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Faculty of 1000 Biology - Reviews  
149



149. Cahalan, M.D. and K.G. Chandy. 2009.
The functional network of ion channels in T lymphocytes.
Immunological Reviews 231: 59-87.  pdf

Faculty Member Comments  




Markus Hoth

Saarland University, Germany
Cell Biology
 Evaluated 03 mar 2010









In their excellent and comprehensive review, Cahalan and Chandy summarize the ion channel network of T lymphocytes. They analyze and discuss the functional implications of all ion channels known so far in T cells. Not many researchers have considered, so far, analyzing ion channels in T cell subsets. Pretty much all labs just analyze 'The T cell', which is usually a T helper cell line. Cahalan and Chandy highlight the importance and, of course, the challenge of subset-specific analysis to fully understand the role of ion channels in T cell function. They furthermore address the importance of ion channel localization in different membrane areas, for instance at the immunological synapse. The potential importance of this point is neglected by most groups. They propose the opportunities for immunotherapy by targeting ion channels (best done differentially in T cell subsets). Here, two of the best experts in lymphocyte ion channel research summarize the know-how of lymphocyte ion channel research accumulated during the last 25 years or so in this exceptionally well-written review. This article reads extremely well and is easy to understand for non-expert readers, but, on the other hand, provides important and detailed information for expert readers in the field. In addition, new and interesting ideas are proposed. It highlights the importance of the fact that ion channel research in T cells is not simply measuring ion currents under defined conditions in the whole-cell patch-clamp configuration under artificial conditions. We need to develop tools and ideas to analyze ion channels under conditions as closely as possible mimicking physiological, and potentially in vivo, conditions. Currently, almost nothing is known about subset-specific ion channel expression in T cells, even less is known about the functional importance of ion channels in T cell subsets, and nothing is known about the potential physiological role of the differential localization of ion channels in T cells. This review sets the stage for future ion channel research and suggests the functional implications of this research for T lymphocyte activation.












145




145. Penna, A., A. Demuro, A.V. Yeromin, S.L. Zhang, O. Safrina, I. Parker, and M.D. Cahalan. 2008.
The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.
Nature 456: 116-120. pdf


Faculty Member Comments  



Stéphane Bécart, Amnon Altman

La Jolla Institute for Allergy and Immunology, USA
Immunology
 Evaluated 28 Oct 2008
Together with a parallel paper (see Ji et al. {1}), this work establishes the stoichiometry of the Ca2+ release-activated Ca2+ (CRAC) channel, a finding critical for understanding the process of CRAC channel assembly and function. The molecular identity of the lymphocyte CRAC channel has long been a mystery, but two essential components, the ER Ca2+ sensor Stim1 and the plasma membrane pore-forming subunit Orai1/CRACM1, were recently identified; however, how these subunits assemble to form an active CRAC channel has been controversial. Using a combination of biochemical approaches and total internal reflection (TIRF) microscopy, the authors demonstrate that under resting conditions Orai1 is predominantly a dimer in the plasma membrane, whereas in the activated state the CRAC channel consists of a tetramer formed by Stim1-induced dimerization of Orai1 dimers. Reference: Ji et al. Proc Natl Acad Sci USA 2008, 105:13668-73 [PMID:18757751].






142

142. Lioudyno, M.I., J.A. Kozak. A. Penna, O. Safrina, S.L. Zhang, D. Sen, J. Roos, K.A. Stauderman, and M.D. Cahalan. 2008.
Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation.
Proceedings of the National Academy of Sciences  U.S.A. 105:2011-2016.  pdf

Faculty Member Comments



Indu Ambudkar

Molecular Physiology and Therapeutics Branch NIDCR, NIH, USA Physiology
 Evaluated04 Mar 2008


This study is very interesting since it demonstrates the molecular basis of spatially regulated calcium signalling that is critical in the activation of T lymphocytes. This study shows that, in T lymphocytes, activation of the antigen receptor provides the cue for recruitment of CRAC channel proteins Orai1 and STIM1, and thus for the assembly of functional CRAC channels, at the immunological synapse -- i.e. the T cell/antigen-presenting cell interface. CRAC channels are generated by association of the pore-forming protein, Orai1, with the regulatory ER-Ca2+ sensor protein, STIM1. Previous studies have shown that STIM1 is recruited to plasma membrane domains when intracellular Ca2+ stores are depleted, where it interacts with and activates store-operated calcium channels such as CRAC. The nature of the plasma membrane domains involved in this recruitment is not yet known. The findings in this study are consistent with accumulation of functional CRAC channel components promoting localized Ca2+ influx at the immunological synapse, which is critical for T cell activation. Finally, long-term calcium signalling at this location induces a feedforward regulation of the channel components, which contributes to increased response strength to subsequent encounters with antigen.
139

139. Matheu, M.P., J.A. Deane, I. Parker, D.A. Fruman, and M.D. Cahalan. 2007.
Class IA phosphoinositide 3-kinase modulates basal lymphocyte motility in the lymph node. Journal of Immunology 179: 2261-2269.
Journal of Immunology 179: 2261-2269. pdf


Faculty Member Comments  








Steve Ward

University of Bath,
United Kingdom
IMMUNOLOGY Evaluated 20 Aug 2007

Using pharmacological and genetic approaches, the authors reveal an unexpected specific contribution of different class IA regulatory isoforms to basal T and B lymphocyte motility. With the aid of two photon cell imaging, this study demonstrates distinct roles for PI3K enzymatic activity and class IA regulatory subunit function during basal lymphocyte motility within lymph nodes. Deletion of the p85alpha regulatory isoform resulted in a striking alteration of B lymphocyte cell motility and morphology, whereas only modest effects were observed in T cells. However, deletion of the p85beta regulatory isoform resulted in a much larger reduction of T cell motility than in B lymphocytes. Complete ablation of all class IA p85 isoforms in T cells resulted in an additive reduction in velocity and a reduced ability to polarise. This study provides an interesting counter-view to that recently proposed elsewhere {1} concerning the role of PI3K in T cell migration within the lymph node. Reference: {1} Asperti-Boursin et al. J Exp Med 2007, 204:1167-79 [PMID:17485513].
135






135. Yeromin, A.V., S.L. Zhang, W. Jiang, Y. Yu, O. Safrina, and M.D. Cahalan. 2006.
Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.
Nature 443:226-229.   pdf

 


Faculty Member Comments  










Ian Hall

University Hospital of Nottingham, UK
Pharm. & Drug Disc.

Evaluated 05 oct 2006

This is one of two papers (see also {1}) in the same issue of Nature describing for the first time the role of Ora1 in contributing to store-operated calcium entry. This mechanism is important for the activation of T cells, and mutations in the gene for Ora1 (also known as olf-186-F) are associated with a severe combined immunodeficiency syndrome. In this paper, the authors show using site-directed mutagenesis that Ora-1 itself is likely to form the Ca2+ selectivity filter of the CRAC channel. Reference: {1} Prakriya et al. Nature 2006, 443:230-233 [PMID:16921383].



Anthony Means

Duke University Medical Center, USA
Cell Biology
 
Evaluated 05 oct 2006

I found this article and the accompanying one by Prakriya et al. {1} fascinating because they identify Orai 1 as the Ca2+-selectivity filter that is essential to the function of the Ca2+ release-activated Ca2+ (CRAC) channels. For many years there has been a quest to identify the proteins required for store-operated Ca2+ influx and Ca2+ release-activated channels that are opened in the plasma membrane upon the release of Ca2+ from the endoplasmic reticulum (ER) and function to refill this essential Ca2+ store. These papers demonstrate that Orai 1 is an essential link between the transmembrane STIM proteins, which sense ER Ca2+ and trigger CRAC activity, because it functions as the Ca2+ selectivity filter of the CRAC channel. Loss of function mutatons in Orai 1 result in severe combined immunodeficiency disease which links CRAC channel function to human disease. Reference: {1} Prakriya et al. Nature 2006, 443:230-233
Angus Nairn

Yale School of Medicine, USA
Neuroscience
 Evaluated 15 Sep 2006
In this and an accompanying paper from Hogan, Rao and coworkers {1}, the recently identified Orai protein is confirmed as the pore forming subunit of the store-operated calcium channel, CRAC. Following on from a recent flurry of high-quality papers on the topic of the molecular identification of Orai and Stim1 as critical components of CRAC, mutational analysis here confirms that Orai contains the critical calcium selectivity filter. Orai is shown to be a membrane protein with four transmembrane-spanning domains and intracellular N- and C- termini. Future studies will presumably address the question of whether CRAC is a multimer of several Orai polypeptides. {1} Prakriya et al. Nature 2006, Aug 20 [PMID:16921383].
Steve Ward

University of Bath,
United Kingdom
IMMUNOLOGY Evaluated 24 Aug 2006
One of two papers (see also {1}) that clinch the identification of Orai (CracM) as a major pore-forming subunit of the Icrac channel responsible for store-operated Ca entry (SOCE) in many cells. Orai is a novel 4 TM spanning protein recently identified by several labs as a strong candidate for the Icrac channel; these papers provide the definitive proof, by showing that point mutations in the putative pore region lead to dramatic changes in the ionic selectivity and other permeation properties of the channel. At the same time these results provide the first structural information on these novel channels. Reference: {1} Prakriya et al. Nature 2006, Aug 20, Epub ahead of print [PMID:16921383].
134

134. Sanna, M.G., S.K. Wang, P.J. Gonzalez-Cabrera, A. Don, D. Marsolais, M.P. Matheu, S.H. Wei, I. Parker, E. Jo, W.C. Cheng, M.D. Cahalan, C.H. Wong, H. Rosen. 2006.
Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo.
Nature Chemical Biology 2:434-441.  pdf


Faculty Member Comments  








Steve Ward

University of Bath,
United Kingdom
IMMUNOLOGY

Evaluated 24 Aug 2006

This paper describes the use of novel chemical probes, namely two enantiomers (R and S) of sphingosine 1-phosphate (S1P) derivatives, to examine the molecular basis underlying S1P-induced lymphopenia. Moreover, it also fuels the debate between the model of S1P "functional antagonism" vs the "endothelial cell gating' model proposed by the authors. Only the R-enantiomer is biologically active as an S1P1 receptor antagonist and vascular administration of the R-enantiomer enhances capillary leakage but does not alter bloodstream lymphocyte numbers. In vivo imaging of lymph nodes demonstrates altered motility of medullary lymphocytes after addition of S1P agonists which is reversed by co-administration of the R-enantiomer antagonist. This work therefore provides insight into therapeutic options and limitations of targeting S1P.
   

   
127  

127. Wei, S.H., H. Rosen, M.P. Matheu, M.G. Sanna, S-K. Wang, E. Jo, C-H Wong, I. Parker, and M.D. Cahalan. 2005.
S1P1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses..
Nature Immunology 6, 1228 - 1235. pdf

 

Faculty Member Comments  
 

Steve Ward

University of Bath,
United Kingdom
IMMUNOLOGY

Evaluated 12 Jan 2006

 
This is an elegant and thought-provoking paper that proposes a novel model for sphingosine-1 phosphate receptor (S1P1)-mediated inhibition of T cell egress in lymphoid tissue. Imaging of T cells in explanted lymph nodes after treatment with S1P1 agonists revealed a marked reduction of T cell migration in the lymph node medulla. This could be reversed by washout of the S1P1 agonist or addition of S1P1 antagonists, such that medullar migration and sinus entry was restored. The authors interpret their data as evidence for the existence of endothelial gates (which are closed by S1P1 agonists) through which lymphocytes pass. This work does not provide definitive evidence for endothelial gates and key questions remain unanswered; however, it certainly provides an interesting alternative view to the popular notion that S1P1 agonists cause sequestration of lymphocytes due to internalisation of the receptors.
   
       

Benjamin Cravatt

The Scripps Research Institute,
United States
CHEMICAL BIOLOGY

Evaluated 10 Jan 2006

 
This paper describes the application of innovative chemical and cell biology tools to delineate the mechanism of action of sphingosine 1-phosphate (S1P) receptors in lymphocyte transendothelial migration. Previous genetic studies had suggested that agonists of S1P receptors might inhibit lymophocyte egress by acting as 'functional antagonists' (i.e. downregulating these receptors). However, here, the authors show, through the use of two-photon imaging of lymphocyte movement in explanted lymph nodes, that S1P agonists cause a rapid block of T cell migration that can be reversed by agonist removal or the inclusion of an S1P receptor antagonist. These data suggest that S1P agonism, rather than functional antagonism, is the cause of blockade of lymphocyte egress. The authors propose a novel and provocative model for S1P agonist function whereby these reagents act on receptors expressed in endothelial cells (rather than lymphocytes) to inhibit lymphocyte egress. These studies also have important biomedical implications, as S1P receptors agonists may serve as useful immunosuppressant drugs.
   
   

   
 

126. Zhang S.L. Y. Yu, J. Roos, J.A. Kozak, T.J. Deerinck, M.H. Ellisman, K.A. Stauderman, and M.D. Cahalan. 2005.
STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane.
Nature 437: 902-905. pdf, supp fig

Faculty Member Comments  
 

Bernd Nilius

Katholieke Universiteit Leuven,
Belgium

PHYSIOLOGY

Evaluated 2 Feb 2006

 
This manuscript reports on the functional impact of the Ca2+ binding protein STIM1 (stromal interaction molecule) on store-operated (or Ca2+ release-activated) Ca2+ influx (SOC, CRAC). STIM1, when located in the membrane of the endoplasmic reticulum (ER), is likely the protein sensing the store Ca2+ content, and depletion of intracellular Ca2+ stores stimulates trafficking of STIM1 from the ER to the plasma membrane where it induces channel forming. Importantly, mutations in the EF-hand Ca2+ binding motif (the Ca2+ sensor in the ER) of STM1 constitutively activate SOC without changing the store Ca2+ content. SOC/CRAC is important for the regulation of a plethora of cells functions, ranging from gene expression to cell proliferation. STIM1 may now open a novel approach to solving the SOC/CRAC puzzle.
   
       

Anjana Rao

Harvard Medical School,
United States

CELL BIOLOGY

 

This paper and another by Liou et al. {1} both show that the EF-hand protein STIM1 is an ER-localised Ca2+ sensor that relocalises intracellularly after depletion of intracellular Ca2+ stores. However, there remains some uncertainty about whether STIM1 actually inserts into the plasma membrane following store depletion, or relocalises to sites near the plasma membrane to form part of plasma-membrane-associated protein complexes that do not dissociate after detergent lysis under non-denaturing conditions. {1} Liou et al. Curr Biol 2005 15:1235-41

Evaluated 10 Nov 2005

   
       
Colin Taylor

University of Cambridge,
United Kingdom

CELL BIOLOGY
 
STIM1 is shown to provide the Ca2+ sensor that allows the Ca2+ content of intracellular stores to regulate the widely expressed store-operated Ca2+ (SOC) entry pathway. STIM1 is required for activation of SOC and it translocates from the ER to the plasma membrane when intracellular Ca2+ stores are emptied. Mutation of the luminal EF-hand-like Ca2+ - binding site of STIM1 causes constitutive activation of SOC.

Evaluated 14 Oct 2005

   
       

Annette Dolphin

University College London,
United Kingdom

CELL BIOLOGY

Evaluated 11 Oct 2005

 
This paper reports the fascinating result that an ER resident transmembrane protein Stim1 moves to the plasma membrane when ER Ca2+ stores are depleted, and may itself represent the elusive CRAC (Ca2+ release activated calcium) channel. Stim1 contains a low affinity EF hand normally facing the ER lumen, and when this is mutated so that it no longer binds Ca2+ , ICRAC becomes constitutively activated, even when the stores are full. Furthermore, this mutant Stim1 is found in the plasma membrane. The authors present two main possibilities, either Stim1 multimerizes to form the CRAC channel or it associates with other proteins that form this channel. Either way the elusive channel is now nearer to being pinned down. Similar conclusions were also reached by Liou et al. (Curr Biol 2005, 15:1235-41 [PMID:16005298]), although the present paper goes a little further in showing convincingly that Stim1 becomes inserted into the plasma membrane.
   
       
 

124. Roos, J. DiGregorio, P.J., A.V. Yeromin, K. Ohlsen, M. Lioudyno, S. Zhang, O. Safrina, J.A. Kozak, S. Wagner, M.D. Cahalan, G. Velicelebi, and K.A. Stauderman. 2005.
STIM1, an essential and conserved component of store-operated calcium channel function.
Journal of Cell Biology 169: 435-445. pdf

Faculty Member Comments  
 
Mitsuhiko Ikura

University of Toronto, Canada
STRUCTURAL BIOLOG
 
A mystery in regulatory mechanisms underlying store-operated Ca2+ (SOC) influx has been solved! Through RNAi screens with 170 genes, the authors identified a gene that gave a substantially reduced activity in thapsigargin-activated Ca2+ entry. This gene encodes the already known membrane protein called stromal interaction molecule-1 (STIM1), which probably exists on the ER-membrane (some on the plasma membrane as well). STIM1 is now shown to play an essential role in SOC influx and may be a common component of SOC and Ca2+ release-activated Ca2+ (CRAC) channels.

Evaluated 23 May 2005
   
       
Roger Hardie

University of Cambridge,
United Kingdom

NEUROSCIENCE
 
Using an RNAi screen to search for genes required for store-operated Ca2+ (SOC) entry, just one gene (out of 170 tested), STIM1 was found to reduce Ca2+ entry in a Drosophila cell line. STIM1 is an ubiquitously expressed novel protein associated with both plasma and ER membranes; with just one transmembrane domain, it is unlikely to represent the elusive SOC channel, but the authors suggest that it may represent a Ca2+ sensor, coupling the ER and plasma membrane with its N-terminal EF hand in the ER lumen. Intriguingly, despite including all the known Drosophila TRP channels in the screen, none of these was able to reduce Ca entry.

Evaluated 9 May 2005
   
       
Colin Taylor

University of Cambridge,
United Kingdom

CELL BIOLOGY
 
This paper reports that STIM1, an integral membrane protein expressed in ER and the plasma membrane, is required for store-operated Ca2+ entry in diverse cell types. An RNAi-based screen of Drosophila S2 cells established that from many candidate proteins, only STIM1 was essential for thapsigargin-activated Ca2+ entry. STIM1 may be a ubiquitous component of store-regulated Ca2+ entry, with its EF-hand perhaps sensing the Ca2+ content of the ER.

Evaluated 4 May 2005
   
       
 

123. Okada, T., M.J. Miller, I. Parker, M.F. Krummel, M. Neighbors, S.B. Hartley, A. O’Garra, M.D. Cahalan, and J.G. Cyster.
Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells.
PLoS, Biology 3: 1047-061. pdf

Faculty Member Comments  
 
James Crowe

Vanderbilt University School of Medicine,
United States

MICROBIOLOGY
 

This paper uses of two-photon microscopy of intact lymph nodes to define the kinetics and anatomy of antigen-specific B-T cell interactions. The work demonstrates that B cells migrate in lymph nodes in a CCR7 gradient-determined fashion. The ability to define the number and duration of cell-cell interactions and the kinetics of chemotaxis leads to some fascinating images and a better understanding of the complexity of in vivo immune responses.

Evaluated 20 Jun 2005

   
       
Peter Openshaw

Imperial College London,
United Kingdom

MICROBIOLOGY
 
B cells and T cells need to engage to make a good immune response, but the way they do so has not been visualised before. Using two-photon microscopy, B cells are now seen to move towards the T cell zone of intact lymph nodes after exposure to antigen. Here, they rest for a day before starting to dance through the T cells at a speedy 9 um/min, touching many but lingering over the few that are antigen-specific. The T cells are then 'monogamous and clingy', hanging on to the B cells that continue to move, searching for yet more partners.

Evaluated 20 May 2005
   
118      
 

118. Yeromin A.V., J. Roos, K.A. Stauderman, and M.D. Cahalan. 2004.
A store-operated calcium channel in Drosophila S2 cells.
Journal of General Physiology 123: 167-182. pdf

Faculty Member Comments  
 
Roger Hardie

University of Cambridge,
United Kingdom
NEUROSCIENCE
 
A detailed electrophysiological characterization of the store-operated Ca (SOC) current in Drosophila S2 cells reveals that its properties are extrmely close to those of mammalian ICRAC. Drosophila S2 cells are ideally suited for high throughput, full genome screening using RNAi gene silencing. This system thus has great potential for the final molecular identification of the elusive SOC channels.

Evaluated 3 Feb 2004
   
       
 

117. Miller, M.J., A.S. Hejazi, S.H. Wei, I. Parker, and M.D. Cahalan. 2004.
T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node.
Proceedings of the National Academy of Sciences U.S.A. 101: 998-1003. pdf

Faculty Member Comments  
  Faculty of 1000 Biology
Andrea Sant

University of Rochester Medical Center,
United States
IMMUNOLOGY
 
This paper deals with the important issue of the mechanisms through which rare naive T cells encounter low numbers of recently emigrated antigen-bearing dendritic cells in peripheral lymph nodes. The authors employ a novel approach to activate and label peripheral dendritic cells and to follow their migration and encounter with T cells in the draining lymph nodes using two photon microscopy. Their results show that extended and rapidly moving dendrites from the dendritic cell body allow a typical dendritic cell to collectively scan up to 5000 T cells per hour. This sweeping scanning pattern of dendritic cells, coupled with rapid and random motility of T cells accounts for the ultimate contact between antigen bearing dendritic cells with rare antigen specific T cells and thus the initiation of an antigen specific T cell response.

Evaluated 10 Aug 2004
   
       
Rick Tarleton

University of Georgia,
United States
IMMUNOLOGY
 
Using two-photon microscopy and injection of CFSE (carboxyfluorescein succinimidyl ester) in alum, this study shows that in vivo labelled dendritic cells (DCs) traffic from the skin to the draining lymph node where the sweeping action of their dendrites and the random movement of T cells allow each DC to make an estimated 5000 T cell contacts per hour. At this rate, as few as 100 antigen-bearing DC were determined to be able to efficiently screen for low frequency antigen-specific T cells (1 in 10e6) in as little as 6 hours.

Evaluated 24 Feb 2004
   
       
Michael Dustin

New York University School of Medicine,
United States
IMMUNOLOGY
 
This paper develops a new and highly effective method for labeling dendritic cells in tissues for later visualization in explanted lymph nodes or detection by flow cytometry. They find that the T cell scanning rate of dendritic cells is 10-fold higher than earlier estimates, probably due to better resolution of fine, dendritic processes which make up much of dendritic cell surface area available for T cell contact. The method consists of mixing microgram amounts of carboxyfluorescein succinimidyl ester (CFSE) with alum and injecting it subcutaneously. The CFSE appears to be trapped in the alum depot such that it does not directly drain to the lymph node, but does label dermal dendritic cells that migrate and reach the lymph node after several hours. Fluorescent protein antigens could also be included in the alum suspension and could be detected associated with the CFSE labeled dendritic cells. This method is analogous to the FITC painting method for labeling Langerhan's cells. An advantage of this approach is that it allows visualization of cell populations that are directly relevant to vaccination strategies. The authors provide additional support for their earlier proposal that T cell repertoire scanning is primarily accomplished by rapid, autonomous migration of T cells through a field of slower migrating dendritic cells elaborating a high surface area to form ~300 contacts at any time and 5000 contacts per hour per dendritic cell.

Evaluated 3 Feb 2004
   
       
110  

110. Miller, M.M., S.H. Wei, I. Parker, and M.D. Cahalan. 2003.
Autonomous T cell trafficking examined in vivo using intravital two-photon microscopy.
Proceedings of the National Academy of Sciences U.S.A. 100: 2604-2609. pdf

Faculty Member Comments  
  Faculty of 1000 Biology


Michael Dustin


New York University School of Medicine,
United States
IMMUNOLOGY

Evaluated 12 Aug 2003

 
This paper reports that T lymphocytes in lymph nodes in vivo display a spectacular motility that can be described as a random walk within the T cell zones. This behavior suggests that lymphocytes encounter antigen-presenting cells through autonomous motility without a need for specific chemoattraction. This study and previous organ culture studies from the same lab have provoked a paradigm shift in thinking about the early moments of the T cell-dendritic cell interaction. T lymphocytes from DO.11 T cell receptor transgenic mice were labeled with carboxyfluorescein succinimidylester (CFSE) and were transferred into 4 week old MHC identical normal recipient mice. The mice were anesthetized and the inguinal lymph node was surgically exposed and mounted in a temperature controlled stage and the interior of the lymph node was imaged with a two-photon laser scanning microscope through natural windows in the adipose tissue surrounding the lymph node in mice of this age. Blood flow was maintained as demonstrated by the visualization of labeled T cells flowing in the microcirculation of the node. Lymph flow was not verified, but the avoidance of surgery near the node to remove adipose tissue means that afferent and efferent lymphatic were likely intact. Z-stacks were obtained of lymph nodes with sufficient time resolution to track the migration of T cells within the T cell zones. The average velocity was 10.2-11.5 micrometers/min with a peak velocity of up to 25 micrometers/min. While cells sometimes briefly paused, there appeared to be no population of immobile cells. The direction of motion appeared to be random in both parallel to the capsule and axially. While the T cell frequently moved in a straight line for ~30 micrometers before changing direction, the movement at longer time scales was well modeled by a random walk where the slot of displacement vs the square root of time was linear. The animals were maintained during anesthesia with 95% oxygen and 5% carbon dioxide, a gas mix that is used to elevate tissue oxygen levels. Therefore the relationship of tissue O2 levels in this system to those in a normal awake mouse is not known. The high rate of T cell autonomous motility described here also provides a clear model for immune surveillance in lymph nodes in which T cells may encounter many potential antigen presenting cells in search of the "antigenic needle in a haystack" to quote the authors.
   
       
Ulrich Von Andrian

Harvard Medical School,
United States
IMMUNOLOGY
 
This in vivo analysis of the migratory dynamics of T cells in inguinal lymph nodes validates earlier in vitro observations by this group in excised lymph nodes, which have shown that naive T cells migrate very rapidly and with apparently random directionality. The finding that T cells act as autonomous agents challenges the concept that interstitial chemokine gradients channel streams of migrating T cells within lymph nodes.

Evaluated 26 Mar 2003
   
       
104  

104. Miller, M.M., S.H. Wei, I. Parker, and M.D. Cahalan. 2002.
Two-photon imaging of lymphocyte motility and dynamic antigen responses in intact lymph node.
Science 296: 1869-1873. pdf

Faculty Member Comments  
  Faculty of 1000 Biology
Casey Weaver

University of Alabama at Birmingham,
United States
IMMUNOLOGY
 
This study introduces two photon-videomicroscopy to the study of T cell and B cell motility, and T cell responses to specific antigen, in explanted lymph nodes. Striking differences in the motilities of T and B cells within the lymph node architecture are directly observed. The kinetics and character of the response of T cells to specific antigen are defined.

Evaluated 1 Jul 2002
   
       
Jonathan Howard

Institut fur Genetik,
Germany
IMMUNOLOGY
 
Modern microscopic methods, especially two-photon laser scanning photography, allow direct visualisation of live fluorochrome-labelled lymphocytes trafficking in lymphoid tissues. Differentially labelled T and B cells move about in complex trajectories after entering their characteristic regions of a lymph node. The presence of an antigen specific for labelled T lymphocytes induces perturbations in their movement. In particular, stationary clusters and dynamic swarms form. Subsequently, the cells enlarge and divide. Without breaking radical new ground conceptually, these results demonstrate the validity of concepts of lymphocyte behaviour obtained with disaggregated cell mixtures. They further demonstrate the extraordinary power of the new microscopy.

Evaluated 26 Jun 2002
   
       
Matthias von Herrath

La Jolla Institute for Allergy and Immunology,
United States
IMMUNOLOGY
 
This is a very impressive advance for the live tracking of ongoing antigen-specific immune responses, and could be a first step in revolutionizing non-invasive imaging techniques for understanding immunity. Direct visualization of antigen-specific T cells and antigen-presenting cells in lymph nodes and possibly other organs in the future will improve our comprehension of the immune system.

Evaluated 14 Jun 2002
   
 
 

Maintained by
Jeff Ingeman
Physiology and Biophysics, University of California,Irvine, CA, USA, 92697-4561
Phone: (949) 824-6754 • Fax: (949) 824-3143 • Email:mcahalan@uci.edu

Modified:
07/27/2011