A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

Bollampalli, Vishnu Priya; Nylén, Susanne; Rothfuchs, Antonio Gigliotti

doi:10.3791/54620

Cited by 10 publications

(18 citation statements)

References 26 publications

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“…Samples were analyzed in an LSR-II flow cytometer (BD Biosciences). For footpad-popliteal LN DC follow-up, mice were anesthetized and footpads were injected with 0.5 mM of 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) ( 33 ) co-administered with 1 × 10 6 PFU of WT or ΔgD-2 in 30 µl final volume. As a negative control, mice were injected with CFSE in PBS.…”

Section: Methodsmentioning

confidence: 99%

US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

et al. 2017

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Herpes simplex virus (HSV) type 1 (HSV-1) and type 2 (HSV-2) produce lifelong infections that are associated with frequent asymptomatic or clinically apparent reactivation. Importantly, HSV express multiple virulence factors that negatively modulate innate and adaptive immune components. Notably, HSV interfere with dendritic cell (DC) viability and function, likely hindering the capacity of the host to mount effective immunity against these viruses. Recently, an HSV-2 virus that was deleted in glycoprotein D was engineered (designated ΔgD-2). The virus is propagated on a complementing cell line that expresses HSV-1 gD, which permits a single round of viral replication. ΔgD-2 is safe, immunogenic, and provided complete protection against vaginal or skin challenges with HSV-1 and HSV-2 in murine models. Here, we sought to assess the interaction of ΔgD-2 with DCs and found that, in contrast to wild-type (WT) virus which induces DC apoptosis, ΔgD-2 promoted their migration and capacity to activate naïve CD8+ and CD4+ T cells in vitro and in vivo. Furthermore, DCs exposed to the WT and ΔgD-2 virus experienced different unfolded protein responses. Mice primed with DCs infected with ΔgD-2 in vitro displayed significantly reduced infection and pathology after genital challenge with virulent HSV-2 compared to non-primed mice, suggesting that DCs play a role in the immune response to the vaccine strain.

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Section: Methodsmentioning

confidence: 99%

US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

et al. 2017

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“…To assess whether protective HSV mutants recover this important DC function, or even enhance their migration, assays that consist on the local injection of virus and tracking dyes in the footpads or hind flank of limbs may be performed. These assays allow for the quantification of DC subsets that migrate from the periphery into the lymph nodes after infection ( 103 ). We expect HSV mutants that elicit protective immunity to promote the migration of DCs from the site of inoculation to the draining lymph nodes, either by increasing the amount of DCs that reach this secondary lymphoid organ or the migration of DC subtypes that are related to enhanced T cell activation ( 100 , 101 , 104 ).…”

Section: Suggested Experimental Testsmentioning

confidence: 99%

A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

et al. 2017

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Herpes simplex virus type 2 (HSV-2) is highly prevalent in the human population producing significant morbidity, mainly because of the generation of genital ulcers and neonatal encephalitis. Additionally, HSV-2 infection significantly increases the susceptibility of the host to acquire HIV and promotes the shedding of the latter in the coinfected. Despite numerous efforts to create a vaccine against HSV-2, no licensed vaccines are currently available. A long-standing strategy, based on few viral glycoproteins combined with adjuvants, recently displayed poor results in a Phase III clinical study fueling exploration on the development of mutant HSV viruses that are attenuated in vivo and elicit protective adaptive immune components, such as antiviral antibodies and T cells. Importantly, such specialized antiviral immune components are likely induced and modulated by dendritic cells, professional antigen presenting cells that process viral antigens and present them to T cells. However, HSV interferes with several functions of DCs and ultimately induces their death. Here, we propose that for an attenuated mutant virus to confer protective immunity against HSV in vivo based on adaptive immune components, such virus should also be attenuated in dendritic cells to promote a robust and effective antiviral response. We provide a background framework for this idea, considerations, as well as the means to assess this hypothesis. Addressing this hypothesis may provide valuable insights for the development of novel, safe, and effective vaccines against herpes simplex viruses.

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“…A CFSE fluorochrome-based migration assay was used to track the movement of migratory skin DCs to dLN. (3, 24). We have used this setup in the past to study early responses to BCG, another live-attenuated vaccine and so included BCG as a comparison to VACV.…”

Section: Resultsmentioning

confidence: 99%

“…(A) Total number CFSE-labeled skin DCs shown. (B) CFSE expression within different defined subsets of skin DCs based on previously published gating strategy (3, 24) shown. LC: Langerhans cells.…”

Section: Resultsmentioning

confidence: 99%

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Vaccinia Virus Infection Inhibits Skin Dendritic Cell Migration to Draining Lymph Node

Aggio

Krmeská

Ferguson

et al. 2020

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18 Despite the success of Vaccinia virus (VACV) against smallpox there remains a paucity of 19 information on Dendritic cell (DC) responses to the virus, especially on the traffic of DCs and 20 VACV to draining LN (dLN). Herein we studied skin DC migration in response to VACV and 21 compared it to the tuberculosis vaccine Mycobacterium bovis Bacille Calmette-Guérin (BCG), 22 another live-attenuated vaccine administered via the skin. In stark contrast to BCG, skin DCs 23 did not relocate to dLN in response to VACV. This happened in spite of virus-induced 24 accumulation of several other innate-immune cell populations in the dLN. UV inactivation of 25 VACV or use of the Modified Vaccinia virus Ankara (MVA) strain promoted DC movement 26 to dLN, indicating that the virus actively interferes with skin DC migration. This active immune 27 suppression by VACV was potent enough to ablate the mobilization of skin DCs in response to 28 BCG, and to reduce the transport of BCG to dLN. Expression of inflammatory mediators 29 associated with BCG-triggered DC migration were absent from virus-injected skin, suggesting 30 that other pathways provoke DC movement in response to replication-deficient VACV. Despite 31 viral suppression of DC migration, VACV was detected in dLN much earlier than BCG, 32 indicating a rapid, alternative route of viral traffic to dLN despite marked blockade of skin DC 33 mobilization from the site of infection. 34 35 Word count: 217 36 37

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A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with <em>Mycobacterium bovis</em> Bacille Calmette-Guérin

Cited by 10 publications

References 26 publications

US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

Vaccinia Virus Infection Inhibits Skin Dendritic Cell Migration to Draining Lymph Node

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