Journal: PLOS Pathogens
Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells
doi: 10.1371/journal.ppat.1014150
Figure Lengend Snippet: (A) Experimental setup for (B-J) : (B, C) 2x10 7 purified T cells obtained from adult eGFP transgenic mice (eGFP) were transferred (Tx) into neonatal mice and analysed after 2 days. (D-G) 10 7 eGFP + T cells were stained with cell proliferation dye eF450 and adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received cells but were not infected. Animals were analysed at 7 days after cell transfer. (H,I) Various numbers of eGFP + T cells were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received no T cells. (J) 8x10 6 purified eGFP + CD44 + T cells obtained from adult non-infected mice were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV or buffer control. Animals were analysed at 8 days post infection (dpi). (B) Representative flow cytometry plots of spleen T cells. (C) Absolute numbers of T cells calculated to animal body weight 2 days after adoptive transfer. Cell numbers of non-treated PND 28 animals are depicted in for comparison. (D) Representative flow cytometry plots of eGFP + T cells isolated from lungs, indicating eF450 signal loss with each proliferation cycle. (E) Quantitative analysis of proliferated eGFP + T cells isolated from lungs. (F) Representative flow cytometry plots of eGFP + CD8 + T cells isolated from lungs simultaneously stained with M38-, M45-, and m139-pMHC tetramers. (G) Quantitative analysis of MCMV-specific tetramer + eGFP + CD8 + T cells isolated from lungs. (H) Immunohistology of lungs indicating localisation of transferred eGFP + T cells in nodular inflammatory foci (NIF). (I) Lung viral loads after adoptive transfer of various numbers of naïve T cells. RLU, relative light units. (J) Lung viral loads after adoptive transfer of CD44 + T cells from adult non-infected mice. (K) Experimental setup for (L-O) : MCMV-2DR contains no additional inserted sequences besides the fluorophore and luciferase reporters, whereas MCMV-4DR encodes for sequences of chicken ovalbumin. Neonatal wildtype or adult Rag2-/-Il2rg-/- mice were infected (104 or 2x105 PFU, respectively) with either MCMV-2DR or MCMV-4DR and received adoptive transfers of 5x10 6 OT-I eCFP and 5x10 6 OT-II eGFP proliferation dye-labelled cells; control animals did not receive T cells. Animals were analysed at 8 dpi. (L) Representative flow cytometry plots of T cells isolated from lung draining lymph nodes indicating eF450 signal loss with each proliferation cycle. (M) Quantitative analysis of proliferated T cells. (N) Immunohistology of MCMV-4DR -infected neonatal lungs, indicating localisation of transferred OT-I eCFP and OT-II eGFP T cells next to MCMV-infected cells in NIF. (O) Lung viral loads after infection with MCMV-4DR (upper panel) or MCMV-2DR (lower panel) and adoptive transfer of OT-I and OT-II cells. Data were acquired from two or more experiments (B and C, n = 5-6; D-G, n = 5; H and I, n = 5-13, J, n = 5-6 L-O, n = 7-11 animals per group). Scale bars, 100 µm. Statistical differences between groups were calculated with Mann-Whitney U (C, E, G, L, and N) or Kruskal-Wallis (I) tests. The p values are provided above each graph. GLuc, Gaussia luciferase; hMIEP, human major immediate early promotor; OVA, chicken ovalbumin; TfR, transferrin receptor sequence.
Article Snippet: Naïve CD44 - T cells were isolated by magnetically depleting CD44 + cells using CD44 Microbeads (Miltenyi Biotec).
Techniques: Purification, Transgenic Assay, Staining, Infection, Control, Flow Cytometry, Adoptive Transfer Assay, Comparison, Isolation, Luciferase, MANN-WHITNEY, Sequencing
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