Current projects

   

The control of T cell fate in the thymus

During thymic development, T cell precursors migrate to the thymus where they proliferate, rearrange their antigen receptor genes, and eventually give rise to the mature T cell subsets. During this process, the thymocytes are subject to a selection process that results in the death of ~99% of the cells and that shapes the mature T cell repertoire. We are investigating the mechanism that lead to positive and negative selection of T cells in the thymus, as well as the signaling events that control to cell fate decisions, including the CD4 versus CD8 cell lineage choice.

In vitro systems for T cell development

We are also developing in vitro systems to support the development of mouse and human T cells from blood stem cells and embryonic stem cells. Such systems should enable us to probe signaling events that control T cell fate decisions in more detail than can be achieved in vivo, and may eventually provide a source for defined human T cells populations for therapeutic purposes. 

Immune responses to parasitic infection

Our lab is investigating host-pathogen interactions using a mouse infection model of the intracellular parasite, Toxoplasma gondii. We have established mouse infection models that enable us to quantitiate immune responses to the parasites in vivo and to visualize immune responses in real-time. Our ongoing efforts in this area are focused on examining CD8 T cell during priming and effector phases of the immune response, and to examining immune protection during chronic infection. 

 

Research

T cell development 

During thymic development, early T cell progenitors arrive from the bloodstream and undergo a series of migration, proliferation and differentiation events in the thymus before returning to the circulation as mature T cells. Each of these T cell–maturation events takes place in a discrete region of the thymus and relies on interactions with specialized resident cells of the thymus found in each of these anatomical regions. The positive selection of T cells occurs mainly in the outer area of the thymus called the cortex and involves weak recognition of self peptide bound to major histocompatibility complex (MHC) proteins expressed on cortical thymic epithelial cells (TECs) by the newly formed  T cell receptors (TCRs) on cortical thymocytes, resulting in thymocyte survival and maturation. The central medullary region of the thymus is the main site for negative selection, which occurs via strong recognition of self peptide–MHC complexes displayed on other cells, including thymic dendritic cells and medullary TECs, resulting in thymocyte apoptosis.

jj

 

Positive and negative selection 

The last couple of years have seen immense progress in our understanding of the cellular and molecular processes occurring during T cell development in the thymus. Now, thanks to two-photon laser scanning microscopy, we know that T cell development is a dynamic process and thymocytes are in constant motion, searching for microenvironments supportive of the next step of their differentiation. The earliest T cell progenitors enter the thymus from the large blood vessels at the border between cortex and medulla and migrate towards the capsule of the organ. Close to the capsule, they undergo rearrangement of their T cell receptor β  (TCRβ) chain and only the ones that successfully do so emerge to proliferate extensively and give rise to                 Compartimentalization of the thymus (from Ladi E, Yin X, Chtanova T & Robey EA, Nat Immunol, 2006)        CD4+CD8+double-positive (DP) thymocytes. DP cells occupy the majority of the cortex and move at low speeds via random walk trying to rearrange their TCRα chain.  The expression of the newly formed TCR on the cell surface is the defining moment in the life of thymocytes.  Depending on its affinity to MHC-peptide complexes present on the surrounding cells three fates await the immature thymocytes.  If the TCR – MHC-peptide interaction is of intermediate affinity the cell undergoes positive selection, speeds up and heads to the medulla.  The TCRs of most DPs do not recognize any MHC-peptide with an appreciable affinity and die by neglect.  They are cleared by the phagocytes in the thymus. In contrast some cells’ TCRs have very high affinity for MHC-peptide complexes.  These cells die through a process called negative selection that ensures that only non-autoreactive T cells will be released in the circulation. The importance of the thymic selection process is illustrated by the cases when it fails.  Then autoimmune diseases develop that are a major source of mortality and morbidity among people. This is why our understanding and ability to manipulate thymic selection is important for two major classes of human diseases – cancer and autoimmunity. 

The application of two-photon microscopy has made it possible to monitor and quantify thymocyte migration and cell-cell interactions in real time in three-dimensional thymic environments. Those studies have provided a dynamic view of the activity of thymocytes in the thymic environment and the relationship between positive and negative selection and thymocyte migration. 

 

See the following bibliography: 

 

Ladi E, Yin X, Chtanova T, Robey EA.

Thymic microenvironments for T cell differentiation and selection.

Nat Immunol. 2006 Apr;7(4):338-43. Review.

 

Yin X, Ladi E, Chan SW, Li O, Killeen N, Kappes DJ, Robey EA.

CCR7 expression in developing thymocytes is linked to the CD4 versus CD8 lineage decision.

J Immunol. 2007 Dec 1;179(11):7358-64. 

 

Ladi E, Schwickert TA, Chtanova T, Chen Y, Herzmark P, Yin X, Aaron H, Chan SW, Lipp M, Roysam B, Robey EA.

Thymocyte-dendritic cell interactions near sources of CCR7 ligands in the thymic cortex.

J Immunol. 2008 Nov 15;181(10):7014-23. 

 

Le Borgne M, Ladi E, Dzhagalov I, Herzmark P, Liao YF, Chakraborty AK, Robey EA.

The impact of negative selection on thymocyte migration in the medulla.

Nat Immunol. 2009 Aug;10(8):823-30. 

 

Dzhagalov I, Robey EA.

Multitasking in the medulla.

Nat Immunol. 2010 Jun; 11(6):461-2.

 

Melichar HJ, Li O, Herzmark P, Padmanabhan RK, Oliaro J, Ludford-Menting MJ, Bousso P, Russell SM, Roysam B, Robey EA.

Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues.

Immunol Cell Biol. 2011 May; 89(4):549-57.

 


 

Immune responses to Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii provides an excellent experimental system for the study of T cell responses during infection. T. gondii infects a wide range of avian and mammalian species and can cause severe disease in humans. The parasite is highly adapted to its mammalian hosts and has evolved mechanisms to modulate host immunity to promote parasite spread and persistence while avoiding excessive mortality. During the acute phase of infection, parasites disseminate widely through the body via lymphatics and circulation, but are eventually brought under control by a robust adaptive immune response. Parasites then persist as cysts in brain and muscle for the lifetime of the host.   

jj

Protection from toxoplasmosis is mediated by CD8+ T cells that play a key protective role in both the acute and chronic phases of infection, despite the fact that parasites reside in a specialized parasitophorous vacuole that limits access of parasite antigens to the host cytosol and the class I presentation pathway.    

 

Proinflammatory cytokines produced by lymphocytes are crucial for controlling T. gondii infection. Cytokines such as tumor necrosis factor, lymphotoxin and interferon mediate protection against the acute and chronic stages of toxoplasmosis. Natural killer cells as well as CD4+ T cells and CD8+ T cells, three lymphocyte subsets that produce these cytokines, have all been suggested to influence T. gondii immunity in mice and humans.

 
 
RFP-labelled T. gondii parasites (red) were observed invading a T cell
(green) during an antigen-dependent contact with another invaded cell in the
lymph node (Aa) and emerging from a rupturing cyst in the brain (Ab).
(from Coombes JL & Robey EA, Nat Rev Immunol., 2010)

 

We are using mouse infection by Toxoplasma gondii as an experimental system for understanding the mammalian immune response to an intracellular pathogen, with particular relevance for the design of vaccines for CD8-mediated protection and oral pathogens. We have identified the natural T cells epitopes recognized in both resistant (BALB/c; H-2d) and sensitive (C57Bl/6 ; H-2b) strains, and have begun to characterize the interactions between parasites, T cells, and potential antigen-presenting cells in lymph nodes.  We are currently investigating the mechanisms that make certain T cell responses effective.  We are also examining the impact of innate immune responses on CD8 T cell responses, and are using a combination of 2-photon imaging approaches and ex vivo quantitation of immune responses to examine the dynamic aspects of immune responses and the fate of parasites.

 

See the following bibliography:

  

Blanchard N, Gonzalez F, Schaeffer M, Joncker NT, Cheng T, Shastri AJ, Robey EA, Shastri N.
Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum.
Nat Immunol. 2008 Aug;9(8):937-44. 
 
Chtanova T, Schaeffer M, Han SJ, van Dooren GG, Nollmann M, Herzmark P, Chan SW, Satija H, Camfield K, Aaron H,                
Striepen B, Robey EA.
Dynamics of neutrophil migration in lymph nodes during infection.
Immunity. 2008 Sep 19;29(3):487-96. 
 
Schaeffer M, Han SJ, Chtanova T, van Dooren GG, Herzmark P, Chen Y, Roysam B, Striepen B, Robey EA.
Dynamic imaging of T cell-parasite interactions in the brains of mice chronically infected with Toxoplasma gondii.
J Immunol. 2009 May 15;182(10):6379-93.
 
Chtanova T, Han SJ, Schaeffer M, van Dooren GG, Herzmark P, Striepen B, Robey EA.
Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node.
Immunity. 2009 Aug 21;31(2):342-55.
 
Coombes JL, Robey EA.
Dynamic imaging of host-pathogen interactions in vivo.
Nat Rev Immunol. 2010 May; 10(5):353-64. Review.