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Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6− circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60–80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

Macrophage-inducible C-type lectin (Mincle)–dependent sensing of pathogens triggers proinflammatory immune responses in professional phagocytes that contribute to protecting the host against pathogen invasion. In this study, we examined whether overexpression of Mincle that is designed to improve early pathogen sensing by professional phagocytes would improve lung-protective immunity against Streptococcus pneumoniae in mice. Proteomic profiling of alveolar macrophages of Mincle transgenic (tg) mice stimulated with the Mincle-specific pneumococcal ligand glucosyl-diacylglycerol (Glc-DAG) revealed increased Nlrp3 inflammasome activation and downstream IL-1β cytokine release that was not observed in Glc-DAG–stimulated Mincle knockout or Nlrp3 knockout macrophages. Along this line, Mincle tg mice also responded with a stronger Nlrp3 expression and early proinflammatory cytokine release after challenge with S. pneumoniae, ultimately leading to fatal pneumonia in the Mincle tg mice. Importantly, Nlrp3 inhibitor treatment of Mincle tg mice significantly mitigated the observed hyperinflammatory response to pneumococcal challenge. Together, we show that overexpression of the pattern recognition receptor Mincle triggers increased Glc-DAG–dependent Nlrp3 inflammasome activation in professional phagocytes leading to fatal pneumococcal pneumonia in mice that is amenable to Nlrp3 inhibitor treatment. These data show that ectopic expression of the Mincle receptor confers increased susceptibility rather than resistance to S. pneumoniae in mice, thus highlighting the importance of an inducible Mincle receptor expression in response to microbial challenge.

Appropriate T cell responses are controlled by strict balance between activatory and inhibitory pathways downstream of TCR. Although mice or humans with impaired TCR signaling develop autoimmunity, the precise molecular mechanisms linking reduced TCR signaling to autoimmunity are not fully understood. Engagement of TCR activates Ca2+ signaling mainly through store-operated Ca2+ entry activated by stromal interaction molecule (Stim) 1 and Stim2. Despite defective T cell activation, mice deficient in both Stim1 and Stim2 in T cells (conditional double knockout [cDKO]) developed lymphoproliferative disorders and skin inflammation with a concomitant increase in serum IgG1 and IgE levels. In cDKO mice, follicular helper T (Tfh) cells were dramatically increased in number, and they produced IL-4 spontaneously. These inflammatory symptoms were abolished by the deletion of IL-4 in cDKO mice. Tfh development and inflammatory symptoms in cDKO mice were abrogated by further deletion of NFAT2 in T cells. These findings suggest that Tfh cells spontaneously developed in the absence of Ca2+ signaling and caused unregulated type 2 responses.

C-type lectin receptors expressed in APCs are recently defined pattern recognition receptors that play a crucial role in immune responses against pathogen-associated molecular patterns. Among pathogen-associated molecular patterns, cord factor (trehalose-6,6′-dimycolate [TDM]) is the most potent immunostimulatory component of the mycobacterial cell wall. Two C-type lectin receptors, macrophage-inducible C-type lectin (Mincle) and macrophage C-type lectin (MCL), are required for immune responses against TDM. Previous studies indicate that MCL is required for TDM-induced Mincle expression. However, the mechanism by which MCL induces Mincle expression has not been fully understood. In this study, we demonstrate that MCL interacts with Mincle to promote its surface expression. After LPS or zymosan stimulation, MCL-deficient bone marrow–derived dendritic cells (BMDCs) had a lower level of Mincle protein expression, although mRNA expression was comparable with wild-type BMDCs. Meanwhile, BMDCs from MCL transgenic mice showed an enhanced level of Mincle expression on the cell surface. MCL was associated with Mincle through the stalk region and this region was necessary and sufficient for the enhancement of Mincle expression. This interaction appeared to be mediated by the hydrophobic repeat of MCL, as substitution of four hydrophobic residues within the stalk region with serine (MCL4S) abolished the function to enhance the surface expression of Mincle. MCL4S mutant failed to restore the defective TDM responses in MCL-deficient BMDCs. These results suggest that MCL positively regulates Mincle expression through protein–protein interaction via its stalk region, thereby magnifying Mincle-mediated signaling.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM−/− mice. CRTAM−/− mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM−/− CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.

The cross-linking of IgE-bound FcεRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcεRIγ signaling to IgE/FcεRI-mediated mast cell responses has not yet been elucidated. In this study FcεRIγ−/− mast cells were reconstituted with either wild-type or mutant FcεRIγ in transgenic mice and transfected mast cells in vitro. We demonstrate that FcεRIγ-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcεRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcεRIγ-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcεRI is independent of FcεRIγ signaling. These results indicate that FcεRIγ-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.

The pre-TCRα (pTα) is exclusively expressed in immature thymocytes and constitutes the pre-TCR complex with TCRβ, which regulates early T cell differentiation. Despite the recent identification of the pTα enhancer, the contribution of the promoter region, the direct DNA-protein interaction, and the regulation of such interaction along with T cell development have not been investigated. We analyzed the pTα promoter region and identified the critical elements for transcription of the pTα gene. The pTα promoter was found to contain two consecutive E-box elements that are critical for pTα transcription. The E-box elements in the promoter region formed the specific DNA-protein complex that was exclusively observed in immature thymocytes, not in mature thymocytes and T cells. The E proteins in this complex were identified as E2A and HeLa E-box binding protein (HEB), and overexpression of E2A and HEB resulted in activation of the pTα promoter. The binding complex in the consecutive E-boxes in the pTα promoter changed along with T cell development, as a distinct DNA-binding complex was observed in mature T cells. Comparing the E-box regions in the enhancer and the promoter, those in the promoter appear to make a greater contribution to pTα gene transcription.

Streptococcus pneumoniae possesses a thick polysaccharide capsule. Host defense to this bacterium is mediated by neutrophilic inflammatory responses, which is critically regulated by innate immune mechanism. S. pneumoniae has been reported to be sensed by pattern-recognition receptors such as TLR2, TLR4 and SIGN-R1. In this study, we examined the role of Dectin-2 in the host defense to S. pneumoniae infection. When bone marrow-derived dendritic cells from wild-type (WT) and Dectin-2KO mice were stimulated with culture supernatants (Sup) of a strain of S. pneumoniae (URF918) or its ConA-bound fraction, IL-12p40 production was abrogated in Dectin-2KO mice. In a NFAT-GFP reporter assay, Sup provided a positive signal in Dectin-2 gene-transfected reporter cells. These results suggest that Dectin-2 senses mannose-containing polysaccharides in S. pneumoniae capsule. Dectin-2KO mice infected intratracheally with S. pneumoniae showed shorter survival and larger bacterial burden and lower IFN-γ production in lungs than WT mice. Although the neutrophilic infiltration in lungs was equivalent in both mice, the ratio of engulfed/un-engulfed neutrophils was lower in KO mice than in WT mice. The levels of anti-pneumococcal polysaccharide Ab in bronchoalveolar lavage fluid and serum were lower in KO mice than WT mice. These results suggest that Dectin-2 plays critical roles in the host defense to S. pneumoniae infection through promoting the Ab-mediated phagocytosis by neutrophils.