Lisa A. Pitcher

Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.

Th17 Cells Induce Ectopic Lymphoid Follicles in Central Nervous System Tissue Inflammation

Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögrens syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Trafficking of B Cell Antigen in Lymph Nodes

The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.

PTPH1 Is a Predominant Protein-tyrosine Phosphatase Capable of Interacting with and Dephosphorylating the T Cell Receptor ζ Subunit

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor ζ subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor ζ subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR ζ substrate (specific activity ranging from 0.23 to 40 pmol/min/μg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-ζ. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ζ ITAMs, with PTPH1 directly dephosphorylating ζ. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR ζ.

The formation and functions of the 21- and 23-kDa tyrosine-phosphorylated TCR zeta subunits.

The interaction between the T cell receptor (TCR) and its cognate antigen/major histocompatibility complex (MHC) complex activates a cascade of intracellular protein phosphorylations within the T cell. The signals are initiated by the specific phosphorylation of two tyrosine residues located in a conserved sequence motif termed an ITAM (immune receptor‐based tyrosine activation motif). There are 10 ITAMs in the TCR complex, and 6 of these ITAMs are present in the TCR ζ homodimer. Following TCR stimulation, the TCR ζ subunit forms two tyrosine‐phosphorylated intermediates of 21‐ and 23‐kDa, respectively. The dramatic and diverse biological responses of T cells are proposed to be partly regulated by the relative ratios of the 21‐ vs. 23‐kDa phosphorylated forms of TCR ζ that are induced following TCR ligation. In this review, we describe a stepwise model of ζ phosphorylation required for the formation of these two phosphorylated derivatives. We describe the kinases and phosphatases controlling these phosphorylation processes. In addition, we present some preliminary findings from ongoing studies that discuss the contributions of each phosphorylated form of ζ on T cell development, TCR signaling, T cell anergy induction, and T cell survival.

Complement-Dependent Transport of Antigen into B Cell Follicles

Since the original proposal by Fearon and Locksley (Fearon and Locksley. 1996. Science 272: 50–53) that the complement system linked innate and adaptive immunity, there has been a rapid expansion of studies on this topic. With the advance of intravital imaging, a number of recent papers revealed an additional novel pathway in which complement C3 and its receptors enhance humoral immunity through delivery of Ag to the B cell compartment. In this review, we discuss this pathway and highlight several novel exceptions recently found with a model influenza vaccine, such as mannose-binding lectin opsonization of influenza and uptake by macrophages, and the capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes.

The membrane-proximal portion of CD3 associates with the serine/threonine kinase GRK2

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 ϵ. Mass spectrometry analysis of CD3 ϵ-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 ϵ within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the α/β TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 ϵ may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.

The CD3 γε/δε signaling module provides normal T cell functions in the absence of the TCR ζ immunoreceptor tyrosine-based activation motifs

T cell receptor (TCR) signal transduction is mediated by the immunoreceptor tyrosine‐based activation motifs (ITAM). The ten ITAM in the TCR complex are distributed in two distinct signaling modules termed TCR ζζ and CD3 γϵ/δϵ. To delineate the specific role of the ζ ITAM in T cell development and TCR signal transmission, we compared the properties of T cells from different TCR ζ‐transgenic lines wherein tyrosine‐to‐phenylalanine substitutions had been introduced in the ζ subunit. These lines lack selected phosphorylated forms of TCR ζ including just p23, both p21 and p23, or all phospho‐ζ derivatives. We report herein that the efficiency of positive selection in HY TCR‐transgenic female mice was directly related to the number of ζ ITAM in the TCR. In contrast, TCR‐mediated signal transmission and T cell proliferative responses following agonist peptide stimulation were similar and independent of the ζ ITAM. Only the duration of MAPK activation was affected by multiple ζ ITAM substitutions. These results strongly suggest that the ITAM in the CD3 γϵ/δϵ module can provide normal TCR signal transmission, with ζ ITAM providing a secondary function facilitating MAPK activation and positive selection.

Uncoupling CD21 and CD19 of the B-cell coreceptor

Complement receptors (CRs) CD21 and CD35 form a coreceptor with CD19 and CD81 on murine B cells that when coligated with the B-cell receptor lowers the threshold of activation by several orders of magnitude. This intrinsic signaling role is thought to explain the impaired humoral immunity of mice bearing deficiency in CRs. However, CRs have additional roles on B cells independent of CD19, such as transport of C3-coated immune complexes and regulation of C4 and C3 convertase. To test whether association of CR with CD19 is necessary for their intrinsic activation-enhancing role, knockin mice expressing mutant receptors, Cr2Δ/Δgfp, that bind C3 ligands but do not signal through CD19 were constructed. We found that uncoupling of CR and CD19 significantly diminishes survival of germinal center B cells and secondary antibody titers. However, B memory is less impaired relative to mice bearing a complete deficiency in CRs on B cells. These findings confirm the importance of interaction of CR and CD19 for coreceptor activity in humoral immunity but identify a role for CR in B-cell memory independent of CD19.