Regulation of B cell function by plasmacytoid dendritic cells

Dendritic cells (DCs) are early sentinels of pathogen exposure and central in the initiation and orchestration of adaptive immune responses. Apart from the prominent role of DCs in the activation of T cells, DCs have been shown to influence humoral B cell mediated responses. DCs are therefore important cells for regulating immune responses to vaccines. Many of the vaccines under development today are against pathogens such as Mycobacterium tuberculosis and HIV-1 that likely require induction of both cellular and humoral responses to cause protection. This generates a need for new safe and effective vaccine adjuvants that can stimulate such responses. One class of adjuvants that has attracted a lot of interest over the last years is TLR (toll-like receptor)-ligands. Selected TLR-ligands can specifically activate subsets of DCs and B cells according to their cognate receptor expression and therefore represent promising candidates to shape vaccine-induced responses. The first aim of this thesis was to investigate the responsiveness of B cells to TLR stimulation to proliferate and differentiate into antibody (Ab) producing cells. Furthermore, the aims were to study, whether the distinctly different myeloid (MDCs) and plasmacytoid (PDCs) can support these responses in a T helper cell-independent or dependent manner. In addition, differences in the responses of B cells and DCs from humans versus non-human primates (NHP) were addressed. In paper I, we established and refined isolation protocols for subsets of primary human PDCs, MDCs and B cells from blood and methods to examine their functions. We found that total B cells responded strongly to engagement of TLR9, less to TLR7/8 and not to engagement of TLR3. Furthermore, PDCs but not MDCs markedly enhanced B cell proliferation and differentiation into Ab producing cells in response to TLR7/8-ligand stimulation and to a lesser extent to TLR9-ligands (CpG ODN classes A, B, and C). PDCs strongly enhanced TLR7/8-ligand-induced proliferation of both memory and naive B cells but were only able to support memory cells to differentiate to CD27high plasmablasts. Type I IFN produced to high levels by PDCs was the principal mediator of the enhanced responses upon TLR7/8 stimulation. This effect may at least in part be explained by the reported upregulation of TLR7 and MyD88 by IFNα. Although MDCs expressed high levels of the known B cell growth factors IL-6, IL-10, and B cell-activating factor (BAFF) in response to TLR7/8 stimulation, they were unable to enhance B cell responses in this system. In paper II, we found that PDCs also had the ability to augment naïve B cell responses induced by BCR engagement and T cell help. The presence of supernatants from TLR-stimulated PDCs increased B cell proliferation, the frequency of B cells that differentiated to CD27high CD38high cells, and secretion of IgM. IFNα produced by PDCs was again instrumental in these processes and increased cell viability or proliferation were not main reasons for the improved B cell function. We found that PDC supernatants or IFNα induced upregulation of the co-stimulatory molecule CD86 on B cells. Further, these B cells showed improved ability to interact with and activate T cells. Thus, increased B cell responsiveness to T cell contact, mediated by PDCs via their production of IFNα, may facilitate B cell proliferation and differentiation into Ab producing cells. In order to further explore the influence of IFNα and PDCs on B cell functions in vivo in humans, models such as NHPs that more closely resemble humans than rodents need to be utilized. NHPs have the advantage that they to a large extent exhibit similar subpopulations of DC and B cell subsets as well as similar TLR expression as humans. However, similarities and potential disparities between the species need to be carefully investigated to facilitate the translation of NHP studies into clinical trials. In paper III, we therefore examined whether the effect of activated PDCs or IFNα enhanced B cell functions was comparable in human and NHP rhesus macaque cells in response to TLR ligands. We found similar responses in human and rhesus cultures to the selected TLR ligands in terms of B cell proliferation. B cell proliferation to the TLR7/8-L and CpG class C showed a significant and comparable increase in presence of IFNα. However, upon stimulation only human B cells acquired high expression of CD27, associated with plasmablast formation, although both human and rhesus B cells produced increased levels of IgM. Instead, rhesus B cell differentiation was associated with a more prominent downregulation of CD20. This validates that rhesus macaques are relevant and appropriate in vivo models to study TLR induced B cell responses although the choice of B cell differentiation markers to measure must be considered. In conclusion, the studies included in this thesis highlight the potential of PDCs and IFNα to shape B cell differentiation to Ab secreting cells. These studies add to the understanding on the role of DCs in modulation of B cell responses, which is crucial information for the design of novel vaccines, adjuvants and immuno-modulatory treatment formulations

Mdscs in Infectious diseases: regulation, roles, and readjustment

Many pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host

MxB is an interferon-induced restriction factor of human herpesviruses

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid

Plasmacytoid dendritic cells respond to Epstein-Barr virus infection with a distinct type I interferon subtype profile

Infectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8$^{+}$ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8$^{+}$ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8$^{+}$ T-cell expansion but are not required to control primary infection

Attenuated immune control of Epstein-Barr virus in humanized mice is associated with the multiple sclerosis risk factor HLA-DR15

Immune responses to Epstein-Barr virus (EBV) infection synergize with the main genetic risk factor HLA-DRB1*15:01 (HLA-DR15) to increase the likelihood to develop the autoimmune disease multiple sclerosis (MS) at least sevenfold. In order to gain insights into this synergy, we investigated HLA-DR15 positive human immune compartments after reconstitution in immune-compromised mice (humanized mice) with and without EBV infection. We detected elevated activation of both CD4$^{+}$ and CD8$^{+}$ T cells in HLA-DR15 donor-reconstituted humanized mice at steady state, even when compared to immune compartments carrying HLA-DRB1*04:01 (HLA-DR4), which is associated with other autoimmune diseases. Increased CD8$^{+}$ T cell expansion and activation was also observed in HLA-DR15 donor-reconstituted humanized mice after EBV infection. Despite this higher immune activation, EBV viral loads were less well controlled in the context of HLA-DR15. Indeed, HLA-DR15-restricted CD4$^{+}$ T cell clones recognized EBV-transformed B cell lines less efficiently and demonstrated cross-reactivity toward allogeneic target cells and one MS autoantigen. These findings suggest that EBV as one of the main environmental risk factors and HLA-DR15 as the main genetic risk factor for MS synergize by priming hyperreactive T-cell compartments, which then control the viral infection less efficiently and contain cross-reactive CD4$^{+}$ T cell clones

Epstein-barr viruses (EBVs) deficient in EBV-encoded RNAs have higher levels of latent membrane protein 2 RNA expression in lymphoblastoid cell lines and efficiently establish persistent infections in humanized mice

Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system

Virus Isolates during Acute and Chronic Human Immunodeficiency Virus Type 1 Infection Show Distinct Patterns of Sensitivity to Entry Inhibitors

We studied the effect of entry inhibitors on 58 virus isolates derived during acute and chronic infection to validate these inhibitors in vitro and to probe whether viruses at early and chronic disease stages exhibit general differences in the interaction with entry receptors. We included members of all types of inhibitors currently identified: (i) agents that block gp120 binding to CD4 (CD4-IgG(2) and monoclonal antibody [MAb] IgG(1)b12), (ii) compounds that block the interaction with CCR5 (the chemokine RANTES/CCL5, the small-molecule inhibitor AD101, and the anti-CCR5 antibody PRO 140), (iii) the fusion inhibitor enfuvirtide (T-20), and (iv) neutralizing antibodies directed against gp120 (MAb 2G12) and gp41 (MAbs 2F5 and 4E10). No differences between viruses from acute and chronic infections in the susceptibility to inhibitors targeting the CD4 binding site, CCR5, or fusion or to MAb 2G12 were apparent, rendering treatment with entry inhibitors feasible across disease stages. The notable exceptions were antibodies 2F5 and 4E10, which were more potent in inhibiting viruses from acute infection (P = 0.0088 and 0.0005, respectively), although epitopes of these MAbs were equally well preserved in both groups. Activities of these MAbs correlated significantly with each other, suggesting that common features of the viral envelope modulate their potencies

MDSCs in infectious diseases: regulation, roles, and readjustment.

International audienceMany pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host

MDSCs in infectious diseases: regulation, roles, and readjustment

Many pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host

IFN-α produced by human plasmacytoid dendritic cells enhances T cell-dependent naïve B cell differentiation

Plasmacytoid dendritic cells via their production of IFN-α render B cells more receptive to T cell contact which facilitates B cell proliferation and differentiation