Protection of mice deficient in mature B cells from West Nile virus infection by passive and active immunization.

B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike µMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient µMT mice, but unlike µMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in µMT mice. Thus, the immature B cells present in BAFFR-/- and not µMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.

Controlling immune responses by targeting antigens to dendritic cell subsets and B cells.

Delivering antigens in vivo by coupling them to mAbs specific for unique receptors on antigen-presenting cells (APCs) is a promising approach for modulating immune responses. Antigen delivery to receptors found on myeloid dendritic cell (DC) subsets, plasmacytoid DCs and B cells has shown them all to be viable targets to stimulate either the cellular or humoral arms of the immune system. It is now evident that antigen-targeting approaches can also be used to invoke antigen-specific inhibition of immune responses. The outcome of activation versus inhibition is determined by a combination of factors that include the choice of APC, the receptor that is targeted, whether to include an adjuvant and, if so, which adjuvant to employ. In addition to their use as a means to modulate immune responses, antigen-targeting systems are also a useful method to investigate the function of DC subsets and the early mechanistic events that underlie the initiation of both cellular and humoral immune responses. In this review, we focus on the literature surrounding the control of B-cell responses when antigen is delivered to various APC subsets.

Development of antigen cross-presentation capacity in dendritic cells.

Cross-presentation by dendritic cells (DCs) of exogenous antigens on MHC class I is important for the generation of immune responses to intracellular pathogens, as well as for maintenance of self tolerance. In mice, the CD8(+) DC lineage is specialised for this role. However, DCs of this lineage are not born with cross-presentation capacity. Several studies have demonstrated that it must be induced as a later developmental step by cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), or by microbial products such as toll-like receptor (TLR) ligands. Increased cross-presentation capacity is thus induced in peripheral CD8 lineage DCs during inflammation or infection. However, this capacity is already fully developed in steady-state thymic CD8(+) DCs, in accordance with their role in the deletion of self-reactive developing T cells.

HSV-1 amplicon vectors launch the production of heterologous rotavirus-like particles and induce rotavirus-specific immune responses in mice.

Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines.

Adeno-associated virus type 2 modulates the host DNA damage response induced by herpes simplex virus 1 during coinfection.

Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.

Thymic but not splenic CD8⁺ DCs can efficiently cross-prime T cells in the absence of licensing factors.

Cross-presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for cross-presentation, little is known about differences in cross-presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady-state thymic CD8(+) DCs can efficiently cross-prime naïve CD8(+) T cells in the absence of costimulation. Surprisingly, cross-priming by splenic CD8(+) DCs was dependent on licensing factors such as GM-CSF. In the absence of GM-CSF, antigen-MHC-class-I complexes were detected on thymic but not on splenic CD8(+) DCs, indicating that the cross-presentation capacity of the thymic subpopulation was higher. The observed cross-priming differences between thymic and splenic CD8(+) DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide-loaded splenic CD8(+) DCs were able to induce CD8(+) T-cell proliferation. The observation that thymic CD8(+) DCs are more efficient than splenic CD8(+) DCs in T-cell cross-priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.

Inhibition of herpes simplex virus type 1 replication by adeno-associated virus rep proteins depends on their combined DNA-binding and ATPase/helicase activities.

Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.

Lentiviral-mediated transcriptional targeting of dendritic cells for induction of T cell tolerance in vivo.

Dendritic cells (DCs) are important APCs able to induce both tolerance and immunity. Therefore, DCs are attractive targets for immune intervention. However, the ex vivo generation and manipulation of DCs at sufficient numbers and without changing their original phenotypic and functional characteristics are major obstacles. To manipulate DCs in vivo, we developed a novel DC-specific self-inactivating lentiviral vector system using the 5' untranslated region from the DC-STAMP gene as a putative promoter region. We show that a gene therapy approach with these DC-STAMP-lentiviral vectors yields long-term and cell-selective transgene expression in vivo. Furthermore, transcriptionally targeted DCs induced functional, Ag-specific CD4 and CD8 T cell tolerance in vivo, which could not be broken by viral immunization. Tolerized CTL were unable to induce autoimmune diabetes in a murine autoimmune model system. Therefore, delivering transgenes specifically to DCs by using viral vectors might be a promising tool in gene therapy.

Transcriptional targeting of B cells for induction of peripheral CD8 T cell tolerance.

Several mechanisms are in place to neutralize autoimmune CD8 T cells by tolerance induction. Developing self-specific CD8 T cells are eliminated in the thymus by Ag-presenting epithelial and dendritic cells (DCs). However, CD8 T cells escaping thymic central tolerance can also be inactivated by tolerance mechanisms in peripheral organs. In contrast to DCs, the role of B cells in generating CD8 T cell tolerance is not well-characterized. To investigate this question in more detail, we transcriptionally targeted Ag to B cells using B cell-specific retroviral vectors in vivo. Although Ag expression could be detected in B cells of thymus, lymph nodes, and spleen, B cells were unable to induce central tolerance of CD8 thymocytes. In contrast, in peripheral organs, we could identify clonal deletion and functional inhibition (anergy) of CD8 T cells as tolerance-inducing mechanisms. Although Ag expressed by B cells was acquired and cross-presented by DCs, B cells were also sufficient to tolerize CD8 T cells directly. These findings suggest exploitation of B cells for Ag-specific immunotherapy of CD8 T cell-mediated autoimmune diseases.

Analysis of two T-cell receptor BV gene segment polymorphisms in caucasoid Brazilian patients with rheumatoid arthritis.

Considering the role of T-lymphocytes in rheumatoid arthritis (RA) and a possible involvement of the TCR in the pathology of this disease we analyzed allelic and genotypic frequencies of variants of two TCRBV gene segments (TCRBV3S1 and TCRBV18) in RA. A total of 95 caucasoid South Brazilian RA patients were genotyped for both TCRBV gene segment variants by restriction fragment length polymorphism preceded by PCR (PCR-RFLP) and the obtained frequencies were compared to those from healthy individuals. Allelic frequencies for the TCRBV3S1 gene segment were, respectively, for RA patients and controls, 0.447 and 0.545 (allele 1) and 0.553 and 0.455 (allele 2). Allelic frequencies for the TCRBV18 gene segment were, respectively, for RA patients and controls, 0.824 and 0.806 (allele 1) and 0.176 and 0.194 (allele 2). Neither allelic frequencies nor genotypic frequencies differ among RA and healthy individuals, suggesting that there is not a direct association among the TCRBV allelic variants studied and the development of RA and thus excluding the possibility of use of these gene segment polymorphisms as RA susceptibility markers.

Linfócitos T, o rearranjo do receptor de células T (TCR) e a utilização de polimorfismos do TCR como marcadores em artrite reumatóide / Tlymphocytes, T cell receptor (TCR) gene rearrangement and usage of TCR polymorphism as markers in rheumatoid arthritis

O repertório de linfócitos T representado pela diversidade dos receptores de células T (TCR) presentes no sangue periférico é essencial para o desenvolvimento e modulação de uma resposta imune. O polimorfismo dos genes que codificam o TCR, em suas porções constante e variável, parece ser um mecanismo de extrema importância na geração de diversidade interpopulacional. Estes polimorfismos devem ser considerados quando estudamos a capacidade de um determinado indivíduo montar uma resposta imune eficiente. No caso de doenças auto-imunes como a artrite reumatóide, a análise do repertório de células T e a identificação de fragmentos de TGR potencialmente associados à doença podem ser instrumentos úteis na compreensão da sua patogênese