Home › Cell Biology › Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.
Cell Biology JoVE (Open Access) Citable · DOI

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

DOI: 10.3791/51207-v
What you'll learn
  • ✓ Set up ex vivo co-culture systems with HIV-1 infected CD4+ T cells and innate sensing cells
  • ✓ Measure type-I interferon release as a readout of pDC innate sensing
  • ✓ Distinguish innate sensing responses to infected cells versus cell-free viral particles
Protocol

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Difficulty
advanced
Total time
~4–6 hours per experiment (including cell preparation, co-culture, and IFN measurement)
Model organism
Human PBMCs, MT4 CD4+ T cells, HEK293-Blue IFN-α/β reporter cells
Biosafety
BSL-2

Steps

1
Prepare primary or MT4 target CD4+ T cells

Culture and prepare HIV-1 infected CD4+ T cells (using primary or MT4 cell lines) as target cells for sensing cell co-culture. Ensure cells are properly infected and viable before use.

▶ 01:24
2
Isolate or prepare pDC and PBMC sensing cells

Isolate plasmacytoid dendritic cells (pDCs) from peripheral blood mononuclear cells (PBMCs) or use whole PBMCs as the innate sensing cell population. Characterize cell purity and viability.

▶ 01:24
3
Co-culture infected CD4+ T cells with sensing cells

Combine HIV-1 infected target T cells with pDCs or PBMCs at defined ratios in culture medium. Incubate for appropriate duration to allow innate sensing and type-I interferon production.

▶ 02:56
4
Quantify bioactive type-I IFN using reporter cells

Transfer co-culture supernatants to HEK-Blue IFN-α/β reporter cells, which produce a measurable output (alkaline phosphatase) proportional to type-I interferon concentration. Measure reporter signal as readout of pDC activation.

▶ 04:02
5
Characterize and validate co-culture results

Analyze freshly isolated PBMC and infected MT4 T cell co-culture data, including cell phenotyping and IFN production profiles. Validate that infected cells trigger pDC sensing more effectively than cell-free virus.

▶ 05:23
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