T cell activation niches-Optimizing T cell effector function in inflamed and infected tissues.

Successful immunity to infection, malignancy, and tissue damage requires the coordinated recruitment of numerous immune cell subsets to target tissues. Once within the target tissue, effector T cells rely on local chemotactic cues and structural cues from the tissue matrix to navigate the tissue, interact with antigen-presenting cells, and release effector cytokines. This highly dynamic process has been "caught on camera" in situ by intravital multiphoton imaging. Initial studies revealed a surprising randomness to the pattern of T cell migration through inflamed tissues, behavior thought to facilitate chance encounters with rare antigen-bearing cells. Subsequent tissue-wide visualization has uncovered a high degree of spatial preference when it comes to T cell activation. Here, we discuss the basic tenants of a successful effector T cell activation niche, taking cues from the dynamics of Tfh positioning in the lymph node germinal center. In peripheral tissues, steady-state microanatomical organization may direct the location of "pop-up" de novo activation niches, often observed as perivascular clusters, that support early effector T cell activation. These perivascular activation niches appear to be regulated by site-specific chemokines that coordinate the recruitment of dendritic cells and other innate cells for local T cell activation, survival, and optimized effector function.

CXCL10+ peripheral activation niches couple preferred sites of Th1 entry with optimal APC encounter.

Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters are "hotspots" for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolongs T-APC interactions and boosts function. Both the frequency and range of these clusters are enhanced via a T helper 1 (Th1)-intrinsic, interferon-gamma (IFNγ)-dependent positive-feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

The CD28 Transmembrane Domain Contains an Essential Dimerization Motif.

CD28 plays a critical role in regulating immune responses both by enhancing effector T cell activation and differentiation and controlling the development and function of regulatory T cells. CD28 is expressed at the cell surface as a disulfide linked homodimer that is thought to bind ligand monovalently. How ligand binding triggers CD28 to induce intracellular signaling as well as the proximal signaling pathways that are induced are not well-understood. In addition, recent data suggest inside-out signaling initiated by the T cell antigen receptor can enhance CD28 ligand binding, possibly by inducing a rearrangement of the CD28 dimer interface to allow for bivalent binding. To understand how possible conformational changes during ligand-induced receptor triggering and inside-out signaling are mediated, we examined the CD28 transmembrane domain. We identified an evolutionarily conserved YxxxxT motif that is shared with CTLA-4 and resembles the transmembrane dimerization motif within CD3ζ. We show that the CD28 transmembrane domain can drive protein dimerization in a bacterial expression system at levels equivalent to the well-known glycophorin A transmembrane dimerization motif. In addition, ectopic expression of the CD28 transmembrane domain into monomeric human CD25 can drive dimerization in murine T cells as detected by an increase in FRET by flow cytometry. Mutation of the polar YxxxxT motif to hydrophobic leucine residues (Y145L/T150L) attenuated CD28 transmembrane mediated dimerization in both the bacterial and mammalian assays. Introduction of the Y145L/T150L mutation of the CD28 transmembrane dimerization motif into the endogenous CD28 locus by CRISPR resulted in a dramatic loss in CD28 cell surface expression. These data suggest that under physiological conditions the YxxxxT dimerization motif within the CD28 transmembrane domain plays a critical role in the assembly and/or expression of stable CD28 dimers at the cell surface.

Hyperspectral multiphoton microscopy for in vivo visualization of multiple, spectrally overlapped fluorescent labels.

The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally-distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely-overlapped fluorophores in samples containing ten different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin with up to eight different cell types or tissue structures distinguished.

Pivotal role for αV integrins in sustained Tfh support of the germinal center response for long-lived plasma cell generation.

CD4+ follicular helper T cells (Tfh) are essential for germinal center (GC) reactions in the lymph node that generate high-affinity, long-lived plasma cells (LLPCs). Temporal GC analysis suggests B memory cells (Bmem) are generated early, while LLPCs are generated late in the GC reaction. Distinct roles for Tfh at these temporally different stages are not yet clear. Tfh entry into the GC is highly dynamic and the signals that maintain Tfh within the GC for support of late LLPC production are poorly understood. The GC is marked by inflammation-induced presentation of specific ECM components. To determine if T cell recognition of these ECM components played a role in Tfh support of the GC, we immunized mice with a T cell-restricted deletion of the ECM-binding integrin αV (αV-CD4 cKO). T cell integrin αV deletion led to a striking defect in the number and size of the GCs following immunization with OVA protein in complete Freund's adjuvant. The GC defect was not due to integrin αV deficiency impeding Tfh generation or follicle entry or the ability of αV-CD4 cKO Tfh to contact and support B cell activation. Instead, integrin αV was essential for T cell-intrinsic accumulation within the GC. Altered Tfh positioning resulted in lower-affinity antibodies and a dramatic loss of LLPCs. Influenza A infection revealed that αV integrin was not required for Tfh support of Bmem but was essential for Tfh support of LLPCs. We highlight an αV integrin-ECM-guided mechanism of Tfh GC accumulation that selectively impacts GC output of LLPCs but not Bmem.

The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance.

T follicular helper (Tfh) cells are a CD4+ T cell subset critical for long-lived humoral immunity. We hypothesized that integrins play a decisive role in Tfh cell biology. Here we show that Tfh cells expressed a highly active form of leukocyte function-associated antigen-1 (LFA-1) that was required for their survival within the germinal center niche. In addition, LFA-1 promoted expression of Bcl-6, a transcriptional repressor critical for Tfh cell differentiation, and inhibition of LFA-1 abolished Tfh cell generation and prevented protective humoral immunity to intestinal helminth infection. Furthermore, we demonstrated that expression of Talin-1, an adaptor protein that regulates LFA-1 affinity, dictated Tfh versus Th2 effector cell differentiation. Collectively, our results define unique functions for LFA-1 in the Tfh cell effector program and suggest that integrin activity is important in lineage decision-making events in the adaptive immune system.

Abundance and specificity of influenza reactive circulating memory follicular helper and non-follicular helper CD4 T cells in healthy adults.

CD4 T-cell responses are functionally complex and regulate many aspects of innate and adaptive immunity. Follicular helper (Tfh) cells are CD4 T cells specialized to support B-cell production of isotype-switched, high-affinity antibody. So far, studies of Tfh cells in humans have focused on their differentiation requirements, with little research devoted to their antigen specificity. Here, after separating circulating human memory CD4 T cells based on expression of CXCR5, a signature marker of Tfh, we have quantified and assayed the influenza protein antigen specificity of blood Tfh cells and CD4 T cells lacking this marker. Through the use of peptide pools derived from nucleoprotein (NP) or haemagglutinin (HA) and a panel of human donors, we have discovered that circulating Tfh cells preferentially recognize peptide epitopes from HA while cells lacking CXCR5 are enriched for specificity toward NP. These studies suggest that reactive CD4 T cells specific for distinct viral antigens may have generalized differences in their functional potential due to their previous stimulation history.

The control of the specificity of CD4 T cell responses: thresholds, breakpoints, and ceilings.

It has been known for over 25 years that CD4 T cell responses are restricted to a finite number of peptide epitopes within pathogens or protein vaccines. These selected peptide epitopes are termed "immunodominant." Other peptides within the antigen that can bind to host MHC molecules and recruit CD4 T cells as single peptides are termed "cryptic" because they fail to induce responses when expressed in complex proteins or when in competition with other peptides during the immune response. In the last decade, our laboratory has evaluated the mechanisms that underlie the preferential specificity of CD4 T cells and have discovered that both intracellular events within antigen presenting cells, particular selective DM editing, and intercellular regulatory pathways, involving IFN-γ, indoleamine 2,3-dioxygenase, and regulatory T cells, play a role in selecting the final peptide specificity of CD4 T cells. In this review, we summarize our findings, discuss the implications of this work on responses to pathogens and vaccines and speculate on the logic of these regulatory events.

The peptide specificity of the endogenous T follicular helper cell repertoire generated after protein immunization.

T follicular helper (Tfh) cells potentiate high-affinity, class-switched antibody responses, the predominant correlate of protection from vaccines. Despite intense interest in understanding both the generation and effector functions of this lineage, little is known about the epitope specificity of Tfh cells generated during polyclonal responses. To date, studies of peptide-specific Tfh cells have relied on either the transfer of TcR transgenic cells or use of peptide:MHC class II tetramers and antibodies to stain TcR and follow limited peptide specificities. In order to comprehensively evaluate polyclonal responses generated from the natural endogenous TcR repertoire, we developed a sorting strategy to separate Tfh cells from non-Tfh cells and found that their epitope-specific responses could be tracked with cytokine-specific ELISPOT assays. The immunodominance hierarchies of Tfh and non-Tfh cells generated in response to immunization with several unrelated protein antigens were remarkably similar. Additionally, increasing the kinetic stability of peptide-MHC class II complexes enhanced the priming of both Tfh and conventional CD4 T cells. These findings may provide us with a strategy to rationally and selectively modulate epitope-specific Tfh responses. By understanding the parameters that control epitope-specific priming, vaccines may be tailored to enhance or focus Tfh responses to facilitate optimal B cell responses.

Generation of MHC class II-peptide ligands for CD4 T-cell allorecognition of MHC class II molecules.

PURPOSE OF REVIEW: The molecular and cellular mechanisms that underlie allorecognition of MHC class II molecules have been the subject of much debate and experimentation in recent decades. In this review, we discuss several aspects of MHC class II structure, peptide acquisition and TcR-MHC-peptide interactions that have particular relevance to recognition of cells bearing allogeneic class II molecules. RECENT FINDINGS: First, MHC polymorphism is heavily biased toward those amino acids that influence stable peptide binding by MHC class II. Second, the peptide repertoire presented by class II molecules is highly diverse and can be edited substantially by the molecular catalyst HLA-DM and by tissue-specific expression of HLA-DO, stress and cytokines. Third, T-cell receptor docking onto MHC peptide consistently involves substantial contacts with the bound peptide in the MHC class II molecule. Finally, there is increasing evidence that T-cell recognition of MHC is, in part, germline encoded through T-cell-receptor V region contacts with MHC class II alpha helices. SUMMARY: Together, these conclusions support the view that allorecognition of MHC class II molecules is likely to parallel key aspects of conventional CD4 T-cell recognition, with allele-dependent variation in peptide representation accounting in large part for the high precursor frequency of alloreactive CD4 T cells.