Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing effective T-independent antibody responses to microbial pathogens, allergens and vaccines.

In T lymphocytes, Ca²⁺ release-activated Ca²⁺ (CRAC) channels composed of Orai1 subunits trigger antigen-induced gene expression and cell proliferation through the NFAT pathway. We evaluated the requirement of CRAC channel function for lymphocyte homing using expression of a dominant-negative Orai1-E106A mutant to suppress Ca²⁺ signaling. To investigate homing and motility of human lymphocytes in immunocompromised mouse hosts, we transferred human lymphocytes either acutely or following stable engraftment after a second transfer from the same blood donor. Human and mouse lymphocyte homing was assessed and cells were tracked within lymph nodes by two-photon microscopy. Our results demonstrate that human T and B lymphocytes home into and migrate within the lymph nodes of immunocompromised NOD.SCID mice similar to murine lymphocytes. Human T and B cells colocalized in atrophied or reconstituted mouse lymph nodes, where T cells migrated in a random walk at velocities of 9-13 μm/min and B cells at 6 μm/min. Expression of Orai1-E106A inhibited CRAC channel function in human and mouse T cells and prevented homing from high endothelial venules into murine lymph nodes. Ca²⁺ signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel-mediated Ca²⁺ influx for T cell homing to lymph nodes mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.

B-cell–induced peripheral T-cell tolerance is characterized by suppression of T-cell proliferation and T-cell–dependent antibody production. However, the cellular interactions that underlie tolerance induction have not been identified. Using two-photon microscopy of lymph nodes we show that tolerogenic LPS-activated membrane-bound ovalbumin (mOVA) B cells (LPS B cells) establish long-lived, highly motile conjugate pairs with responding antigen-specific OTII T cells but not with antigen-irrelevant T cells. Treatment with anti–CTLA-4 disrupts persistent B-cell–T-cell (B–T) contacts and suppresses antigen-specific tolerance. Nontolerogenic CpG-activated mOVA B cells (CpG B cells) also form prolonged, motile conjugates with responding OTII T cells when transferred separately. However, when both tolerogenic and nontolerogenic B-cell populations are present, LPS B cells suppress long-lived CpG B–OTII T-cell interactions and exhibit tolerogenic dominance. Contact of LPS B cells with previously established B–T pairs resulted in partner-swapping events in which LPS B cells preferentially migrate toward and disrupt nontolerogenic CpG mOVA B-cell–OTII T-cell pairs. Our results demonstrate that establishment of peripheral T-cell tolerance involves physical engagement of B cells with the responding T-cell population, acting in a directed and competitive manner to alter the functional outcome of B–T interactions.

Ca²⁺ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca²⁺ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca²⁺ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca²⁺ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca²⁺ transients: short-duration 'sparkles' and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca²⁺ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (T_H1) and T_H17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-β (TGFβ) signaling and an expanded pool of regulatory T (T_reg) cells. Moreover, mice with deletion of Piezo1 specifically in T_reg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains T_reg cells, without influencing activation events or effector T cell functions.