Autoimmune diseases are caused by immune cells destroying tissues in the body. There are about 80 different autoimmune diseases. They include multiple sclerosis, type-1 diabetes mellitus, rheumatoid arthritis, psoriasis and many more.|~|/files/powerpoints_images/node4189/Slide2.JPG|~|563|~|422|~|0
Image
2
Autoimmune diseases are caused by immune cells destroying tissues in ...
Immune cells (autoreactive cells) that recognize molecules in the myelin sheath, or insulin in the pancreas, or proteins in the joint, are found in all humans. The frequency of these potentially destructive self-reactive immune cells is the same in healthy individuals as in patients with autoimmune disease. The reason healthy individuals do not develop autoimmune diseases is because the self-reactive cells are kept suppressed, maintained in a naive and uninitiated state, by another group of immune cells called regulatory T cells. The initiation of autoimmune diseases involves an \'awakening\' process, whereby the cells escape regulation. If the immune cell that awakens recognizes molecules in the myelin sheath, the patient will develop multiple sclerosis, a disease of the nervous system. If the cell that awakens recognizes insulin, the patient will develop type-1 diabetes mellitus (destruction of insulin-producing cells). Similarly, awakening of an immune cell that recognizes proteins in the joint will lead to joint destruction and rheumatoid arthritis. In this article, we will discuss a new class of drugs that can suppress the disease-causing immune cells without compromising the immune response to viruses and bacteria. |~|/files/powerpoints_images/node4189/Slide3.JPG|~|563|~|422|~|0
Image
3
Immune cells (autoreactive cells) that recognize molecules in the mye...
In order to understand the mechanisms underlying autoimmune disease pathogenesis, we have to introduce the concept of central memory and effector memory T cells. Lanzavecchi and his colleagues in 1999 (Nature. 1999;401:708-12) described two types of memory cells that could be distinguished by the expression on the cell surface of CCR7 (chemokine receptor) and CD45RA (a phosphatase). The cell surface expression of CCR7 and CD45RA on naive and memory cells is shown in the figureNaive T cells (both CD4 and CD8 cells) express both CCR7 and CD45RA. Central memory T cells express CCR7 but not CD45RA. Effector memory T cells express neither CCR7 nor CD45RA. |~|/files/powerpoints_images/node4189/Slide4.JPG|~|563|~|422|~|0
Image
4
In order to understand the mechanisms underlying autoimmune disease p...
Disease-related autoreactive T cells in many different autoimmune diseases are CCR7- CD45RA- effector memory T cells. In multiple sclerosis, T cells that recognize to proteins (myelin antigens) in the myelin sheath are mainly effector memory T cells. These myelin-specific effector memory T cells destroy the myelin sheath contributing the pathogenesis of multiple sclerosis. In patients with type-1 diabetes mellitus, disease-associated T cells that recognize insulin and glutamic acid decarboxylase (GAD65), are effector memory T cells. Therapies that target effector memory T cells might ameliorate autoimmune diseases. In this review, we provide evidence that suggests the voltage-gated Kv1.3 potassium channel in effector memory T cells may be an attractive target for selective suppression of effector memory T cells. |~|/files/powerpoints_images/node4189/Slide5.JPG|~|563|~|422|~|0
Image
5
Disease-related autoreactive T cells in many different autoimmune dis...
In 1984, we (Thomas DeCoursey, George Chandy, Sudhir Gupta and Michael Cahalan) discovered voltage-gated potassium channels in human T cells (Nature. 1984, 307:465-8). Rick Matteson and Carol Deutsch at the University of Pennsylvania independently discovered this channel (Nature. 1984, 307:468-71). Over the next few years, we characterized this channel and demonstrated its role in T cell proliferation (J Exp Med. 1984, 160:369-385; J Physiol. 1985, 358:197-237). We cloned the Kv1.3 gene encoding the voltage-gated channel in 1990 (Science. 1990, 247:973-5; Proc Natl Acad Sci U S A. 1990, 87:9411-5). A calcium-activated potassium channel was discovered by Michael Cahalan\'s group in the early 1990s (J Gen Physiol. 1992, 99:63-84; J Gen Physiol. 1993, 102:601-30) and the KCa3.1 / KCNN4 gene encoding this channel was cloned in 1997 (Proc Natl Acad Sci U S A. 1997, 94:11651-6; Proc Natl Acad Sci U S A. 1997, 94:11013-8; J Biol Chem. 1997, 272:32723-6; J Biol Chem. 1999, 274:5746-54; Genomics. 1998, 51:160-1). The image on the left shows T cells and the flame shaped object is a patch pipette. With the patch clamp method, we are able to measure ionic currents traversing the cell membrane through a single open ion channel.The image in the middle is of a naive T cell stained for CCR7 (green) and CD45RA (red). The cells appear yellow because they express both CCR7 and CD45RA. The flame shaped object is the patch pipette. The figures on the right show the two potassium channels expressed in human T cells. There are 76 potassium channels in the human genome and only Kv1.3 and KCa3.1 are expressed in human T cells. Both proteins have six transmembrane segments with the N- and the C-termini lying intracellularly. The functional channels in both cases are formed by four subunits (i.e. the channels are homotetramers). Kv1.3 is voltage-activated while KCa3.1 is calcium-activated. The calcium sensor for KCa3.1 is calmodulin, which is attached to the c-terminus of the channel; four calmodulin molecules are present per channel tetramer (J Biol Chem. 1999, 274:5746-54).|~|/files/powerpoints_images/node4189/Slide7.JPG|~|563|~|422|~|0
Image
7
In 1984, we (Thomas DeCoursey, George Chandy, Sudhir Gupta and Micha...
In this diagram, an antigen-presenting cell (APC) at the top right corner is presenting an antigen to a T cell (in blue) via the T-cell\'s antigen receptor complex. An early step during T cell activation is the formation of inositol tri-phosphate (IP3). IP3 causes the release of calcium from internal stores (e.g. endoplasmic reticulum) into the T cell cytoplasm. The basal calcium level is about 150 nM and this increases to about 250-300 nM after the internal stores are emptied. As stores empty, a protein called Stim1 on the ER membrane interacts with a protein called Orai on the cell membrane (J Cell Biol. 2006, 174:803-13; J Cell Biol. 2006, 174:815-25). The Stim1/Orai complex constitutes the CRAC (calcium-release activated calcium) channel through which calcium from the external milieu enters the T cell down the electrochemical gradient (Nature. 2008, 456:116-20; Nature. 2006, 443:226-9; Nature. 2005, 437:902-5; Nature. 2006, 441:179-85. Nature. 2006, 443:230-3). Intracellular calcium level rises to micromolar concentrations. Efflux of potassium through Kv1.3 or KCa3.1 channels hyperpolarizes the T cell and promotes calcium entry into the cell (Trends Pharmacol Sci. 2004, 25:280-9). The tight interplay between the calcium entry mechanism and the potassium efflux mechanism contributes to the calcium oscillations that are seen in human T cells ((Cell Regul. 1989, 1:99-112; J Immunol. 1993, 150:2620-33; Nature. 1998, 392:933-6; Nature. 1997, 386:855-8). The elevated intracellular calcium level initiates downstream signaling cascades which lead to cytokine production and cell proliferation. Blockade of the potassium channels with pharmacological agents inhibits the calcium signaling response, cytokine production and cell proliferation. |~|/files/powerpoints_images/node4189/Slide9.JPG|~|563|~|422|~|0
Image
9
In this diagram, an antigen-presenting cell (APC) at the top right co...
The C-terminal end of Kv1.3 is attached via the residues ThrAspVal to a PDZ-domain protein called SAP97 (a.k.a. hDlg) (J Biol Chem. 1997, 272:26899-904). SAP97 in turn is bound to p56lck, a tyrosine kinase, which is attached to CD4 (or CD8) (J Biol Chem. 1997, 272:26899-904). The N-terminus of Kv1.3 in T cells is bound to Kvbeta2 (J Neuroimmunol. 1997, 77:8-16; J Biol Chem. 1999, 274:20123-6). Each Kv1.3 subunit in the tetramer is bound to a Kvbeta2 molecule. Kvbeta2 in turn is bound to a protein called ZIP (Science. 1999, 285:1565-9), which binds to the Src-homology domain of p56lck (Proc Natl Acad Sci U S A. 1995, 92:12338-42, Proc Natl Acad Sci U S A. 1996, 93:5991-5, J Biol Chem. 1996, 271:20235-7). Thus, Kv1.3 is part of a large signaling complex consisting of the T cell receptor, CD4, p56lck, adaptor proteins and Kv1.3 (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Fluorescence energy transfer experiments show that Kv1.3 and CD3 lie within close proximity to one another (Proc Natl Acad Sci U S A. 2003, 100:2592-7). Kv1.3 is also physically coupled to beta1 integrin (J Exp Med. 2000, 191:1167-76). |~|/files/powerpoints_images/node4189/Slide10.JPG|~|563|~|422|~|0
Image
10
The C-terminal end of Kv1.3 is attached via the residues ThrAspVal to...
Kv1.3 localizes at the immunological synapse in T cells (Proc Natl Acad Sci U S A. 2004, 101:1285-90; Proc Natl Acad Sci U S A. 2006, 103:17414-9). In the figure on the left, a DAPI-stained APC (blue) pulsed with the GAD65 antigen is conjugated to a GAD65-specific CD4 effector memory T cell. Within 20 minutes of the conjugation, both CD4 (red) and Kv1.3 (green) cluster at the point of contact (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Other proteins that are physically attached to Kv1.3 (SAP97, Kvbeta2, ZIP, p56lck) traffic to the immunological synapse. This clustering is antigen specific since CD4, Kv1.3 and the attached adaptor proteins do not redistribute if the wrong antigen (MBP) is used (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Co-capping experiments confirm that Kv1.3, SAP97, Kvbeta2 and ZIP are part of a large signaling complex (Proc Natl Acad Sci U S A. 2006, 103:17414-9). KCa3.1 also traffics to the immunological synapse during antigen presentation (Am J Physiol Cell Physiol. 2007 Apr;292(4):C1431-9). Orai has recently been reported to cluster at the immunological synapse as well (Proc Natl Acad Sci U S A. 2008, 105:2011-6).Thus, all three T cell ion channels localize at the point of contact between the T cell and the APC. Blockade of Kv1.3 does not prevent immune synapse formation (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Rather, the Kv1.3 and KCa3.1 channels regulate calcium signaling and downstream events.|~|/files/powerpoints_images/node4189/Slide11.JPG|~|563|~|422|~|0
Image
11
Kv1.3 localizes at the immunological synapse in T cells (Proc Natl Ac...
Resting T cells, be they naive, central memory or effector memory, express ~250 Kv1.3 channels per cell and ~10-20 KCa3.1 channels per cell (J Clin Invest. 2003, 111:1703-13). Upon activation into effectors, naive and central memory T cells up-regulate KCa3.1 channels, whereas effector memory T cells up-regulate Kv1.3 when they change into TEM-effectors (J Biol Chem. 2000, 275:37137-49; J Clin Invest. 2003, 111:1703-13). |~|/files/powerpoints_images/node4189/Slide13.JPG|~|563|~|422|~|0
Image
13
Resting T cells, be they naive, central memory or effector memory, ex...
The numbers of Kv1.3 and KCa3.1 channels per T or B lymphocyte are shown. Naive and early B and T cells up-regulate KCa3.1 channels upon activation, whereas late memory B and T cells up-regulate Kv1.3 channels. |~|/files/powerpoints_images/node4189/Slide14.JPG|~|563|~|422|~|0
Image
14
The numbers of Kv1.3 and KCa3.1 channels per T or B lymphocyte are sh...
Insulin- or myelin-specific T cell lines were generated from the blood of healthy individuals, or patients with type-1 diabetes mellitus or patients with multiple sclerosis (J Clin Invest. 2003, 111:1703-13; Proc Natl Acad Sci U S A. 2006, 103:17414-9). The antigen-specific T cells were patch-clamped. Kv1.3 currents from patched cells are shown in the figure. Type-1 diabetes mellitus-associated insulin-specific T cells from a diabetes patient exhibited Kv1.3 currents of large amplitude, whereas myelin-specific T cells from this patient exhibited Kv1.3 currents that were similar in amplitude to that seen in T cells from a healthy individual (Proc Natl Acad Sci U S A. 2006, 103:17414-9). In contrast, multiple sclerosis-associated myelin-specific T cells from a patient with multiple sclerosis exhibited Kv1.3 currents of large amplitude, whereas insulin-specific T cells from this patient exhibited small Kv1.3 currents like that seen in control T cells (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Thus, disease-associated T cells appear to exhibit large Kv1.3 currents. Analysis of large numbers of cells from multiple patients confirmed this finding (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Flow cytometry and confocal imaging studies in parallel demonstrated that T cells with high numbers of Kv1.3 channels were CCR7- effector memory T cells (Proc Natl Acad Sci U S A. 2006, 103:17414-9). |~|/files/powerpoints_images/node4189/Slide15.JPG|~|563|~|422|~|0
Image
15
Insulin- or myelin-specific T cell lines were generated from the bloo...
T cells isolated from the synovial fluid of seven patients with rheumatoid arthritis or seven patients with osteoarthritis (non-autoimmune control) were activated for 48 hours with anti-CD3 antibody and patch-clamped. Synovial fluid T cells from rheumatoid arthritis patients expressed high numbers of Kv1.3 channels, whereas synovial fluid T cells from patients with osteoarthritis did not (Proc Natl Acad Sci U S A. 2006, 103:17414-9). T cells from the peripheral blood of three of the rheumatoid arthritis patients expressed \'normal\' numbers of Kv1.3 channels, presumably because the disease-causing cells are very infrequent and are unlikely to be detected by a single cell method such as patch clamp (Proc Natl Acad Sci U S A. 2006, 103:17414-9). Flow cytomtery showed that the T cells with elevated Kv1.3 levels in the synovial fluid of rheumatoid arthritis patients were CCR7- effector memory T cells. Immunohistochemistry of synovial biopsies from rheumatoid arthritis patients confirmed that the T cells invading the synovium were CD3 Kv1.3 and CCR7- (Proc Natl Acad Sci U S A. 2006, 103:17414-9). |~|/files/powerpoints_images/node4189/Slide16.JPG|~|563|~|422|~|0
Image
16
T cells isolated from the synovial fluid of seven patients with rheum...
These data demonstrate that disease-associated T cells in three autoimmune diseases, multiple sclerosis, type-1 diabetes mellitus and rheumatoid arthritis, are TEM-effector cells with elevated Kv1.3 expression. T cells with other antigen specificities are CCR7 KCa3.1-dependent naive or central memory T cells. Targeting Kv1.3 channels with pharmacological agents may shut down the disease-relevant effector memory cells, while sparing naive and central memory T cells that provide protection against infections and cancers. |~|/files/powerpoints_images/node4189/Slide17.JPG|~|563|~|422|~|0
Image
17
These data demonstrate that disease-associated T cells in three autoi...
The ShK toxin was discovered by Professor Olga Castaneda at the University of Havana in Cuba and her collaborators in Sweden. Dr. Michael Pennington at Bachem Biosciences developed the method for synthetic production of the peptide, and Professor Raymond Norton in Australia determined the 3D structure of the peptide by NMR. |~|/files/powerpoints_images/node4189/Slide21.JPG|~|563|~|422|~|0
Image
21
The ShK toxin was discovered by Professor Olga Castaneda at the Unive...
ShK toxin structure on the left. The surface of the peptide that interacts with Kv1.3 is shown in orange, with lysine 22 (K22) in red. K22 occludes the Kv1.3\'s ion conduction pathway. On the right, ShK docked in the Kv1.3 pore, The peptide interacts with all four subunits in the Kv1.3 tetramer. The Kv1.3 subunit in the front is removed so you can see the channel pore. K22 (in blue) sticks into the channel pore and blocks it like a cork in a wine bottle. |~|/files/powerpoints_images/node4189/Slide22.JPG|~|563|~|422|~|0
Image
22
ShK toxin structure on the left. The surface of the peptide that inte...
ShK (from the sea anemone) and margatoxin (from a scorpion) are both potent blockers of Kv1.3. Their relative potencies on Kv1.3 and related potassium channels are shown. ShK blocks neuronal channels (Kv1.1, Kv1.6) with picomolar potency. We spent nearly 10 years generating ShK analogs that would be selective for Kv1.3. We were not very successful. |~|/files/powerpoints_images/node4189/Slide23.JPG|~|563|~|422|~|0
Image
23
ShK (from the sea anemone) and margatoxin (from a scorpion) are both ...
We got a clue about how we could develop a specific Kv1.3-blocking ShK analog when we discovered that the attachment of a fluorescein to the N-terminus of ShK enhanced selectivity for Kv1.3. Fluorescein is negatively charged and may be interacting with a positively charged residue on Kv1.3. |~|/files/powerpoints_images/node4189/Slide24.JPG|~|563|~|422|~|0
Image
24
We got a clue about how we could develop a specific Kv1.3-blocking Sh...
Based on this result, we introduced several negatively charged residues at the N-terminus of ShK. Attachment of L-phosphotyroine via a hydrophilic AEEA linker resulted in a peptide, ShK(L5), with 100-fold selectivity for Kv1.3 over Kv1.1, and greater than 200-fold selectivity over other channels. |~|/files/powerpoints_images/node4189/Slide25.JPG|~|563|~|422|~|0
Image
25
Based on this result, we introduced several negatively charged residu...
Ruta tea is reported to be effective in MS. Dr. Heike Wulff, Dr. Hansel and their colleagues at the University of Kiel screened extracts from Ruta for potassium channel blocking activity. 5-methoxy psoralen (5-MOP) from the plant was found to block potassium channels with a IC50 value of 100 uM. 5-MOP was approved for use as a therapeutic for psoriasis in Germany. Dr. Hansel and colleagues therefore did a small clinical study of 5-MOP in multiple sclerosis patients with visual field defects. The drug was effective. Subsequently, Dr. Wulff and her colleagues generated a potent and selective 5-MOP analog called PAP-1, which blocks Kv1.3 with an IC50 value of 2 nM, and shows greater than 20-fold selectivity over other channels. |~|/files/powerpoints_images/node4189/Slide27.JPG|~|563|~|422|~|0
Image
27
Ruta tea is reported to be effective in MS. Dr. Heike Wulff, Dr. Hans...
PAP-1 blocks Kv1.3 with a Hill coefficient of 2. It is the most potent small molecule inhibitor of Kv1.3 |~|/files/powerpoints_images/node4189/Slide28.JPG|~|563|~|422|~|0
Image
28
PAP-1 blocks Kv1.3 with a Hill coefficient of 2. It is the most poten...
Naive T cells express ~250 Kv1.3 channels and 10-20 KCa3.1 channels. Kv1.3 is therefore the main channel in quiescent naive T cells. Consequently, Kv1.3-specific blockers suppress the proliferation of naive T cells induced by anti-CD3 antibody (solid line) at concentrations that block the channel (dotted line). In contrast, KCa3.1-specific blockers are not effective (solid). Once naive T cells are activated into naive-effectors, they up-regulate KCa3.1 channels to 500 channels per cell. In these cells, KCa3.1-specific blockers are effective in suppressing anti-CD3 antibody-induced proliferation, whereas Kv1.3-specific blockers are ineffective. |~|/files/powerpoints_images/node4189/Slide35.JPG|~|563|~|422|~|0
Image
35
Naive T cells express ~250 Kv1.3 channels and 10-20 KCa3.1 channels. ...
In subsequent studies we extended the findings shown in the previous slide. The Kv1.3-specific inhibitor, ShK-186, was more effective in suppressing anti-CD3 antibody proliferation of effector memory T cells than naive or central memory T cells (top right graph). This is because naive T cells up-regulate KCa3.1 channels and therefore become less sensitive to the Kv1.3 blocker. Naive-effectors and central memory-effectors with up-regulated KCa3.1 expression are resistant to Kv1.3 blockers, while effector memory T cells that up-regulate Kv1.3 upon activation are sensitive to ShK-186 blockade. |~|/files/powerpoints_images/node4189/Slide36.JPG|~|563|~|422|~|0
Image
36
In subsequent studies we extended the findings shown in the previous ...
PAP-1 and ShK-186 are more effective in suppressing interleukin-2 and interferon-gamma production by effector memory T cells than naive and/or central memory T cells. Interestingly, tumor necrosis factor-alpha production is not suppressed in either cell population by Kv1.3 blockers. |~|/files/powerpoints_images/node4189/Slide37.JPG|~|563|~|422|~|0
Image
37
PAP-1 and ShK-186 are more effective in suppressing interleukin-2 and...
ShK-186 suppressed anti-CD3 antibody induced calcium signaling in human effector memory T cells. |~|/files/powerpoints_images/node4189/Slide38.JPG|~|563|~|422|~|0
Image
38
ShK-186 suppressed anti-CD3 antibody induced calcium signaling in hum...
We visualized the migration of effector memory T cells in inflamed ear tissues of rats undergoing a delayed type hypersensitivity response. ShK-186 administered by subcutaneous injection (100 ug/kg) once a day immobilized effector memory T cells at the site of inflammation, but it did not prevent the cells from reaching the tissue. This effect of ShK-186 is different from other immunosuppressants such as Tysabri and FTYY720. The paralyzed effector memory T cells are unable to interact with antigen presenting cells in the ear tissue and therefore do not undergo activation. In contrast, effector memory T cells from the ear tissue of rats administered saline get enlarged as a consequence of activation. ShK-186 did not affect the homing to or the motility within lymph nodes of naive and central memory T cells. |~|/files/powerpoints_images/node4189/Slide39.JPG|~|563|~|422|~|0
Image
39
We visualized the migration of effector memory T cells in inflamed ea...
Myelin-specific effector memory T cells with elevated Kv1.3 expression was adoptively transferred into the peritoneal cavity of healthy Lewis rats. The animals developed a paralytic disease called \'adoptive experimental autoimmune encephalomyelitis\' within 4-7 days. Rats administered saline (blue) got severe disease. Treatment with ShK (yellow) from the day of cell transfer (prevention, right) or once the animals developed minimal paralysis, ameliorated the disease. |~|/files/powerpoints_images/node4189/Slide41.JPG|~|563|~|422|~|0
Image
41
Myelin-specific effector memory T cells with elevated Kv1.3 expressio...
Dark Agouti rats challenged with autologous spinal cord and complete Freund\'s adjuvant develop a chronic relapsing-remitting EAE within about 10 days. Treatment with ShK-186 (100 ug/kg administered once/day by subcutaneous injection) significantly ameliorated the disease, reduced inflammatory infiltrate in the CNS and reduced demyelination. |~|/files/powerpoints_images/node4189/Slide42.JPG|~|563|~|422|~|0
Image
42
Dark Agouti rats challenged with autologous spinal cord and complete ...
ShK-186 (100 ug/kg) administered once/day by subcutaneous injection decreased the number of affected joints in DA rats with pristane-induced arthritis. |~|/files/powerpoints_images/node4189/Slide43.JPG|~|563|~|422|~|0
Image
43
ShK-186 (100 ug/kg) administered once/day by subcutaneous injection d...
Saline treated rats (left) had soft tissue damage, bone malformation and damage to the joint. ShK-186-treated rats (right) did not show these findings. |~|/files/powerpoints_images/node4189/Slide44.JPG|~|563|~|422|~|0
Image
44
Saline treated rats (left) had soft tissue damage, bone malformation ...
PAP-1 administered orally (50 mg/kg, once/day) from day 35 to diabetic-prone BB rats significantly reduced the incidence of experimental autoimmune diabetes mellitus. Treatment decreased islet destruction and infiltration of islets by immune cells. |~|/files/powerpoints_images/node4189/Slide46.JPG|~|563|~|422|~|0
Image
46
PAP-1 administered orally (50 mg/kg, once/day) from day 35 to diabeti...
PAP-1 administered by intraperitoneal injection or topically suppressed acute contact dermatitis in rats. |~|/files/powerpoints_images/node4189/Slide47.JPG|~|563|~|422|~|0
Image
47
PAP-1 administered by intraperitoneal injection or topically suppress...
In this review, we summarize findings that suggest that the voltage-gated Kv1.3 channel in human effector-memory T cells is an attractive and safe molecular target for the treatment of diverse autoimmune diseases.
Brief Description