M. Cahalan - Academia.edu (original) (raw)

Papers by M. Cahalan

The Journal of Immunology

Antigen presentation by B cells can generate either long-lived antigen-specific CD4+ T cell toler... more Antigen presentation by B cells can generate either long-lived antigen-specific CD4+ T cell tolerance or activate T cells for additional B cell help during the humoral response. In our model, both naïve and LPS-activated B cells mOVA B cells expressing ovalbumin induced long-lived peripheral T cell tolerance, suppressing OTII T cell proliferation and T-cell-dependent antibody production in vivo. In contrast, CpG-activated mOVA B cells did not induce tolerance to OVA. Using two-photon microscopy we compared T-B cell interactions in the lymph node during tolerance induction or priming of OTII T cells mOVA B cells. Naïve, LPS- or CpG-activated mOVA B cells were individually capable of establishing long-lived interactions with OTII-T cells. Disruption of CTLA-4 / B7 interactions prevented establishment of long-lived T-B cognates and inhibited tolerance induced by LPS-treated B cells in vivo. In competition experiments with co-transferred LPS- and CpG-activated B cells, LPS- activated ...

The FASEB Journal, 2008

B cells, functioning as APC's, can provide signals to T cells either activating immunity or i... more B cells, functioning as APC's, can provide signals to T cells either activating immunity or inducing a specific hyporesponsive state (tolerance). Our laboratory has shown that B cells, when retrovirally transduced with peptide‐IgG, are highly tolerogenic for epitopes fused with the IgG. In this system, a DNA sequence encoding a full‐length protein or an immunodominant epitope is inserted into the N‐terminus of the murine IgG1 heavy chain. After injection into immunocompetent mice, peptide‐IgG transduced B cells are able to induce specific tolerance to the IgG associated antigen. This gene therapy approach has been applied to several animal models of autoimmune diseases, including EAU, EAE, and diabetes, as well as in hemophilia. In these disease models, specific tolerance was observed in both Th1 and Th2 responses. Moreover, our data indicate that the IgG heavy chain is critical for efficient tolerance induction and trafficking to endosomal processing pathways. We recently repor...

Brain, Behavior, and Immunity, 2019

Science, 1988

ministered in vivo, somatotropin induces amounts of O2similar to those produced by IFN-y at a con... more ministered in vivo, somatotropin induces amounts of O2similar to those produced by IFN-y at a concentration that is optimal for macrophage activation (18). Thus, so- matotropin shares at least one biologic activ- ity with IFN-,y, priming macrophages to produce augmented amounts of O2-It will be important to learn whether other macro- phage-activating properties of IFN-y are also shared by somatotropin. These data expand earlier results that have shown that somatotropin is involved in the regulation of immune events in vivo (10)(11). Macrophages are central to the induc- tion and expression of many immune re- sponses, so perhaps a fundamental mechanism of action of somatotropin is at the level of macrophages. Since we used animals that had their pituitary source of somatotropin removed, these results may be particularly important for growth hormone-deficient children who receive exogenous recombi- nant somatotropin to stimulate growth. Although excessive amounts of reactive oxy- gen metabolites can damage host tissues and can kill intracellular bacteria (20), we believe that this discovery of macrophages as one target for the action of somatotropin is important for understanding reciprocal rela- tionships between the immune and endo- crine systems (21).

The Journal of general physiology, 1993

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes... more Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from -100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance ...

Molecular Mechanisms …, 1980

Cellular Physiology and Biochemistry, 1997

Volume-regulated anion channels (VRAC) are ubiquitous ion channels that are normally nonconductin... more Volume-regulated anion channels (VRAC) are ubiquitous ion channels that are normally nonconducting, but can be opened upon cell swelling. Described initially in T lymphocytes, VRAC are characterized by activation in response to a hypotonic challenge, outward rectification of the current-voltage relationship, and a selectivity of I– > NO–3 > Br- > C1- > propionate > gluconate. Currents carried by these

eLife, Dec 14, 2017

Calcium is an essential cellular messenger that regulates numerous functions in living organisms.... more Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca, and a transgenic Careporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Casignals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Casignals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Casignals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Catransients ('sparkles') as cells migrate.

Trends in Neurosciences, 1988

Abstract Patch-clamp recording techniques have made possible the detailed study of ion channels i... more Abstract Patch-clamp recording techniques have made possible the detailed study of ion channels in cells of the immune system. These studies have revealed a number of interesting parallels between channels in the immune and nervous systems, perhaps the most intriguing of which is that channel expression and function is modifiable. Channel plasticity in immune cells takes several forms. The ensemble of K + channels expressed by T lymphocytes changes in a stereotyped way during development and after, as mature cells interact with mitogens. Adhesion to a solid substrate alters the electrical properties of macrophages. During cell activation, Ca 2+ - permeable channels are opened by inositol 1,4,5-trisphosphate (IP 3 ) in T cells and by intracellular Ca 2+ in neutrophils. These studies indicate that, both during the primary phase of differentiation and during subsequent responses to environmental stimuli, the ‘electrical phenotype' of cells in the immune system undergoes changes that may be related to cellular behavior.

The Journal of Immunology

Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice w... more Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice with experimental autoimmune arthritis was studied with the patch-clamp whole-cell recording technique in combination with fluorescence microscopy. CD4+CD8- Th cells from DBA/1 LacJ mice with type II collagen arthritis expressed low levels of type n K+ channels, and CD4-CD8+ T cells (cytotoxic) showed small numbers of type l or n' K+ channels, like their phenotypic counterparts in normal mice. CD4-CD8-Thy-1.2+ (double negative or DN) T cells from the diseased mice, however, displayed an abundance of type l K+ channels compared to DN T cells in normal mice, or mice immunized with CFA. Furthermore, the aberrant expression of type l K+ channels correlated with the presence of active disease. DN T cells from mice with SLE, type-1 diabetes mellitus, and experimental allergic encephalomyelitis, also exhibited a high number of type l K+ channels. These results suggest that expression of nume...

Biophysical Journal, 2015

Calcium signaling is often localized in spatially restricted ''microdomains,'' which may involve ... more Calcium signaling is often localized in spatially restricted ''microdomains,'' which may involve only 1-100 calcium ions. Fluctuations in the local calcium concentration can arise from calcium influx and association/dissociation with calcium buffers [Weinberg and Smith. Biophys J 106(12): 2693 (2014)]. However it is unclear to what extent these fluctuations alter calcium-dependent signaling cascades. We construct a Markov model of a calcium-dependent signaling cascade and compare the first hitting time distribution for a Markov model that accounts for calcium fluctuations, a phase-type distribution that can be calculated from the infinitesimal generator matrix, with the corresponding model that neglects these fluctuations. In general, when calcium fluctuations are much faster than the characteristic time for the signaling cascade, the distributions for the two processes are similar. However, when the time scale of calcium fluctuations is on the same order as the signaling cascade or slower, the mean and variance of the hitting time is increased, in particular when the number of calcium ions is small, a consequence of a long-tailed hitting time distribution. These ''rare events'' comprising the long tail can be significant and have a physiological impact. We further study calcium fluctuations in two settings: calcium-dependent synaptic vesicle release [Bollmann et al. Science 289, 953 (2000)] and a calcium-release site model composed of calcium-activated calcium channels [DeRemigio and Smith. Cell Calcium 38: 73 (2005)]. In these models, we demonstrate the conditions for which calcium fluctuations alter the distribution, mean, and variance of the timing for synaptic vesicle release and calcium-release site activation, respectively. Under physiological conditions, the mean hitting time can be increased orders of magnitude when calcium fluctuations are accounted for, demonstrating a significant influence on intracellular signaling.

Nature Communications, 2015

Foxp3 þ regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain in... more Foxp3 þ regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain incompletely defined. Here by two-photon (2P) imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node (LN). In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigeninduced DC:Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T-cell priming.

Science, 1986

The observation that voltage-dependent K+ channels are required for activation of human T lymphoc... more The observation that voltage-dependent K+ channels are required for activation of human T lymphocytes suggests that pathological conditions involving abnormal mitogen responses might be reflected in ion channel abnormalities. Gigaohm seal techniques were used to study T cells from MRL/MpJ-lpr/lpr mice; these mice develop generalized lymphoproliferation of functionally and phenotypically abnormal T cells and a disease resembling human systemic lupus erythematosus. The number and predominant type of K+ channels in T cells from these mice differ dramatically from those in T cells from control strains and a congenic strain lacking the lpr gene locus. Thus an abnormal pattern of ion channel expression has now been associated with a genetic defect in cells of the immune system.

Proceedings of the National Academy of Sciences, 1990

We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3)... more We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3) with coding regions contained in single uninterrupted exons. Here we have used patch-clamp recordings from Xenopus oocytes injected with mRNA to show that MK3 encodes a channel with biophysical and pharmacological properties indistinguishable from those of voltage-gated type n K+ channels in T cells. In addition, we used the polymerase chain reaction to demonstrate the presence of MK3 mRNA in T cells. These data suggest that MK3 may encode the T-cell voltage-gated type n K+ channel. We also show that MK3 and MK2 are localized on human chromosomes 13 and 12, respectively.

Proceedings of the National Academy of Sciences, 2000

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activate... more The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K + channel, IKCa1 , is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The “ IKCa1 -pharmacophore” consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar π-electron-rich heterocycle (pyrazole or tetrazole) or a −C 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 N group, whereas cytochrome P450 inhibition absol...

Proceedings of the National Academy of Sciences, 2001

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multip... more Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4 + T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype (“chronically activated”) with large numbers of Kv1.3 voltage-gated channels (≈1500 per cell) and small numbers of IKCa1 Ca 2+ -activated K + channels (≈50–120 per cell). In contrast, resting T cells displayed 0–10 Kv1.3 and 10–20 IKCa1 channels per cell (“quiescent” phenotype), whereas T cells stimulated once or twice expressed ≈200 Kv1.3 and ≈350 IKCa1 channels per cell (“acutely activated” phenotype). Consistent with their channel phenotype, [ 3 H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap 22 ) engineered to be highly specific for Kv1.3, but not by a sele...

Nature, 1984

Membrane receptors and ion transport mechanisms probably have an important role in lymphocyte act... more Membrane receptors and ion transport mechanisms probably have an important role in lymphocyte activation leading to T-lymphocyte proliferation in the immune response. Here we have applied a gigaohm-seal patch clamp technique to reveal the identity and properties of ion channels in human T lymphocytes. A voltage-dependent potassium channel bearing a resemblance to the delayed rectifier of nerve and muscle cells was found to be the predominant ion channel in these cells. In the whole cell recording conformation, the channels open with sigmoid kinetics during depolarizing voltage steps, reaching a maximum K+ conductance of 3-5 nS. The current subsequently becomes almost completely inactivated during a long-lasting depolarization. Currents through single K+ channels recorded in whole cell and outside-out patch recording conformations reveal a unitary channel conductance of about 16 pS in normal Ringer solution. Thus, the peak current corresponds to approximately 200-300 conducting K+ channels per cell. Phytohaemagglutinin (PHA), at concentrations that produce mitogenesis, alters K+ channel gating within 1 min of addition to the bathing solution, causing channels to open more rapidly and at more negative membrane potentials. 3H-thymidine incorporation by T lymphocytes following PHA stimulation is inhibited by the 'classical' K+ channel blockers tetraethylammonium and 4-aminopyridine, and also by quinine, at doses found to block the K+ channel in voltage-clamped T lymphocytes, suggesting that K+ channels may play a part in mitogenesis.

The Journal of Immunology

Antigen presentation by B cells can generate either long-lived antigen-specific CD4+ T cell toler... more Antigen presentation by B cells can generate either long-lived antigen-specific CD4+ T cell tolerance or activate T cells for additional B cell help during the humoral response. In our model, both naïve and LPS-activated B cells mOVA B cells expressing ovalbumin induced long-lived peripheral T cell tolerance, suppressing OTII T cell proliferation and T-cell-dependent antibody production in vivo. In contrast, CpG-activated mOVA B cells did not induce tolerance to OVA. Using two-photon microscopy we compared T-B cell interactions in the lymph node during tolerance induction or priming of OTII T cells mOVA B cells. Naïve, LPS- or CpG-activated mOVA B cells were individually capable of establishing long-lived interactions with OTII-T cells. Disruption of CTLA-4 / B7 interactions prevented establishment of long-lived T-B cognates and inhibited tolerance induced by LPS-treated B cells in vivo. In competition experiments with co-transferred LPS- and CpG-activated B cells, LPS- activated ...

The FASEB Journal, 2008

B cells, functioning as APC's, can provide signals to T cells either activating immunity or i... more B cells, functioning as APC's, can provide signals to T cells either activating immunity or inducing a specific hyporesponsive state (tolerance). Our laboratory has shown that B cells, when retrovirally transduced with peptide‐IgG, are highly tolerogenic for epitopes fused with the IgG. In this system, a DNA sequence encoding a full‐length protein or an immunodominant epitope is inserted into the N‐terminus of the murine IgG1 heavy chain. After injection into immunocompetent mice, peptide‐IgG transduced B cells are able to induce specific tolerance to the IgG associated antigen. This gene therapy approach has been applied to several animal models of autoimmune diseases, including EAU, EAE, and diabetes, as well as in hemophilia. In these disease models, specific tolerance was observed in both Th1 and Th2 responses. Moreover, our data indicate that the IgG heavy chain is critical for efficient tolerance induction and trafficking to endosomal processing pathways. We recently repor...

Brain, Behavior, and Immunity, 2019

Science, 1988

ministered in vivo, somatotropin induces amounts of O2similar to those produced by IFN-y at a con... more ministered in vivo, somatotropin induces amounts of O2similar to those produced by IFN-y at a concentration that is optimal for macrophage activation (18). Thus, so- matotropin shares at least one biologic activ- ity with IFN-,y, priming macrophages to produce augmented amounts of O2-It will be important to learn whether other macro- phage-activating properties of IFN-y are also shared by somatotropin. These data expand earlier results that have shown that somatotropin is involved in the regulation of immune events in vivo (10)(11). Macrophages are central to the induc- tion and expression of many immune re- sponses, so perhaps a fundamental mechanism of action of somatotropin is at the level of macrophages. Since we used animals that had their pituitary source of somatotropin removed, these results may be particularly important for growth hormone-deficient children who receive exogenous recombi- nant somatotropin to stimulate growth. Although excessive amounts of reactive oxy- gen metabolites can damage host tissues and can kill intracellular bacteria (20), we believe that this discovery of macrophages as one target for the action of somatotropin is important for understanding reciprocal rela- tionships between the immune and endo- crine systems (21).

The Journal of general physiology, 1993

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes... more Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from -100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance ...

Molecular Mechanisms …, 1980

Cellular Physiology and Biochemistry, 1997

Volume-regulated anion channels (VRAC) are ubiquitous ion channels that are normally nonconductin... more Volume-regulated anion channels (VRAC) are ubiquitous ion channels that are normally nonconducting, but can be opened upon cell swelling. Described initially in T lymphocytes, VRAC are characterized by activation in response to a hypotonic challenge, outward rectification of the current-voltage relationship, and a selectivity of I– > NO–3 > Br- > C1- > propionate > gluconate. Currents carried by these

eLife, Dec 14, 2017

Calcium is an essential cellular messenger that regulates numerous functions in living organisms.... more Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca, and a transgenic Careporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Casignals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Casignals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Casignals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Catransients ('sparkles') as cells migrate.

Trends in Neurosciences, 1988

Abstract Patch-clamp recording techniques have made possible the detailed study of ion channels i... more Abstract Patch-clamp recording techniques have made possible the detailed study of ion channels in cells of the immune system. These studies have revealed a number of interesting parallels between channels in the immune and nervous systems, perhaps the most intriguing of which is that channel expression and function is modifiable. Channel plasticity in immune cells takes several forms. The ensemble of K + channels expressed by T lymphocytes changes in a stereotyped way during development and after, as mature cells interact with mitogens. Adhesion to a solid substrate alters the electrical properties of macrophages. During cell activation, Ca 2+ - permeable channels are opened by inositol 1,4,5-trisphosphate (IP 3 ) in T cells and by intracellular Ca 2+ in neutrophils. These studies indicate that, both during the primary phase of differentiation and during subsequent responses to environmental stimuli, the ‘electrical phenotype' of cells in the immune system undergoes changes that may be related to cellular behavior.

The Journal of Immunology

Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice w... more Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice with experimental autoimmune arthritis was studied with the patch-clamp whole-cell recording technique in combination with fluorescence microscopy. CD4+CD8- Th cells from DBA/1 LacJ mice with type II collagen arthritis expressed low levels of type n K+ channels, and CD4-CD8+ T cells (cytotoxic) showed small numbers of type l or n' K+ channels, like their phenotypic counterparts in normal mice. CD4-CD8-Thy-1.2+ (double negative or DN) T cells from the diseased mice, however, displayed an abundance of type l K+ channels compared to DN T cells in normal mice, or mice immunized with CFA. Furthermore, the aberrant expression of type l K+ channels correlated with the presence of active disease. DN T cells from mice with SLE, type-1 diabetes mellitus, and experimental allergic encephalomyelitis, also exhibited a high number of type l K+ channels. These results suggest that expression of nume...

Biophysical Journal, 2015

Calcium signaling is often localized in spatially restricted ''microdomains,'' which may involve ... more Calcium signaling is often localized in spatially restricted ''microdomains,'' which may involve only 1-100 calcium ions. Fluctuations in the local calcium concentration can arise from calcium influx and association/dissociation with calcium buffers [Weinberg and Smith. Biophys J 106(12): 2693 (2014)]. However it is unclear to what extent these fluctuations alter calcium-dependent signaling cascades. We construct a Markov model of a calcium-dependent signaling cascade and compare the first hitting time distribution for a Markov model that accounts for calcium fluctuations, a phase-type distribution that can be calculated from the infinitesimal generator matrix, with the corresponding model that neglects these fluctuations. In general, when calcium fluctuations are much faster than the characteristic time for the signaling cascade, the distributions for the two processes are similar. However, when the time scale of calcium fluctuations is on the same order as the signaling cascade or slower, the mean and variance of the hitting time is increased, in particular when the number of calcium ions is small, a consequence of a long-tailed hitting time distribution. These ''rare events'' comprising the long tail can be significant and have a physiological impact. We further study calcium fluctuations in two settings: calcium-dependent synaptic vesicle release [Bollmann et al. Science 289, 953 (2000)] and a calcium-release site model composed of calcium-activated calcium channels [DeRemigio and Smith. Cell Calcium 38: 73 (2005)]. In these models, we demonstrate the conditions for which calcium fluctuations alter the distribution, mean, and variance of the timing for synaptic vesicle release and calcium-release site activation, respectively. Under physiological conditions, the mean hitting time can be increased orders of magnitude when calcium fluctuations are accounted for, demonstrating a significant influence on intracellular signaling.

Nature Communications, 2015

Foxp3 þ regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain in... more Foxp3 þ regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain incompletely defined. Here by two-photon (2P) imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node (LN). In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigeninduced DC:Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T-cell priming.

Science, 1986

The observation that voltage-dependent K+ channels are required for activation of human T lymphoc... more The observation that voltage-dependent K+ channels are required for activation of human T lymphocytes suggests that pathological conditions involving abnormal mitogen responses might be reflected in ion channel abnormalities. Gigaohm seal techniques were used to study T cells from MRL/MpJ-lpr/lpr mice; these mice develop generalized lymphoproliferation of functionally and phenotypically abnormal T cells and a disease resembling human systemic lupus erythematosus. The number and predominant type of K+ channels in T cells from these mice differ dramatically from those in T cells from control strains and a congenic strain lacking the lpr gene locus. Thus an abnormal pattern of ion channel expression has now been associated with a genetic defect in cells of the immune system.

Proceedings of the National Academy of Sciences, 1990

We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3)... more We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3) with coding regions contained in single uninterrupted exons. Here we have used patch-clamp recordings from Xenopus oocytes injected with mRNA to show that MK3 encodes a channel with biophysical and pharmacological properties indistinguishable from those of voltage-gated type n K+ channels in T cells. In addition, we used the polymerase chain reaction to demonstrate the presence of MK3 mRNA in T cells. These data suggest that MK3 may encode the T-cell voltage-gated type n K+ channel. We also show that MK3 and MK2 are localized on human chromosomes 13 and 12, respectively.

Proceedings of the National Academy of Sciences, 2000

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activate... more The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K + channel, IKCa1 , is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The “ IKCa1 -pharmacophore” consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar π-electron-rich heterocycle (pyrazole or tetrazole) or a −C 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 N group, whereas cytochrome P450 inhibition absol...

Proceedings of the National Academy of Sciences, 2001

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multip... more Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4 + T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype (“chronically activated”) with large numbers of Kv1.3 voltage-gated channels (≈1500 per cell) and small numbers of IKCa1 Ca 2+ -activated K + channels (≈50–120 per cell). In contrast, resting T cells displayed 0–10 Kv1.3 and 10–20 IKCa1 channels per cell (“quiescent” phenotype), whereas T cells stimulated once or twice expressed ≈200 Kv1.3 and ≈350 IKCa1 channels per cell (“acutely activated” phenotype). Consistent with their channel phenotype, [ 3 H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap 22 ) engineered to be highly specific for Kv1.3, but not by a sele...

Nature, 1984

Membrane receptors and ion transport mechanisms probably have an important role in lymphocyte act... more Membrane receptors and ion transport mechanisms probably have an important role in lymphocyte activation leading to T-lymphocyte proliferation in the immune response. Here we have applied a gigaohm-seal patch clamp technique to reveal the identity and properties of ion channels in human T lymphocytes. A voltage-dependent potassium channel bearing a resemblance to the delayed rectifier of nerve and muscle cells was found to be the predominant ion channel in these cells. In the whole cell recording conformation, the channels open with sigmoid kinetics during depolarizing voltage steps, reaching a maximum K+ conductance of 3-5 nS. The current subsequently becomes almost completely inactivated during a long-lasting depolarization. Currents through single K+ channels recorded in whole cell and outside-out patch recording conformations reveal a unitary channel conductance of about 16 pS in normal Ringer solution. Thus, the peak current corresponds to approximately 200-300 conducting K+ channels per cell. Phytohaemagglutinin (PHA), at concentrations that produce mitogenesis, alters K+ channel gating within 1 min of addition to the bathing solution, causing channels to open more rapidly and at more negative membrane potentials. 3H-thymidine incorporation by T lymphocytes following PHA stimulation is inhibited by the 'classical' K+ channel blockers tetraethylammonium and 4-aminopyridine, and also by quinine, at doses found to block the K+ channel in voltage-clamped T lymphocytes, suggesting that K+ channels may play a part in mitogenesis.