Rachel Friedman - Academia.edu (original) (raw)

Papers by Rachel Friedman

The Journal of Immunology

Defining processes of islet infiltration and destruction in Type 1 diabetes (T1D) is important fo... more Defining processes of islet infiltration and destruction in Type 1 diabetes (T1D) is important for its prevention and preventing destruction of transplanted islets in diabetic individuals. We hypothesize that T1D onset and persistence is maintained by T cell-dendritic cell (DC) interactions in both the draining pancreatic lymph node (PLN) and the islets. In the steady-state, we found that intra-islet DCs are a uniform semi-mature population that constitutively take up β cell antigens in preparation for antigen presentation. Islet-antigen specific CD8 T cells activated in the PLN trafficked to the islets where they induced maturation of islet DCs. Leukocytic infiltration of the islets and islet DC maturation were dependent on IFN-γ production by T cells. Blockade of antigen processing following T cell activation in the PLN also blocked islet DC maturation but not islet infiltration. This suggests that the DCs require productive antigen-presenting interactions with T cells in the isle...

The Journal of Immunology

Real-time in vivo imaging has given insight into the cellular dynamics of T cell activation, but ... more Real-time in vivo imaging has given insight into the cellular dynamics of T cell activation, but less is known about receptor and signaling dynamics in vivo. Live imaging of subcellular signaling complexes has been challenging in living tissues due to low physiological molecular densities, inherent limitations of fluorophores, and strong autofluorescence of tissues. Our goal was to characterize T cell receptor (TCR) dynamics in naïve T cells in the lymph node during their first encounter with dendritic cells (DCs) bearing cognate antigen. To do so we developed a mouse expressing a GFP tagged OT-I TCR to track surface and intracellular TCR, and used image processing facilitated by co-labeling of the T cells to isolate specific fluorescence. In the absence of antigen, the TCR was mainly confined to the cell surface without consistent polarity; however, a small percentage of T cells transiently clustered their TCR. Within 30 min of antigen recognition, many T cells stopped crawling an...

The Journal of Immunology

CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell deat... more CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell death. Either granzyme A or B can act with perforin to trigger apoptosis. Granzyme B activates a ubiquitous apoptotic cascade induced by caspase cleavage, but the granzyme A pathway is largely unknown. Using affinity chromatography with recombinant mutant inactive granzyme A, we previously isolated two granzyme A-binding proteins, PHAP (putative HLA-associated protein) I and II. PHAP II, a substrate of granzyme A, is degraded within minutes of CTL attack. Two additional cytoplasmic proteins of 27 and 53 kDa bind strongly to the mutant granzyme A column, requiring 6 M urea to elute. Sequencing identified these as the monomer and dimer of hsp27, a small heat shock protein up-regulated by stress and cellular activation. Hsp27 coprecipitates with granzyme A from cytoplasmic lysates and is not a substrate of the enzyme. Hsp27 translocates to the detergent-insoluble fraction of target cells and re...

Journal of Immunology, Apr 1, 2009

Blood, 2000

Down-modulation of CD3ζ expression on CD8 T lymphocytes occurs, independently of other T-cell rec... more Down-modulation of CD3ζ expression on CD8 T lymphocytes occurs, independently of other T-cell receptor (TCR)-CD3 components, in tumor-infiltrating lymphocytes, human immunodeficiency virus infection, and autoimmune disease. These associations suggest that it might be related to chronic antigenic stimulation. CD3ζ down-modulation was found, however, in CD8 T cells that proliferate in response to acute viral infections. In 3 otherwise healthy donors with acute gastroenteritis, infectious mononucleosis, and Epstein–Barr virus/cytomegalovirus/mononucleosis, 30% to 60% of circulating CD8 T cells had down-modulated CD3ζ to below the level of detection. The CD3ζ-T cells were also CD28− but expressed the activation markers HLA-DR and CD57. CD3ζ–CD28– T cells are effector CTL because they express perforin and produce IFN-γ, but not IL-2, on activation and contain the viral-specific cytotoxic T lymphocyte (CTL). However, CD3ζ–CD28–T cells generally do not express CD25 after anti-CD3 and anti-...

Clinical and Experimental Immunology, 2019

Summary Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1... more Summary Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1D) has therapeutic potential, since targeting of T cell and B cell trafficking has been clinically effective in other autoimmune diseases. Trafficking to the islets is characterized by redundancy in adhesion molecule and chemokine usage, which has not enabled effective targeting to date. Additionally, cognate antigen is not consistently required for T cell entry into the islets throughout the progression of disease. However, myeloid cells are required to enable T cell and B cell entry into the islets, and may serve as a convergence point in the pathways controlling this process. In this review we describe current knowledge of the factors that mediate immune cell trafficking to pancreatic islets during T1D progression.

Understanding mechanisms of immune regulation is key to developing effective immunotherapies for ... more Understanding mechanisms of immune regulation is key to developing effective immunotherapies for autoimmunity and cancer; however, many regulatory mechanisms have not been elucidated. By analyzing T cell motility and activation at the disease site as well as disease progression, we examined the role of mononuclear phagocytes in driving regulation of effector T cells in type 1 diabetes and melanoma. We report that mononuclear phagocytes in the islets impair T cell responsiveness to antigen by preventing antigen-mediated T cell arrest. Mononuclear phagocytes in the autoimmune lesion express the TAM family receptor tyrosine kinase Mertk which functions in efferocytosis. Inhibition or deficiency of Mertk led to a release from T cell regulation characterized by enhanced T cell arrest in pre-diabetic islets and at the tumor site. This T cell arrest was accompanied by increased T cell-antigen presenting cell interactions as well as increased antigen experience and effector function by T ce...

Frontiers in Immunology, 2019

Nature communications, Jan 11, 2017

Antigens derived from viral infection or vaccination can persist within a host for many weeks aft... more Antigens derived from viral infection or vaccination can persist within a host for many weeks after resolution of the infection or vaccine responses. We previously identified lymphatic endothelial cells (LEC) as the repository for this antigen archival, yet LECs are unable to present their archived antigens to CD8+ T cells, and instead transfer their antigens to CD11c+ antigen-presenting cells (APC). Here we show that the exchange of archived antigens between LECs and APCs is mediated by migratory dendritic cells (DC). After vaccination, both migratory basic leucine zipper ATF-like transcription factor 3 (BatF3)-dependent and BatF3-independent DCs are responsible for antigen exchange and cross-presentation. However, exchange of archived viral antigens is mediated only by BatF3-dependent migratory DCs potentially acquiring apoptotic LECs. In conclusion, LEC-archived antigens are exchanged with migratory DCs, both directly and through LEC apoptosis, to cross-present archived antigens ...

Proceedings of the National Academy of Sciences of the United States of America, Apr 4, 2017

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector pr... more Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the im...

Journal of Clinical Medicine, 2016

B cells have been strongly implicated in the development of human type 1 diabetes and are require... more B cells have been strongly implicated in the development of human type 1 diabetes and are required for disease in the NOD mouse model. These functions are dependent on B cell antigen receptor (BCR) specificity and expression of MHC, implicating linked autoantigen recognition and presentation to effector T cells. BCR-antigen affinity requirements for participation in disease are unclear. We hypothesized that BCR affinity for the autoantigen insulin differentially affects lymphocyte functionality, including tolerance modality and the ability to acquire and become activated in the diabetogenic environment. Using combined transgenic and retrogenic heavy and light chain to create multiple insulin-binding BCRs, we demonstrate that affinity for insulin is a critical determinant of the function of these autoreactive cells. We show that both BCR affinity for insulin and genetic background affect tolerance induction in immature B cells. We also find new evidence that may explain the enigmatic ability of B cells expressing 125 anti-insulin BCR to support development of TID in NOD mice despite a reported affinity beneath requirements for binding insulin at in vivo concentrations. We report that when expressed as an antigen receptor the affinity of 125 is much higher than determined by measurements of the soluble form. Finally, we show that in vivo acquisition of insulin requires both sufficient BCR affinity and permissive host/tissue environment. We propose that a confluence of BCR affinity, pancreas environment, and B cell tolerance-regulating genes in the NOD animal allows acquisition of insulin and autoimmunity.

Journal of immunology (Baltimore, Md. : 1950), 2016

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabe... more T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the β cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA(+/+) mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.

Journal of immunology (Baltimore, Md. : 1950), 2015

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the genera... more In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c(+) B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c(+) B cells form significantly more stable interactions with T cells than...

Current opinion in cell biology, 2014

T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subseq... more T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subsequently, they must navigate to effector sites, which they enter through the process of trans-endothelial migration (TEM). During interstitial migration, T cells migrate according to one of two modes that are distinguished by the strength and sequence of adhesions and the requirement for Myosin-IIA. In contrast during TEM, T cells require Myosin-IIA for the final process of pushing their nucleus through the endothelium. A generalized model emerges with dual roles for Myosin-IIA: This motor protein acts like a tensioning or expansion spring, transmitting force across the cell cortex to sites of surface contact and also optimizing the frictional coupling with substrata by modulating the surface area of the contact. The phosphorylation and deactivation of this motor following TCR engagement can allow T cells to rapidly alter the degree to which they adhere to surfaces and to switch to a mode ...

Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2014

Defining the processes of autoimmune attack of tissues is important for inhibiting continued tiss... more Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8(+) T cells. During early-islet infiltration, T-cell interactions with CD11c(+) antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell-APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling ...

Current Topics in Microbiology and Immunology, 2009

The Journal of Immunology, 2014

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention beca... more In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention because immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration, we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune NOD mice. Using both intravital and explanted two-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was Ag dependent and due, at least in part, to Ag recognition through sustained interactions with CD11c+ APCs. As islet infiltration progressed, T cell motility bec...

Nature Immunology, 2010

During trafficking through tissues, T cells fine-tune their motility to balance the extent and du... more During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts with the need to traverse an entire organ. In vivo, Myosin-IIAdeficient T cells exhibited a triad of defects including over-adherence to high-endothelial venules, reduced interstitial migration, and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of 3-dimensional motility in microchannels revealed that the degree of confinement and Myosin-IIA function, rather than integrin adhesion as proposed by the haptokinetic model, optimize motility rate. This occurs via a Myosin-IIA-dependent rapid 'walking' motility mode using multiple small and simultaneous adhesions to the substrate, which prevent spurious and prolonged adhesions. Adhesion discrimination provided by Myosin-IIA is thus necessary for optimizing motility through complex tissues.

Seminars in Immunology, 2005

T lymphocytes are capable of rapid motility in vitro and in vivo. Upon antigen recognition, they ... more T lymphocytes are capable of rapid motility in vitro and in vivo. Upon antigen recognition, they may stop crawling and form a stable cell-cell contact called the 'immunological synapse' (IS). However, it is becoming clear that this outcome may not occur with the reliability that was once presumed. T cells, particularly naïve cells, are apparently triggered partly 'on the fly' during short contacts with peptide-MHC (pMHC) bearing antigen-presenting cells (APCs) and are also influenced in both activity and synapse duration by a multitude of external cues. Underlying the emerging issues is a paucity of data concerning the cell biology of T lymphocytes. Here, we review the molecular mechanisms of crawling and adhesion versus the various potential modes of 'stopping' in T lymphocytes. Both motility and arrest involve similar processes: adhesion, actin elongation and internal tension control, but with different coordination. We will attempt to integrate this with the known and potential external cues that signal for T cell motility versus stopping to form a synapse in vivo. Finally, we discuss how this interplay may give rise to unexpectedly complex motile and morphological behavior.

Science, 2008

The prevention of autoimmunity requires the elimination of self-reactive T cells during their dev... more The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator ( Aire ) gene. Here we report the identification of extrathymic Aire -expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen–expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

The Journal of Immunology

Defining processes of islet infiltration and destruction in Type 1 diabetes (T1D) is important fo... more Defining processes of islet infiltration and destruction in Type 1 diabetes (T1D) is important for its prevention and preventing destruction of transplanted islets in diabetic individuals. We hypothesize that T1D onset and persistence is maintained by T cell-dendritic cell (DC) interactions in both the draining pancreatic lymph node (PLN) and the islets. In the steady-state, we found that intra-islet DCs are a uniform semi-mature population that constitutively take up β cell antigens in preparation for antigen presentation. Islet-antigen specific CD8 T cells activated in the PLN trafficked to the islets where they induced maturation of islet DCs. Leukocytic infiltration of the islets and islet DC maturation were dependent on IFN-γ production by T cells. Blockade of antigen processing following T cell activation in the PLN also blocked islet DC maturation but not islet infiltration. This suggests that the DCs require productive antigen-presenting interactions with T cells in the isle...

The Journal of Immunology

Real-time in vivo imaging has given insight into the cellular dynamics of T cell activation, but ... more Real-time in vivo imaging has given insight into the cellular dynamics of T cell activation, but less is known about receptor and signaling dynamics in vivo. Live imaging of subcellular signaling complexes has been challenging in living tissues due to low physiological molecular densities, inherent limitations of fluorophores, and strong autofluorescence of tissues. Our goal was to characterize T cell receptor (TCR) dynamics in naïve T cells in the lymph node during their first encounter with dendritic cells (DCs) bearing cognate antigen. To do so we developed a mouse expressing a GFP tagged OT-I TCR to track surface and intracellular TCR, and used image processing facilitated by co-labeling of the T cells to isolate specific fluorescence. In the absence of antigen, the TCR was mainly confined to the cell surface without consistent polarity; however, a small percentage of T cells transiently clustered their TCR. Within 30 min of antigen recognition, many T cells stopped crawling an...

The Journal of Immunology

CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell deat... more CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell death. Either granzyme A or B can act with perforin to trigger apoptosis. Granzyme B activates a ubiquitous apoptotic cascade induced by caspase cleavage, but the granzyme A pathway is largely unknown. Using affinity chromatography with recombinant mutant inactive granzyme A, we previously isolated two granzyme A-binding proteins, PHAP (putative HLA-associated protein) I and II. PHAP II, a substrate of granzyme A, is degraded within minutes of CTL attack. Two additional cytoplasmic proteins of 27 and 53 kDa bind strongly to the mutant granzyme A column, requiring 6 M urea to elute. Sequencing identified these as the monomer and dimer of hsp27, a small heat shock protein up-regulated by stress and cellular activation. Hsp27 coprecipitates with granzyme A from cytoplasmic lysates and is not a substrate of the enzyme. Hsp27 translocates to the detergent-insoluble fraction of target cells and re...

Journal of Immunology, Apr 1, 2009

Blood, 2000

Down-modulation of CD3ζ expression on CD8 T lymphocytes occurs, independently of other T-cell rec... more Down-modulation of CD3ζ expression on CD8 T lymphocytes occurs, independently of other T-cell receptor (TCR)-CD3 components, in tumor-infiltrating lymphocytes, human immunodeficiency virus infection, and autoimmune disease. These associations suggest that it might be related to chronic antigenic stimulation. CD3ζ down-modulation was found, however, in CD8 T cells that proliferate in response to acute viral infections. In 3 otherwise healthy donors with acute gastroenteritis, infectious mononucleosis, and Epstein–Barr virus/cytomegalovirus/mononucleosis, 30% to 60% of circulating CD8 T cells had down-modulated CD3ζ to below the level of detection. The CD3ζ-T cells were also CD28− but expressed the activation markers HLA-DR and CD57. CD3ζ–CD28– T cells are effector CTL because they express perforin and produce IFN-γ, but not IL-2, on activation and contain the viral-specific cytotoxic T lymphocyte (CTL). However, CD3ζ–CD28–T cells generally do not express CD25 after anti-CD3 and anti-...

Clinical and Experimental Immunology, 2019

Summary Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1... more Summary Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1D) has therapeutic potential, since targeting of T cell and B cell trafficking has been clinically effective in other autoimmune diseases. Trafficking to the islets is characterized by redundancy in adhesion molecule and chemokine usage, which has not enabled effective targeting to date. Additionally, cognate antigen is not consistently required for T cell entry into the islets throughout the progression of disease. However, myeloid cells are required to enable T cell and B cell entry into the islets, and may serve as a convergence point in the pathways controlling this process. In this review we describe current knowledge of the factors that mediate immune cell trafficking to pancreatic islets during T1D progression.

Understanding mechanisms of immune regulation is key to developing effective immunotherapies for ... more Understanding mechanisms of immune regulation is key to developing effective immunotherapies for autoimmunity and cancer; however, many regulatory mechanisms have not been elucidated. By analyzing T cell motility and activation at the disease site as well as disease progression, we examined the role of mononuclear phagocytes in driving regulation of effector T cells in type 1 diabetes and melanoma. We report that mononuclear phagocytes in the islets impair T cell responsiveness to antigen by preventing antigen-mediated T cell arrest. Mononuclear phagocytes in the autoimmune lesion express the TAM family receptor tyrosine kinase Mertk which functions in efferocytosis. Inhibition or deficiency of Mertk led to a release from T cell regulation characterized by enhanced T cell arrest in pre-diabetic islets and at the tumor site. This T cell arrest was accompanied by increased T cell-antigen presenting cell interactions as well as increased antigen experience and effector function by T ce...

Frontiers in Immunology, 2019

Nature communications, Jan 11, 2017

Antigens derived from viral infection or vaccination can persist within a host for many weeks aft... more Antigens derived from viral infection or vaccination can persist within a host for many weeks after resolution of the infection or vaccine responses. We previously identified lymphatic endothelial cells (LEC) as the repository for this antigen archival, yet LECs are unable to present their archived antigens to CD8+ T cells, and instead transfer their antigens to CD11c+ antigen-presenting cells (APC). Here we show that the exchange of archived antigens between LECs and APCs is mediated by migratory dendritic cells (DC). After vaccination, both migratory basic leucine zipper ATF-like transcription factor 3 (BatF3)-dependent and BatF3-independent DCs are responsible for antigen exchange and cross-presentation. However, exchange of archived viral antigens is mediated only by BatF3-dependent migratory DCs potentially acquiring apoptotic LECs. In conclusion, LEC-archived antigens are exchanged with migratory DCs, both directly and through LEC apoptosis, to cross-present archived antigens ...

Proceedings of the National Academy of Sciences of the United States of America, Apr 4, 2017

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector pr... more Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the im...

Journal of Clinical Medicine, 2016

B cells have been strongly implicated in the development of human type 1 diabetes and are require... more B cells have been strongly implicated in the development of human type 1 diabetes and are required for disease in the NOD mouse model. These functions are dependent on B cell antigen receptor (BCR) specificity and expression of MHC, implicating linked autoantigen recognition and presentation to effector T cells. BCR-antigen affinity requirements for participation in disease are unclear. We hypothesized that BCR affinity for the autoantigen insulin differentially affects lymphocyte functionality, including tolerance modality and the ability to acquire and become activated in the diabetogenic environment. Using combined transgenic and retrogenic heavy and light chain to create multiple insulin-binding BCRs, we demonstrate that affinity for insulin is a critical determinant of the function of these autoreactive cells. We show that both BCR affinity for insulin and genetic background affect tolerance induction in immature B cells. We also find new evidence that may explain the enigmatic ability of B cells expressing 125 anti-insulin BCR to support development of TID in NOD mice despite a reported affinity beneath requirements for binding insulin at in vivo concentrations. We report that when expressed as an antigen receptor the affinity of 125 is much higher than determined by measurements of the soluble form. Finally, we show that in vivo acquisition of insulin requires both sufficient BCR affinity and permissive host/tissue environment. We propose that a confluence of BCR affinity, pancreas environment, and B cell tolerance-regulating genes in the NOD animal allows acquisition of insulin and autoimmunity.

Journal of immunology (Baltimore, Md. : 1950), 2016

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabe... more T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the β cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA(+/+) mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.

Journal of immunology (Baltimore, Md. : 1950), 2015

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the genera... more In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c(+) B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c(+) B cells form significantly more stable interactions with T cells than...

Current opinion in cell biology, 2014

T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subseq... more T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subsequently, they must navigate to effector sites, which they enter through the process of trans-endothelial migration (TEM). During interstitial migration, T cells migrate according to one of two modes that are distinguished by the strength and sequence of adhesions and the requirement for Myosin-IIA. In contrast during TEM, T cells require Myosin-IIA for the final process of pushing their nucleus through the endothelium. A generalized model emerges with dual roles for Myosin-IIA: This motor protein acts like a tensioning or expansion spring, transmitting force across the cell cortex to sites of surface contact and also optimizing the frictional coupling with substrata by modulating the surface area of the contact. The phosphorylation and deactivation of this motor following TCR engagement can allow T cells to rapidly alter the degree to which they adhere to surfaces and to switch to a mode ...

Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2014

Defining the processes of autoimmune attack of tissues is important for inhibiting continued tiss... more Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8(+) T cells. During early-islet infiltration, T-cell interactions with CD11c(+) antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell-APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling ...

Current Topics in Microbiology and Immunology, 2009

The Journal of Immunology, 2014

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention beca... more In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention because immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration, we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune NOD mice. Using both intravital and explanted two-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was Ag dependent and due, at least in part, to Ag recognition through sustained interactions with CD11c+ APCs. As islet infiltration progressed, T cell motility bec...

Nature Immunology, 2010

During trafficking through tissues, T cells fine-tune their motility to balance the extent and du... more During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts with the need to traverse an entire organ. In vivo, Myosin-IIAdeficient T cells exhibited a triad of defects including over-adherence to high-endothelial venules, reduced interstitial migration, and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of 3-dimensional motility in microchannels revealed that the degree of confinement and Myosin-IIA function, rather than integrin adhesion as proposed by the haptokinetic model, optimize motility rate. This occurs via a Myosin-IIA-dependent rapid 'walking' motility mode using multiple small and simultaneous adhesions to the substrate, which prevent spurious and prolonged adhesions. Adhesion discrimination provided by Myosin-IIA is thus necessary for optimizing motility through complex tissues.

Seminars in Immunology, 2005

T lymphocytes are capable of rapid motility in vitro and in vivo. Upon antigen recognition, they ... more T lymphocytes are capable of rapid motility in vitro and in vivo. Upon antigen recognition, they may stop crawling and form a stable cell-cell contact called the 'immunological synapse' (IS). However, it is becoming clear that this outcome may not occur with the reliability that was once presumed. T cells, particularly naïve cells, are apparently triggered partly 'on the fly' during short contacts with peptide-MHC (pMHC) bearing antigen-presenting cells (APCs) and are also influenced in both activity and synapse duration by a multitude of external cues. Underlying the emerging issues is a paucity of data concerning the cell biology of T lymphocytes. Here, we review the molecular mechanisms of crawling and adhesion versus the various potential modes of 'stopping' in T lymphocytes. Both motility and arrest involve similar processes: adhesion, actin elongation and internal tension control, but with different coordination. We will attempt to integrate this with the known and potential external cues that signal for T cell motility versus stopping to form a synapse in vivo. Finally, we discuss how this interplay may give rise to unexpectedly complex motile and morphological behavior.

Science, 2008

The prevention of autoimmunity requires the elimination of self-reactive T cells during their dev... more The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator ( Aire ) gene. Here we report the identification of extrathymic Aire -expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen–expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.