Elena Tonti

Invariant NKT cells sustain specific B cell responses and memory

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as α-galactosylceramide (αGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and αGC develop antibody titers 1–2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with αGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4+ T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.

Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus

Lymph nodes (LNs) capture microorganisms that breach the body’s external barriers and enter draining lymphatics, limiting the systemic spread of pathogens. Recent work has shown that CD11b+CD169+ macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for the clearance of viruses from the lymph and for initiating antiviral humoral immune responses. Here we show, using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the central nervous system (CNS). On local depletion of LN macrophages, about 60% of mice developed ascending paralysis and died 7–10 days after subcutaneous infection with a small dose of VSV, whereas macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system through peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially in SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but decreased type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I-producing plasmacytoid dendritic cells to the SCS and in addition were a major source of IFN-I themselves. Experiments in bone marrow chimaeric mice revealed that IFN-I must act on both haematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal infection with VSV. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral invasion of the nervous system on peripheral infection.

Systematic Discovery of TLR Signaling Components Delineates Viral-Sensing Circuits

Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.

B Cell Maintenance of Subcapsular Sinus Macrophages Protects against a Fatal Viral Infection Independent of Adaptive Immunity

Neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (VSV). Utilizing mice that possess B cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) VSV challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. However, B cells were absolutely required to provide lymphotoxin (LT) α1β2, which maintained a protective subcapsular sinus (SCS) macrophage phenotype within virus draining lymph nodes (LNs). Macrophages within the SCS of B cell-deficient LNs, or of mice that lack LTα1β2 selectively in B cells, displayed an aberrant phenotype, failed to replicate VSV, and therefore did not produce type I interferons, which were required to prevent fatal VSV invasion of intranodal nerves. Thus, although B cells are essential for survival during VSV infection, their contribution involves the provision of innate differentiation and maintenance signals to macrophages, rather than adaptive immune mechanisms.

NKT-cell help to B lymphocytes can occur independently of cognate interaction

CD4(+) T (Th)-cell help to B lymphocytes requires cognate interaction and CD40 engagement. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that recognize alphagalactosylceramide (alphaGalCer) presented by CD1d, and can help B-cell responses. We asked whether alphaGalCer-activated iNKT cells help B lymphocytes through cognate interaction, or indirectly, via enhancement of Th-B-cell interaction. After immunization with protein Ags and alphaGalCer, antibody titers were assessed in wild-type or splenectomized mice, and in bone marrow radiation chimeras lacking CD1d or CD40 expression on B lymphocytes, or expressing CD1d or MHC II disjointly on antigen-presenting cells (APCs). We find that alphaGalCer-dependent enhancement of B-cell response (1) can occur when B cells do not express CD1d but express CD40; (2) requires that iNKT and Th cells interact with the same APCs that coexpress both CD1d and MHC-II; and (3) takes place without spleen. These findings demonstrate alphaGalCer-induced help for antibody responses can occur without cognate iNKT/B-cell interaction, and suggest this help entails activation of APCs by iNKT cells, which in turn activate Th cells and their helper functions for B cells. Thus, the alphaGalCer-induced help recapitulates the function of classical adjuvants that stimulate the innate immune system to support adaptive immune responses.

Follicular Helper NKT Cells Induce Limited B Cell Responses and Germinal Center Formation in the Absence of CD4+ T Cell Help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4+ T follicular helper (TFH) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4+ T cells (MHC II−/−) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II−/− mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II−/− mice display a typical CXCR5hiprogrammed death-1hiICOShiBcl-6hi TFH phenotype and induce germinal centers. The specific IgG response induced in MHC II−/− mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKTFH cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKTFH and CD4+ TFH cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.

Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR α-chain CDR3 loop

A significant fraction of CD1d‐restricted T cells express an invariant T cell receptor (TCR) α‐chain. These highly conserved invariant NKT (iNKT) populations are important regulators of a wide spectrum of immune responses. The ability to directly identify and manipulate iNKT cells is essential to understanding their function and to exploit their therapeutic potential. To this end, we sought monoclonal and polyclonal antibodies specific for iNKT cells by immunizing CD1d KO mice, which lack iNKT cells, with a cyclic peptide modeled after the TCRα CDR3 loop. One mAb (6B11) was specific for cloned and primary human but not rodent iNKT cells and the human invariant TCRα, as shown by transfection and reactivity with human invariant TCRα transgenic T cells ex vivo and in situ. 6B11 was utilized to identify, purify, and expand iNKT cells from an otherwise minor component of human peripheral blood lymphocytes and to specifically identify human iNKT cells in tissue. Thus, we report a novel and general strategy for the generation of mAb specific for the CDR3 loop encoded by the TCR of interest. Specifically, an anti‐Vα24Jα18 CDR3 loop clonotypic TCR mAb is available for the enumeration and therapeutic manipulation of human and non‐human primate iNKT populations.

Targeted Expression of Human CD1d in Transgenic Mice Reveals Independent Roles for Thymocytes and Thymic APCs in Positive and Negative Selection of Vα14i NKT Cells

CD1d-dependent invariant Vα14 (Vα14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Vα14-Jα18 TCR α-chain paired mostly with Vβ8.2 and Vβ7. The cellular requirements for thymic positive and negative selection of Vα14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Vα14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Vα14i NKT cells that proved functional when activated ex vivo with the Ag α-galactosyl ceramide. Vα14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vβ8.2. The low number of Vβ8.2+ Vα14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vβ7+ Vα14i NKT cells. Vβ8.2+, but not Vβ7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Vα14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.

Bisphosphonates Target B Cells to Enhance Humoral Immune Responses

Bisphosphonates are a class of drugs that are widely used to inhibit loss of bone mass in patients. We show here that the administration of clinically relevant doses of bisphosphonates in mice increases antibody responses to live and inactive viruses, proteins, haptens, and existing commercial vaccine formulations. Bisphosphonates exert this adjuvant-like activity in the absence of CD4(+) and γδ T cells, neutrophils, or dendritic cells, and their effect does not rely on local macrophage depletion, Toll-like receptor signaling, or the inflammasome. Rather, bisphosphonates target directly B cells and enhance B cell expansion and antibody production upon antigen encounter. These data establish bisphosphonates as an additional class of adjuvants that boost humoral immune responses.