often there is a strong synergistic interaction between these 2 cytokines for induced synthesis of chemokines). This situation potentially

11 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 11 creates a bit of a paradox in that antigen-reactive cells would initially be confined to the cutaneous vasculature unless T cells reactive with that antigen had already exited into the dermis (T cells release cytokines only when activated by the pathway outlined in Fig 4). However, one other chemokine, CTACK, is now known to be expressed constitutively in skin tissue, where it serves to permit background entry of T cells into uninflamed skin for surveillance of potential reactive antigens. 77 At that point, dermal T cells could be activated by cutaneous antigens. Release of IFN-γ and TNF-α from T cells in the dermis will then stimulate the synthesis of numerous chemokines that are found in psoriatic lesions. Fig 5 diagrams specific chemokines and chemokine receptors that are thought to influence the accumulation of type 1 T cells in skin lesions. CCR6 is expressed selectively on CLA + T cells, and its activating ligand, MIP- 3α, is highly expressed in psoriatic lesional tissue. 78 Likewise, RANTES is overexpressed in psoriatic lesional skin, while activation of CCR5 by RANTES activates type 1 T cells T cells bearing CCR4 and CXCR3 receptors are selectively increased in psoriatic skin lesions. 83 Chemokines that activate these receptors (MDC, TARC, MIG and IP-10) are also increased in psoriatic lesions (see Fig 5 for binding specificity of these ligands). Once activated by cytokines, dermal microvascular endothelial cells can synthesize TARC, MIG, and IP-10 chemokines, 83 which would directly attract CCR4 + and CXCR3 + T cells into inflamed skin. The subsequent trafficking of Tc1 into the epidermis of psoriatic lesions (Fig 5, step 5) is also likely to be regulated, at least in part, by chemokine gradients set up during inflammation. CXCR3 + CD8 + T cells are increased by 10-fold in psoriatic epidermis compared with the frequency of these cells in peripheral blood of patients with psoriasis. 83 Importantly, activating ligands for CXCR3, MIG, and IP-10 (Fig 5) are synthesized by epidermal keratinocytes in response to IFNγ, which provides not only direct means of attracting Tc1 cells toward the epidermis, but also a positive feedback loop for the ongoing recruitment of new lymphocytes. The interaction of T H 1 and Tc1 T cells with epidermal keratinocytes and other resident cell types to initiate pathologic inflammation is the subject of the next section. However, it should be noted that many cell types need to become activated to produce cellular pathology typical of psoriasis vulgaris. In this way, genetic susceptibility to psoriasis might ultimately program hyperresponsiveness of resident skin cells (eg, epidermal keratinocytes) to a given immune stimulus. While immune activation might also be influenced in an aberrant fashion by genetic differences in patients with psoriasis, to date only expression of otherwise normal immune circuits has been found in psoriatic lesions. In this way, psoriasis vulgaris can be taken as a model disease for chronic activation of otherwise normal T H 1 and Tc1 effector immune pathways in peripheral tissues. Effector immune responses and T-cell mediated inflammation in skin lesions It is now certain that cellular changes in skin-resident cells that define psoriasis pathology, as well as clinical disease activity, are dependent on the continued presence of T lymphocytes within the epidermis and dermis of skin lesions. The exact immunologic mechanisms that trigger the psoriatic phenotype are not proven, but we have an understanding of some basic elements in this pathogenic process. Accordingly, some of the T-cell dependent pathways that contribute to epidermal alterations, angiogenesis, and leukocyte-mediated inflammation in psoriatic skin lesions are diagrammed in Fig 6. The pathogenic scheme outlined in this figure begins with recruitment of T H 1 and Tc1 T cells into the dermis from the cutaneous vasculature. T H 1 and Tc1 cells are programmed to release high levels of IFN-γ and TNFα upon antigen-driven activation (which would be enabled by mature Langerhans cells in the dermis of early lesions). These cytokines are potent inducers of ICAM-1, CD40, and MHC-II proteins on epidermal keratinocytes, whereas IFN-γ serves as a potent stimulator of TNF-α release from dermal macrophages or monocytes (Fig 6, step 1). In fact, one of the first observations that suggested that T-cell products contribute to pathologic keratinocyte changes in psoriasis was detection of HLA-DR molecules on epidermal keratinocytes in psoriatic plaques. 13 Once keratinocytes have been induced to synthesize ICAM-1, it would be possible for LFA-1 + T cells to migrate into inflamed epidermis through LFA-1/ICAM-1 interactions (Fig 7). However, even before this pathway is enabled, T cells may be able to migrate into epidermis by adhesive interactions between αeβ7 integrin and E-cadherin, which is constitutively synthesized by epidermal keratinocytes. Expression of αeβ7 integrin is greatly increased on epidemal Tc1 cells compared with T cells in circulation, and it is generally considered to enable intraepithelial T-cell trafficking in other organs. 83 Existing data are most consistent with the idea that intraepidermal T cells trigger keratinocyte hyperproliferation, which accelerates epidermal growth occurring in the regenerative differentiation pathway. 8 Some inflammatory cytokines (eg, IL-1 and IL-6) have been shown to be direct keratinocyte mitogens, so elaboration of cytokines from intraepidemal T cells could directly stimulate keratinocyte proliferation. It has also been demonstrated that IFN-γ is a trigger for epidermal hyperplasia

12 12 Krueger J AM ACAD DERMATOL JANUARY 2002 Fig 7. T-cell migration into the epidermis is enabled by adhesion proteins (ICAM-1 and E-cadherin) expressed on keratinocytes. Migration disrupts the epidermal basement membrane focally and severs desmosome connections between spinous keratinocytes. It is hypothesized that resulting epidermal injury triggers regenerative epidermal growth, a woundrepair growth program of keratinocytes. when injected into skin, although this cytokine inhibits the growth of cultured keratinocytes in vitro. Human skin xenografts injected with defined cytokines also show increased epidermal growth, but the degree of hyperplasia is less than that seen with psoriasis. 32 Hence additional mechanisms may be operative for stimulating keratinocyte hyperplasia to the extent seen in psoriasis. Another mechanism has been suggested by our ultrastructural investigations of psoriatic epidermis (L. Austin and J. Krueger, unpublished studies). We have observed that migrating lymphocytes in the epidermis disrupt desmosome connections between adjacent keratinocytes in a traumatic fashion, that is, cell processes that contain both hemidesmosome pairs appear to be severed by migrating lymphocytes and these pairs are then associated with only one keratinocyte (see Fig 7). The disruption or severing of a cell process would necessarily lead to a transient membrane defect that could be viewed by the keratinocyte as injury. In addition, entry of T cells into the epidermal compartment means that the epidermal basement membrane must be broached. It seems likely that basement membrane defects in the epidermis 84 are likely to have resulted from leukocyte migration into the epidermis, as diagrammed in Fig 7. In these ways, an injury response program (regenerative maturation of the epidermis) could be triggered. In turn, numerous mitogenic cytokines and receptors on keratinocytes (EGF, IGF-1, and KGF pathways, at least, are implicated in regenerative hyperplasia and psoriasis) would be stimulated as part of the wound repair response. By ongoing release of inflammatory cytokines from activated T cells and ongoing migration of T cells in the epidermis, there would be a continuous set of signals for chronic epidermal hyperplasia in psoriatic lesions. Finally, it is well recognized that chronic lesions of psoriasis vulgaris contain prominent neutrophil accumulations in the epidermis and elongated, ectatic blood vessels in the papillary dermis. Step 3 in Fig 6 diagrams pathways that could lead to these sets of cellular changes. Release of inflammatory

13 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 13 cytokines from intraepidermal T cells can trigger more differentiated keratinocytes to synthesize and release IL This cytokine/chemokine probably serves as the main chemotactic signal for recruitment of neutrophils into the epidermis from vascular stores. Given that neutrophils have a short lifespan, persistence of neutrophils in the epidermis implies ongoing trafficking of cells into the epidermis with the potential to further injure basement membranes and keratinocyte junctions. It is likely that T-cell triggered release of vascular endothelial growth factor (VEGF) 86 and possibly other angiogenic cytokines regulates vascular growth and remodeling to the extent that this is seen in psoriasis vulgaris lesions (Fig 6). Although an extensive discussion of vascular abnormalities in psoriatic lesions is beyond the scope of this review, it should be mentioned that endothelial cells in psoriasis lesions express a number of leukocyte-related adhesion proteins that are normally associated with high endothelial venules in lymph nodes (Fig 6). These molecules, which include CD31, CD34, and peripheral lymph node addressin, could potentially allow for entry of some naive T cells in chronic lesions of psoriasis vulgaris. Hence, through association with mature Langerhans cells, some naive T cells could be converted to memory cells in secondary lymphoid tissues of the skin. A large increase in CD11c + APCs in psoriasis 45 also supports this potential because APCs that express this integrin are normally found only in primary lymphoid tissues. VEGF may be an important link between angiogenesis and cell-mediated inflammation in psoriasis, because leukocytes show increased adhesion to selectins and VCAM expressed on new vessels stimulated by this growth factor in skin; that is, VEGF is indirectly a proinflammatory cytokine. 87 IMMUNE-ALTERING BIOLOGIC THERAPY IN PSORIASIS Novel therapeutic agents Biologic therapeutics are mainly proteins that are designed to bind to extracellular targets, usually with the intent of blocking molecular activation in one of the cellular pathways outlined in Fig 3. Our review of immune pathways in cutaneous inflammation has focused mainly on extracellular adhesion proteins, receptors, cytokines, and chemokines because these molecules are the main pharmacologic targets of new biologic therapeutics. Protein-based therapeutics fall into 3 classes of agents: antibodies, fusion proteins, and recombinant cytokines. In the beginning, therapeutic antibodies were murine monoclonals that could be used for shortterm administration to humans because antimurine human antibodies soon developed that blocked activity of the murine reagents. One murine antibody, the anti-cd3 clone OKT3, is still available to use as a short-term therapeutic in acute organ rejection settings. The remarkable effectiveness of OKT3, which could reverse rejection that was uncontrollable with conventional immunosuppressive drugs, led to the development of new types of monoclonal antibodies with more human characteristics. Today, antibodies in therapeutic trials are mainly chimeric (fused segments of mouse and human antibodies), humanized (individual amino acids in a human backbone replaced with specific binding sequences derived from a murine monoclonal), or human sequence (generated in genetically engineered mice). Most therapeutic antibodies in trials are humanized because this technology allows for more flexibility in design of IgG isotypes and it permits reengineering of some antibody characteristics (eg, modification of Fc receptor binding). Fusion proteins are a more diverse set of molecules. Often, the receptor domain of a human protein is fused to constant region sequences of human IgG so that the fusion protein has binding specificity for a particular ligand or co-receptor (such as the variable region of an antibody) and so that the fusion protein is soluble in plasma (such as native IgG). Fig 8 displays a comparison of the structure of humanized antibodies to IgG-fusion proteins. Examples of fusion proteins with this construction are CTLA4Ig, LFA3 tip (alefacept), and TNFRIg (etanercept). Alternatively, human proteins have been combined with bacterial toxins through genetic engineering. The fusion protein DAB 389 IL-2 has already been introduced. This molecule is composed of human IL- 2 sequences that are fused to a fragment of diphtheria toxin, rendering a toxin that binds to cells having only IL-2R. Therapeutic strategies The main goal of biologic therapy is to prevent pathologic effector immune responses in skin tissue. This can be accomplished by altering cellular activation or T-cell trafficking at one of the steps outlined in Fig 3. Examples of new biologic agents that target specific immune-activation steps will be discussed in some detail later in this article. However, in conceptual terms, it is desirable to derive strategies that yield effective clinical control of psoriasis with the least amount of global immune suppression that can be attained. It should be realized that biologic therapies for psoriasis are very new and that efficacy and safety information from clinical trials is just becoming available. Dermatologists now have an opportunity that has not previ-

14 14 Krueger J AM ACAD DERMATOL JANUARY 2002 Fig 8. Schematic drawings of antibodies and fusion proteins used as immunotherapeutics. A, Chimeric antibody. B, Humanized antibody. C, Human antibody. D, Fusion protein. A and B, Murine-derived amino acids are indicated by yellow, whereas human sequences are shown in gray. D, Receptor domains (blue) are shown fused to constant-region sequences of human IgG (gray). The region of each molecule that binds to a target antigen is shown by light-gray background shading. ously existed with a human autoimmune disease, which is to compare a very broad range of targeted immunologic interventions for therapeutic activity and safety in a single disease, psoriasis vulgaris. Presently, we cannot say which immune-targeting approach or approaches will ultimately satisfy longterm efficacy and safety requirements for treating this chronic disease. The discussion that follows is designed to introduce readers to new biologic agents in clinical trials in the context of cellular and molecular immune activation pathways that were outlined in the prior section of this review. Targeting initial T-cell activation The first 2 steps in generating a cellular immune response (Fig 3) are Langerhans cell maturation and T-cell activation, as directed by the molecular interactions that are illustrated in Fig 4. At present, there are no direct ways to prevent maturation of Langerhans cells after antigen capture (but approaches might become available). Accordingly, the T-cell activation steps outlined in Fig 4 are the earliest therapeutic targets in the cellular immune response cascade. Given that LFA-1/ICAM-1 binding is the initial interaction of a T cell with a Langerhans cell (or other types of dendritic APCs), blockade of this interaction with humanized anti-cd11a (efalizumab, Genentech, Inc, South San Francisco, Calif, and Xoma, Inc, Berkeley, Calif) is the initial approach to mention. LFA-1 is a T-cell surface integrin that is a heterodimeric molecule composed of an α-chain (CD11a) and a β-chain (CD18). Blocking the α-chain with a humanized antibody to an extracellular region of CD11a prevents binding of the heterodimer to ICAM molecules. CD11a antibodies are extremely effective in preventing rejection of organ allografts in experimental systems, probably because of blockade of the function of LFA-1 in initial T-cell activation. However, LFA-1 is also an important adhesion molecule in T-cell trafficking and T-cell adhesion to keratinocytes. Hence clinical experiments with anti-cd11a are discussed later in the article, when approaches to block trafficking and effector immune responses are considered. The first T-cell activation signal is delivered by the TCR complex after a full agonist peptide is recognized. There are data in some experimental systems to suggest that partial agonist peptides presented on MHC molecules produce defective signaling that can lead to T-cell anergy (failure to activate when then exposed to a full agonist peptide or antigen). 88 Therefore, when the antigens involved in psoriasis have been positively identified, it may be possible to modify T-cell activation by synthetic partial agonists. However, without that knowledge, one is left with several other strategies to modulate the primary T- cell signal. Modulation or blockade of TCR stimulation If the TCR α or β families that are responding to an antigen can be identified, the T cells that harbor those specific TCR subtypes (representing the disease-mediating cells) could also be identified and eliminated. This would allow for disease suppression without producing generalized immunosuppression. This approach has been successful in reducing autoimmune encephalitis in mice, a disease elicited in response to myelin basic protein by T cells that express the Vβ8 TCR chain. Mice immunized with

15 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 15 synthetic peptides homologous to Vβ8 demonstrate delayed onset and reduced severity of encephalitis after challenge with myelin basic protein. 89 Such a TCR peptide vaccination approach has also shown great promise in rheumatoid arthritis. 90 In psoriasis, T cells infiltrating the skin express Vβ3 and Vβ and, consequently, synthetic peptides to Vβ3 and Vβ13.1 have been tested in patients with psoriasis in preliminary studies. Unfortunately, the short peptides that were administered did not produce good immunization responses, and a decision as to whether this is a viable approach must await additional tests of more immunogenic preparations. Antibodies to various protein subunits of the TCR complex can be used for therapeutic immune modulation. Anti-CD3 monoclonal antibodies (directed to ε-subunit) have been used for many years in treatment of acute rejection of organ transplants in patients. Initially, anti-cd3 monoclonal antibodies were thought to be effective through depleting effects on T lymphocytes. However, new data using a humanized anti-cd3 monoclonal antibody (HuM291, 92,93 Protein Design Labs, Fremont, Calif) suggest a more specific mechanism. First, it has been found that after binding to CD3 (ε-subunit) HuM291 only partly activates the TCR complex. This results in anergic T cells that are similar to those induced by partial agonist antigens. 94 Second, it has been demonstrated that antibodies with the same binding specificity as HuM291 will selectively deplete antigen-reactive T cells through an apoptotic mechanism that involves activation-induced cell death. 95 An early case report showed activity of murine anti- CD3 monoclonal antibodies in alleviating psoriasis, 96 and an initial study of HuM291 in patients with psoriasis is presently ongoing. HuM291 has shown remarkably good activity in treating patients with refractory graft-versus-host disease, where it rapidly depletes pathogenic CD8 + T cells from cutaneous lesions. 97 Likewise, CD4 is another protein in the TCR complex that can be used to modulate T-cell activity (but it has direct effects only on CD4 + T cells). Administration of murine anti-cd4 + monoclonal antibodies to small numbers of patients with severe psoriasis did induce a rapid reduction of lesions, 98 although antibodies against the murine antibodies were detected in all patients. More recent studies have reported reductions in psoriatic lesional area and severity in patients after therapy with humanized anti-cd4 monoclonal antibodies. 99,100 Both CD4 + and CD8 + T cells were diminished in skin lesions of patients by anti-cd4, which suggests a potential interaction of these 2 T-cell types. For example, we previously discussed a role for CD40L (expressed only by activated CD4 + T cells) in directing the differentiation of CD8 + T cells through dendritic cell bridges. Blocking accessory signals or costimulation Studies indicate that with TCR stimulation the absence of CD28 costimulation leads to an abortive T-cell proliferative response to antigen 101,102 and that blockade of the CD28/B7 interaction (Fig 4) interferes with T-cell activation, proliferation, cytokine production, and overall survival. 103,104 Importantly, for this approach to be successful, no knowledge of the primary antigenic trigger is required. In experimental systems, T-cell costimulation can be reduced or blocked by monoclonal antibodies to CD80 and CD86, as well as by the fusion protein CTLA4Ig CTLA4Ig is a fusion protein that combines the extracellular domain of CTLA4 with the IgG heavy-chain sequence to create a soluble, antibody-like protein that binds with high affinity to both CD80 and CD86. Clinically, reductions in psoriasis have been observed with CTLA4Ig-based therapy. 109 Among 43 patients with stable psoriasis vulgaris who received 4 infusions of CTLA4Ig, 46% demonstrated a sustained improvement of 50% or more, with progressively greater effects observed in the groups receiving the highest doses. Importantly, reductions in skin-infiltrating T cells were correlated with reductions in epidermal hyperplasia. A favorable safety profile was observed with this treatment. Furthermore, this activation pathway is relatively cyclosporine resistant, 110 further indicating the potential advantages of CTLA4Ig therapy. The mechanism of T-cell costimulation blockade induced by CTLA4Ig has been recently explored in more detail, 45 demonstrating a reduction in the expression of CD40, CD54, and MHC class II HLA-DR antigens on lesional keratinocytes, together with concurrent reductions in B7-1 (CD80), B7-2 (CD86), CD40, MHC class II, CD83, DC-LAMP, and CD11c expression on lesional dendritic cells after treatment with CTLA4Ig. The overall number of activated dendritic cells within the psoriatic lesions was also reduced. These data clearly suggest that ongoing T- cell costimulation is important in sustaining disease activity. Furthermore, these data establish that proteins expressed on activated dendritic cells are relevant therapeutic targets in psoriasis. This B7/CD28 costimulatory pathway has also been exploited clinically with IDEC-114, a primatized anti B7-1 monoclonal antibody. In early clinical studies, IDEC-114 was well tolerated, with most adverse events mild to moderate in severity. 111 In the higher dose groups ( 15 mg/kg), there was evidence of clinical activity. IDEC-114 specifically blocked the B7-1 (CD80)-CD28 interaction without affecting interactions with CTLA4 (Fig 4). This suggests that optimal

16 16 Krueger J AM ACAD DERMATOL JANUARY 2002 blockade might require the infusion of both anti B7-1 (CD80) and anti B7-2 (CD86) antibodies. There are experimental data to suggest that levels of B7-2 (CD86) on skin-derived dendritic cells, as well as T-cell stimulating capacity, can be significantly reduced by IL Indeed, in a recently reported phase II trial of recombinant IL-10 in 10 patients with psoriasis, 113 efficacy was observed in 9 patients, with a significant decrease in lesional area and severity index. Importantly, treatment with IL-10 was well tolerated. As illustrated in Fig 4, there are several other accessory proteins on APCs and T cells that interact to promote T-cell activation and, hence, are potential therapeutic targets. In experimental models it has been observed that although CTLA4Ig prolongs allogeneic organ transplantation, rejection, although delayed, still occurs. In contrast, simultaneous blockade of B7-mediated costimulation with CTLA4Ig and CD40-CD40L interaction by monoclonal antibodies or fusion proteins has produced long-term allogeneic organ engraftment. 114,115 Clinical studies have been conducted in organ transplants with antibodies to CD40L, and psoriasis may be another clinical use of these antibodies, especially since CD40 is strongly expressed in psoriatic lesions. The LFA-3/CD2 signaling pathway is also being targeted (Fig 6). MEDI-507 is a humanized anti-cd2 monoclonal antibody developed by MedImmune, Inc (Gaithersburg, Md) that leads to a selective deletion of antigen-specific activated T cells. 116 A phase II trial in psoriasis is currently under way. Another molecule that targets CD2 + T cells is alefacept (Biogen, Inc, Cambridge, Mass). Alefacept is an LFA-3Ig fusion protein (also termed LFA-3 tip because it contains only the external domain of LFA- 3) that binds to CD2 on T cells. Alefacept has demonstrated encouraging activity in a phase II trial in 229 patients with moderate to severe psoriasis. 117 Clinical clearing of psoriasis was noted in a significant number of patients treated with alefacept. Although therapeutic activity could be partly due to costimulation blockade, there was significant depletion of circulating memory (CD4 + CD45RO + ) T cells in alefacept-treated patients. Hence it has been proposed that this fusion protein depletes CD2 + memory T cells through an apoptotic mechanism that involves T-cell lysis by NK cells. The model proposes that NK cells bind to T lymphocytes through an alefacept bridge (NK cells can bind a portion of the IgG tail of this fusion protein through Fc receptors, whereas T cells bind the tip portion via CD2) and that release of cytotoxic granule proteins from NK cells induces death in the bridged T cell. Clearly, CD2-targeted biologics that work mainly through a cytotoxic mechanism should not be thought of as costimulation inhibitors that are equiv- alent to other agents that block T-cell activation without inducing apoptotic death. Successful phase III studies have now been completed with alefacept, so it is hoped that this agent will soon become available for treatment of patients with psoriasis vulgaris. Modulation of T-cell proliferative signals The third step in T-cell activation (which occurs many hours after the primary stimulus) is binding of mitogenic/differentiation regulating cytokines to extracellular receptors (Fig 4, step 3). The proliferation of T cells after activation is largely thought to be under the control of the IL-2R. 118 Within hours of activation, there is an increased synthesis of IL-2 and the α protein subunit (termed CD25) of the IL-2 receptor that confers high-affinity IL-2 binding. A humanized antibody to CD25, termed daclizumab (Zenapax; Hoffman-La Roche, Inc, Nutley, NJ), has been developed and has demonstrated significant activity in preventing renal allograft rejection. 119 Protein Design Labs, Inc (Fremont, Calif) is conducting studies to examine the use of daclizumab in psoriasis. In a recent study of 19 patients with psoriasis who received daclizumab (initial dose of 2 mg/kg, then 1 mg/kg at weeks 2, 4, 8, and 12), the data clearly demonstrated the complete blockade of CD25 on T cells, the tolerability and safety of daclizumab therapy, and the correlation of reductions in disease with CD25 blockade. 5 When dosing was reduced to every 4 weeks (compared with the initial regimen of every 2 weeks), variable receptor desaturation was observed, which correlated with a reversal in disease improvement. Given that reduction in disease does appear to correlate with receptor blockade, it may be that daclizumab might be most effective if used to maintain disease clearing induced by another immunosuppressive agent. Basiliximab (Simulect; Novartis Pharmaceuticals Corporation, Basel, Switzerland), another agent targeted to the CD25 subunit of the IL-2R and indicated for the prevention of graft rejection, has also proved effective in the treatment of severe psoriasis in several small case studies. 120,121 It should be noted that the IL-2R is only one of several related receptors that share a common γ-chain. 122 These IL-2 family receptors (IL-4R, IL-7R, IL-9R, and IL-15R) also deliver mitogenic signals to T cells, but they have not been well characterized in psoriatic lesions. 69 However, the IL-12R, which is mitogenic for T cells but is not part of the IL-2 family, is also up-regulated in psoriatic lesions. 69 Potentially, T-cell activation through the IL-12R (which includes stimulation of type 1 T-cell growth and IFN-γ production) can be blocked by a neutralizing antibody to IL-12. Since several humanized antibodies to IL-12 exist and are being tested in other human autoimmune diseases, clinical

17 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 17 studies in patients with psoriasis vulgaris should soon be forthcoming. Another biologic agent specific for IL-2R + (activated) T cells is the fusion protein DAB 389 IL-2, in which the cell-binding domain of diphtheria toxin has been replaced with human IL-2, resulting in a molecule that binds specifically to activated T cells. 31 After receptor-mediated endocytosis, these molecules release an enzymatic fragment of the diphtheria toxin that inhibits protein synthesis, eventually leading to apoptosis. Clinically, administration of DAB 389 IL-2 led to a reduction in activated T cells within psoriatic lesions and a parallel clearing of disease. 31,123 However, in the multicenter phase II trial, 10 of 41 patients discontinued therapy because of adverse events, typically involving flu-like symptoms. 123 Particularly at the higher doses, this agent is thought to be too problematic for routine use in patients with psoriasis. Cytokines as therapeutic drugs and anticytokine therapeutics Manipulation of T-cell differentiation by recombinant cytokines ( immune deviation ). As discussed earlier, activated T cells not only proliferate in a clonal fashion, but they differentiate into type 1 or type 2 cells (Fig 3, step 3). Psoriasis is a type 1 disease, characterized by type 1 cytokines (eg, IFNγ) that drive keratinocyte hyperproliferation. 15,65 The manipulation of the T H 1/T H 2 and Tc1/Tc2 balance by exogenously administered cytokines (or other biologic agents) is a therapeutic strategy generally termed immune deviation. In psoriasis, the differentiation of type 1 cytokine-producing T cells potentially can be deviated to the production of type 2 cytokines by the administration of exogenous IL-4, IL-10, or IL-11, because these cytokines influence endogenous differentiation of type 2 T lymphocytes in normal immune responses (Fig 3, step 3). In a recent phase II trial, recombinant IL-10 was administered to 10 patients with psoriasis over a 7- week period, 113,124 and significant antipsoriatic effects were demonstrated in 9 patients. Disease reduction by IL-10 is linked to reduction in messenger (m)rna levels for inflammatory type 1 cytokines in skin lesions. 125 As noted earlier, IL-10 can also exert antipsoriatic effects by reducing the levels of B7-2 (CD86) on skin-derived dendritic cells and a 50% reduction in their T-cell stimulating capacity has been noted. 112 Recombinant IL-11 has also shown interesting antipsoriatic effects in a phase I clinical trial. 69 Seven of 12 patients demonstrated a reduction of disease, including reduced keratinocyte proliferation, normalization of ICAM-1 and K16 expression by keratinocytes, reduced cutaneous inflammation, and decreased expression of mrna levels for several proinflammatory cytokines, including IL-12, IFN-γ, and IL-8. Impressive antipsoriatic effects of recombinant IL-4 have also been noted in an intital study. 126 The ability to modulate production of effector (type 1) cytokines or the differentiation of type 1 T cells by the administration of counter-regulatory type 2 cytokines is appealing, because there is production of excessive numbers of type 1 T cells in psoriasis. 17 Hence suppression of the type 1 T-cell axis can be viewed more as normalization of an intrinsic defect rather than as a global immunosuppressive strategy. However, the initial clinical results from administration of IL-10 and IL-11 suggest that therapeutic activity of these agents is less than that seen with more immunosuppressive agents. A variety of experimental evidence suggests that immune deviation may be most successful when IL-12 is inactivated simultaneously with the administration of type 2 cytokines. Hence future attempts to exploit this approach may be more successful if IL-12 antagonists (eg, humanized IL-12 antibodies) are given in conjunction with recombinant deviating cytokines. Furthermore, this type of strategy might be even better applied to control disease relapses after agents that decrease the overall number of type 1 T cells (ie, T-cell depleting therapies) since disease recurrence then probably is determined by clonal re-expansion of effector T H 1 and Tc1 lymphocytes. Blockade of effector cytokines. Because cytokines secreted by activated T cells drive inflammatory responses and end-stage (effector) immune responses, another therapeutic strategy is to selectively deactivate specific cytokines by antibodies or fusion proteins. As this approach will not prevent underlying T-cell activation, therapeutic activity would depend on maintaining excess of unbound antagonists. Given that effector cytokines are fairly downstream or distal in the inflammatory cascade, therapeutic blockade might be expected to show clinical benefits rather rapidly. As such, these agents may be particularly suited for short-term management of an inflammatory crisis. Based on increased expression of IFN-γ induced chemokines in psoriatic lesions (illustrated in Fig 5), as well as up-regulation of STAT1 (transduces interferon signals) and numerous other interferon-regulated mrnas in psoriatic lesions, 127 much of the pathogenic inflammation in psoriasis is potentially due to release of this cytokine from activated T cells in skin lesions. Protein Design Labs has developed a humanized IgG1 antibody against human IFN-γ (HuZAF), and clinical studies in patients with psoriasis and other type 1 diseases (eg, Crohn s disease)

18 18 Krueger J AM ACAD DERMATOL JANUARY 2002 are now in progress. If successful in the clinic, this approach is extremely exciting because global immune suppression is not produced by blocking this cytokine. Anti TNF-α therapy has demonstrated efficacy in the treatment of rheumatoid arthritis Like IFNγ, TNF-α is expressed at increased levels in psoriatic lesions and expression of several genes regulated by nuclear factor κ β(transduces TNF-α signals) is also increased. Two types of biologic TNF antagonists exist: infliximab, a chimeric anti TNF-α monoclonal antibody (Centocor, Inc, Malvern, Pa), and etanercept, a TNF-receptor-Ig fusion protein (Immunex, Inc, Seattle, Wash). Observed clearing of psoriasis skin lesions in a patient treated with infliximab for inflammatory bowel disease 131 led to a small phase II trial in patients with psoriasis vulgaris. In this trial, good to excellent reduction or clearing of disease was noted in the majority of infliximab-treated patients. 132 In a trial of etanercept in patients with psoriatic arthritis, significant reduction in cutaneous lesions of psoriasis was noted as was alleviation of arthritis. 133 Likewise, infliximab has produced alleviation of psoriatic arthritis and associated cutaneous disease. 134 According to the pathogenic model drawn in Fig 6, IL-8 is a chemokine that amplifies T-cell driven inflammation by recruiting neutrophils into psoriasis lesions. Although this chemokine might also affect T- cell recruitment into lesions, there is no evidence for selectivity of its receptor (CCR1) in regulating type 1 T-cell responses. ABX IL-8 (Abgenix, Inc, Fremont, Calif), a fully human anti IL-8 antibody that neutralizes this chemokine. Moderate clinical improvements observed in most patients with psoriasis treated with anti IL support the role of this chemokine as a part of an inflammatory cascade, but not as a sole mediator. There is also a difficult anatomic problem with respect to IL-8 neutralization in that upper spinous keratinocytes synthesize large amounts of this chemokine, whereas penetration of large proteins (anti IL-8 antibodies) into the epidermis is likely to be quite limited. In addition, a general problem with antagonizing single chemokines is that considerable redundancy exists. For example, both IL-8 and Gro-α are neutrophil chemoattractants that bind to surface receptors CXCR1 or CXCR2. Gro-α is highly expressed in psoriatic lesions 85 and could still stimulate neutrophil trafficking even though IL-8 is fully neutralized by an antibody. The situation in T lymphocytes is similar in that multiple chemokines control T-cell migration responses. However, there is additional redundancy in that some receptors bind 2 or more chemokine ligands (Fig 5). Despite these problems, chemokines and chemokine receptors are attractive therapeutic targets because highly specific immune blockade can be obtained with producing generalized immune suppression. T-cell adhesion and migration as therapeutic targets According to the pathogenic models discussed in section II (especially Figs 6-8), T lymphocytes must infiltrate the dermis and then adhere to keratinocytes to produce a psoriatic plaque. Hence molecules regulating T-cell adhesion and trafficking become tenable therapeutic targets. Intravascular adhesion events can theoretically be inhibited by blocking initial adhesion to endothelial cells (CLA/selectin interaction; Fig 5, step 1), by blocking chemokine triggering (Fig 5, step 2), or by blocking integrin binding (LFA-1 to ICAM-1 and VLA-4 to VCAM; Fig 5, step 3). Integrin blockade has been accomplished in psoriasis patients with a humanized antibody to CD11a, efalizumab (or Hu1124 by a prior designation). At therapeutic doses, efalizumab not only blocks binding of LFA-1 to ICAM, but it down-regulates (decreases) surface expression of CD11a by about 90%. 136 VLA-4, a structurally unrelated integrin, is also significantly down-regulated on the surface of circulating T cells by efalizumab, 137 a effect that is termed transmodulation. A marked lymphocytosis that occurs within days of efalizumab administration is believed to reflect reduced binding of T cells to inflamed endothelium in the skin. Reductions in the number of dermal T cells after efalizumab administration also suggest reduced trafficking of T cells from vascular stores. After therapeutic administration of efalizumab, CD11a on dermal and epidermal T cells within psoriatic lesions is also blocked and down-regulated. This effect on intralesional T cells is linked to therapeutic reductions seen in psoriatic lesions. CD11a blockade also reduces the number of T cells in the epidermis of chronic lesions. Binding of T cells to cytokine-activated epidermis was previously shown to be dependent on binding of LFA-1 to ICAM-1 on keratinocytes. 138 Hence LFA-1/ICAM interactions are also implicated in trafficking of T cells from the dermis to the epidermis (Fig 5, step 5). It should also be noted that LFA-1 functions to enable fully differentiated CD8 + T lymphocytes to serve as cytotoxic effectors (by allowing T cells to bind tightly to target cells), whereas antibody-mediated blockade of LFA-1 prevents target-cell killing. 139 Hence another important part of efalizumab s therapeutic mechanism may be through blockade of damaging interactions of Tc1 cells with keratinocytes in lesional epidermis (Fig 8). Phase II studies of efalizumab in patients with psoriasis show clear dose-related reductions in disease activity 136 and a significant benefit over placebo treatment, based on both clinical and histologic outcome measures. 140 Successful phase III studies have

19 J AM ACAD DERMATOL VOLUME 46, NUMBER 1 Krueger 19 now been completed with efalizumab, so it is hoped that this monoclonal antibody will soon become available for routine treatment of patients with psoriasis vulgaris. It is worth mentioning some other approaches that might also be successful at blocking T-cell migration into inflamed skin. Since CLA is expressed only on skin-homing T cells, its antagonism might be expected to selectively inhibit T-cell mediated inflammation in skin. Presently only a murine antibody to CLA exists, and it has not been tested in clinical studies. However, a humanized antibody to its binding partner, E-selectin, has been developed and was given as a single infusion to patients with psoriasis vulgaris. Thirteen patients with psoriasis were enrolled in this study and received a humanized anti E-selectin antibody or placebo. 141 Therapy was well tolerated, but clinically there were no reductions in disease severity. There was no significant reduction in the number of CLA + cells, but the treatment period was probably too short to have a significant impact on T-cell numbers in skin lesions. Studies are also under way with antisense oligonucleotides to ICAM-1. Inhibition of ICAM-1 expression results in enhanced graft survival in animal models of lung transplantation 142 and significantly decreased inflammation in a model of acute contact hypersensitivity. 143 Although nucleotidebased therapeutics are significantly different from the protein-based therapeutics that have been discussed throughout this review, they are worth mentioning because they target biologic pathways inside the cell. As such, there is potential for a class of therapeutics that affect intracellular signaling pathways that cannot be reached by protein-based agents. We know a great deal about intracellular signal transduction pathways in various types of leukocytes, 48 so there is great opportunity to further expand molecular targets in inflammatory skin diseases. SUMMARY On the basis of the clinical effectiveness of experimental therapeutics that selectively target T lymphocytes, including DAB 389 IL-2 and the CTLA4Ig fusion protein, the activated T cell has become the major focus for new therapies in psoriasis. However, to conceptualize the scientific basis for new immune-targeted therapeutics, it is necessary to understand T-cell mediated inflammation as a multiple-step activation cascade in which sequential activation of Langerhans cells and naive T cells generates memory T-lymphocyte subsets that home specifically to inflamed skin. After exiting cutaneous postcapillary venules, differentiated T H 1 and Tc1 lymphocytes in skin orchestrate inflammation by activating cytokine and chemokine pathways that, sequentially, amplify cellular immune responses. The clinical phenotype of psoriasis is, in turn, dependent on epidermal hyperplasia and vascular changes that result from chronic T-cell mediated inflammation within the cutaneous compartment. This review explains molecular and cellular control points in the generation of cellular immunity and the ways in which more than 20 new biologic therapeutics being tested in psoriasis target these pathways. Note added in proof: The selective expression of α E β7 integrin on CD8 + T cells in psoriatic lesions has now been confirmed in a recent study (Pauls K, Schon M, Kubitza RC, Homey B, Wiesenborn A, Lehmann P, et al. Role of integrin α E (CD103)β7 for tissue-specific epidermal localization of CD8 + T lymphocytes. J Invest Dermatol 2001;117:569-75). 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