Marcella Bothwell

CXCL13 is a plasma biomarker of germinal center activity

Significance A major challenge for vaccine science is that there is no way to measure germinal center activity in humans. This challenge is particularly acute for human clinical trials of candidate vaccines (and most nonhuman primate studies of candidate vaccines), because germinal centers are the engines of Ab affinity maturation, and generation of highly affinity-matured Ab responses is the goal of all Ab-eliciting vaccines. Here, we report that we have identified the chemokine CXCL13 [chemokine (C-X-C motif) ligand 13] as a biomarker of germinal center activity. We show explicit relationships between plasma CXCL13 concentrations and germinal center frequencies in lymph nodes in a series of different conditions, including licensed and experimental vaccines, and in humans, nonhuman primates, and mice. Significantly higher levels of plasma CXCL13 [chemokine (C-X-C motif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4+ T follicular helper (GC Tfh) cells problematic. The CXCL13–CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS+ (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.

BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms

Crotty and colleagues define the gene targets of BCL6 in primary human follicular T helper cells, revealing its primary role as a gene repressor. BCL6 bound to some loci directly and to others by interacting with AP1 and being recruited to canonical AP1-binding sites.

A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood

Detection of Ag-specific CD4+ T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of Ag-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be stingy cytokine producers, fundamentally different from Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of Ag-specific cells by intracellular cytokine staining relies on the ability of the CD4+ T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation-induced marker (AIM) methodology to identify Ag-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus–specific GC Tfh cells produced minimal detectable cytokines by intracellular cytokine staining, the AIM method identified 85-fold more Ag-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare Ag-specific CD4+ T cells in human peripheral blood. Dengue, tuberculosis, and pertussis vaccine–specific CD4+ T cells were readily detectable by AIM. In summary, cytokine assays missed 98% of Ag-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by coexpression of TCR-dependent activation markers.

Cytokine-Independent Detection of Antigen-Specific Germinal Center T Follicular Helper Cells in Immunized Nonhuman Primates Using a Live Cell Activation-Induced Marker Technique

A range of current candidate AIDS vaccine regimens are focused on generating protective HIV-neutralizing Ab responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective Ab responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GCs) are the engines of Ab affinity maturation. GC T follicular helper (Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as Ag-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining. Cytokine production by GC Tfh cells may be intrinsically limited in comparison with other Th effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify Ag-specific GC Tfh cells. RNA sequencing was performed using TCR-stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL-2Rα) and OX40 to be highly upregulated activation-induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison with intracellular cytokine staining, the AIM assay identified >10-fold more Ag-specific GC Tfh cells in HIV Env protein–immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In summary, AIM demonstrates that Ag-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function.

Novel use of polymethyl methacrylate (PMMA) microspheres in the treatment of infraorbital rhytids.

To demonstrate safety with the use of polymethyl methacrylate (PMMA) microspheres in the infraorbital eyelid area using a deliberate conservative injection in the treatment of rhytids.

Evidence that recurrent Group A streptococcus tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant Tfh cells

One Sentence Summary Recurrent tonsillitis is a multifactorial disease associated with an aberrant tonsillar germinal center response to Group A Streptococcus. ABSTRACT Recurrent Group A Streptococcus (GAS) tonsillitis (RT) is a common indication for pediatric tonsillectomy. ‘Strep throat’ is highly prevalent among children; yet, it is unknown why some children develop RT. To gain insights into this classic childhood disease, we performed phenotypic, genotypic, and functional studies on pediatric GAS RT and non-RT tonsils. We observed significantly smaller germinal centers in GAS RT tonsils, and underrepresentation of GAS-specific germinal center follicular helper (GC Tfh) CD4+ T cells. RT children exhibited reduced antibody responses to GAS virulence factor SpeA. Risk and protective HLA Class II alleles for RT were identified. Finally, SpeA induced granzyme B+ GC Tfh cells in RT tonsils that had capacity to kill B cells. Together, these observations suggest that RT susceptibility can occur due to genetic differences that can result in aberrant GC Tfh cells and poor antibody responses to GAS SpeA.

Long-term outcomes of tonsillectomy in children with periodic fever, aphthous stomatitis, pharyngitis, adenitis (PFAPA) syndrome

Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is an inflammatory disorder of childhood classically characterized by recurrent fevers, pharyngitis, stomatitis, cervical adenitis and leukocytosis. Little is known about the true incidence, natural course, pathogenesis, and appropriate therapy in this recently described syndrome. While the mechanism is unclear, previous studies have shown that tonsillectomy can be a therapeutic option with improvement in quality of life in many patients with PFAPA, but long-term clinical follow up is lacking.

Immune dysregulation in Periodic Fever, Aphthous atomatitis, Pharyngitis, Adenitis (PFAPA) syndrome

Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is an autoinflammatory disorder of childhood and little is known about the underlying etiology, pathogenesis or the reason behind the success of tonsillectomy. Numerous immune cell types are present in the tonsillar microenvironment, each of which may contribute to the pattern of symptoms observed in PFAPA patients.

PW03-009 – Genetics of PFAPA syndrome.

Periodic Fever, Aphthous stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome is an autoinflammatory disorder of childhood and little is known about the underlying etiology. While mutations involving the IL-1 pathway have been identified in other recurrent fever disorders, including TNF-receptor associated periodic syndrome (TRAPS) and cryopyrin-associated periodic syndrome (CAPS), PFAPA syndrome is not traditionally considered to be a hereditary fever disorder.