Elissa K. Deenick

The good, the bad and the ugly - TFH cells in human health and disease.

Antibody production is an important feature of the vertebrate immune system. Antibodies neutralize and clear pathogens, thereby protecting against infectious diseases. Such humoral immunity has great longevity, often persisting for the hosts lifetime. Long-lived humoral immunity depends on help provided by CD4+ T cells, namely T follicular helper (TFH) cells, which support the differentiation of antigen-specific B cells into memory and plasma cells. TFH cells are stringently regulated, as aberrant TFH cell activity is involved in immunopathologies such as autoimmunity, immunodeficiencies and lymphomas. The elucidation of the mechanisms that regulate TFH cell differentiation, function and fate should highlight targets for novel therapeutics.

The origins, function, and regulation of T follicular helper cells

The generation of high-affinity antibodies (Abs) plays a critical role in the neutralization and clearance of pathogens and subsequent host survival after natural infection with a variety of microorganisms. Most currently available vaccines rely on the induction of long-lived protective humoral immune responses by memory B cells and plasma cells, underscoring the importance of Abs in host protection. Ab responses against most antigens (Ags) require interactions between B cells and CD4+ T helper cells, and it is now well recognized that T follicular helper cells (Tfh) specialize in providing cognate help to B cells and are fundamentally required for the generation of T cell–dependent B cell responses. Perturbations in the development and/or function of Tfh cells can manifest as immunopathologies, such as immunodeficiency, autoimmunity, and malignancy. Unraveling the cellular and molecular requirements underlying Tfh cell formation and maintenance will help to identify molecules that could be targeted for the treatment of immunological diseases that are characterized by insufficient or excessive Ab responses.

B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans.

Engagement of cytokine receptors by specific ligands activate Janus kinase–signal transducer and activator of transcription (STAT) signaling pathways. The exact roles of STATs in human lymphocyte behavior remain incompletely defined. Interleukin (IL)-21 activates STAT1 and STAT3 and has emerged as a potent regulator of B cell differentiation. We have studied patients with inactivating mutations in STAT1 or STAT3 to dissect their contribution to B cell function in vivo and in response to IL-21 in vitro. STAT3 mutations dramatically reduced the number of functional, antigen (Ag)-specific memory B cells and abolished the ability of IL-21 to induce naive B cells to differentiate into plasma cells (PCs). This resulted from impaired activation of the molecular machinery required for PC generation. In contrast, STAT1 deficiency had no effect on memory B cell formation in vivo or IL-21–induced immunoglobulin secretion in vitro. Thus, STAT3 plays a critical role in generating effector B cells from naive precursors in humans. STAT3-activating cytokines such as IL-21 thus underpin Ag-specific humoral immune responses and provide a mechanism for the functional antibody deficit in STAT3-deficient patients.

Early commitment of naïve human CD4 + T cells to the T follicular helper (T FH ) cell lineage is induced by IL-12

T follicular helper (TFH) cells are a specialized subset of CD4+ T cells that localize to B‐cell follicles, where they are positioned to provide help for the induction of optimal humoral immune responses. Key features of TFH cells are the expressions of CXCR5, ICOS, interleukin (IL)‐21 and BCL‐6. The requirements for human TFH cell development are unknown. Here we show that IL‐6, IL‐12, IL‐21 and IL‐23 are capable of inducing IL‐21 expression in naïve CD4+ T cells isolated from human tonsils, peripheral blood and cord blood. However, only IL‐12 induced sustained expressions of CXCR5 and ICOS on these activated naïve CD4+ T cells, and endowed them with the ability to provide increased help to B cells for their differentiation into immunoglobulin‐secreting cells. The effects of IL‐12 were independent of interferon‐γ and T‐bet, and associated with upregulation of BCL‐6 expression. Thus, these cytokines, particularly IL‐12, are likely to act at an early stage during dendritic cell‐mediated priming of naïve CD4+ T cells into a TFH cell fate, and thus underpin antibody‐mediated immunity.

Intrinsic Differences in the Proliferation of Naive and Memory Human B Cells as a Mechanism for Enhanced Secondary Immune Responses

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with IL-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division 20–30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38+ blasts in a manner that increased with consecutive division number. These differentiated CD38+ B cells divided faster than did CD38− memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38+ and CD38− cells present in cultures of memory B cells was increased by IL-10. In contrast, IL-2 enhanced proliferation of CD38− and CD38+ memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.

Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production

To identify genes and mechanisms involved in humoral immunity, we did a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified had loss-of-function mutations in the gene encoding a previously obscure member of a family of Rho-Rac GTP-exchange factors, DOCK8. DOCK8-mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutations disrupted accumulation of the integrin ligand ICAM-1 in the B cell immunological synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to Dock8 mutation provides evidence that organization of the immunological synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.

Functional STAT3 deficiency compromises the generation of human T follicular helper cells

T follicular helper (Tfh) cells are critical for providing the necessary signals to induce differentiation of B cells into memory and Ab-secreting cells. Accordingly, it is important to identify the molecular requirements for Tfh cell development and function. We previously found that IL-12 mediates the differentiation of human CD4(+) T cells to the Tfh lineage, because IL-12 induces naive human CD4(+) T cells to acquire expression of IL-21, BCL6, ICOS, and CXCR5, which typify Tfh cells. We have now examined CD4(+) T cells from patients deficient in IL-12Rβ1, TYK2, STAT1, and STAT3 to further explore the pathways involved in human Tfh cell differentiation. Although STAT1 was dispensable, mutations in IL12RB1, TYK2, or STAT3 compromised IL-12-induced expression of IL-21 by human CD4(+) T cells. Defective expression of IL-21 by STAT3-deficient CD4(+) T cells resulted in diminished B-cell helper activity in vitro. Importantly, mutations in STAT3, but not IL12RB1 or TYK2, also reduced Tfh cell generation in vivo, evidenced by decreased circulating CD4(+)CXCR5(+) T cells. These results highlight the nonredundant role of STAT3 in human Tfh cell differentiation and suggest that defective Tfh cell development and/or function contributes to the humoral defects observed in STAT3-deficient patients.

Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester

Mature T and B lymphocytes respond to receptor‐delivered signals received during and following activation. These signals regulate the rates of cell death, growth, differentiation and migration that ultimately establish the behaviour patterns collectively referred to as immune regulation. We have been pursuing the philosophy that in vitro systems of lymphocyte stimulation, when analysed quantitatively, help reveal the logical attributes of lymphocyte behaviour. The development of carboxyfluorescein diacetate succinimidyl ester (CFSE) to track division has enabled the variable of division number to be incorporated into these quantitative analyses. Our studies with CFSE have established a fundamental link between differentiation and division number. Isotype switching, expression of T cell cytokines, surface receptor alterations and changes to intracellular signalling components all display independent patterns of change with division number. The stochastic aspects of these changes and the ability of external signals to independently regulate them argue for a probabilistic modelling framework for describing and understanding immune regulation.

TCR affinity and negative regulation limit autoimmunity

Autoimmune diseases are often mediated by self-reactive T cells, which must be activated to cause immunopathology. One mechanism, known as molecular mimicry, proposes that self-reactive T cells may be activated by pathogens expressing crossreactive ligands. Here we have developed a model to investigate how the affinity of the T-cell receptor (TCR) for the activating agent influences autoimmunity. Our model shows that an approximately fivefold difference in the TCR affinity for the activating ligand results in a 50% reduction in the incidence of autoimmunity. A reduction in TCR-ligand affinity to approximately 20 times lower than normal does not induce autoimmunity despite the unexpected induction of cytotoxic T lymphocytes (CTLs) and insulitis. Furthermore, in the absence of a key negative regulatory molecule, Cbl-b, 100% of mice develop autoimmunity upon infection with viruses encoding the lower-affinity ligand. Therefore, autoimmune disease is sensitive both to the affinity of the activating ligand and to normal mechanisms that negatively regulate the immune response.

Stochastic Model of T Cell Proliferation: A Calculus Revealing IL-2 Regulation of Precursor Frequencies, Cell Cycle Time, and Survival

The outcome of Ag exposure is dictated by complex regulation of T cell proliferation. The rates of proliferation and survival are altered by numerous signals that the cell receives and integrates to achieve a net response. We have illustrated previously how small changes in kinetic parameters can lead to large differences, even under conditions of saturating IL-2. In this study, we examine the effect of varying IL-2 concentration on T cell response and develop a model incorporating additional parameters of proliferation and survival. Strikingly, the proportion of cells that enter the first division, but not the time at which they enter, is dramatically altered by IL-2. Furthermore, the survival and average division time of cells in later divisions are also altered by IL-2 concentration. Together, the small simultaneous effects on these parameters result in large differences in total cell number. These results reveal how in vitro systems may exaggerate the contribution of IL-2, and thus how costimuli or additional helper cells that alter IL-2 concentration, even by relatively small amounts, will generate large in vitro differences in cell number and therefore appear obligatory. Furthermore, they illustrate how a quantitative model of T cell activation can clarify how complex signal integration is handled by T cells in situ, and therefore more appropriately aid development of a theory of behavior.