Role of LFA-1 integrin in the control of a lymphocytic choriomeningitis virus (LCMV) infection

Figures & data

Figure 1. Effect of anti-LFA-1 Mab treatment on anti-viral T_EFF burst size

Total numbers of CD8 (a) and CD4 (b) T cells assessed in the blood of LCMV-infected mice at day 9 post-infection (p.i.). (c,d) Frequency of immunodominant (NP396, GP33) and subdominant (NP205, GP206) LCMV-specific CD8+ T_EFF in the blood of infected mice at day 9 p.i. detected by ex vivo re-stimulation with different LCMV peptides. See also Figure S1.* p < 0.05, ** p < 0.001, *** p < 0.0001. Error bars represent mean±SEM. Three or more independent experiments were performed including three mice per group with similar results.

Figure 2. Strain-specific effects of anti-LFA-1 Mab treatment on anti-viral T_EFF differentiation

(a,b) Differentiation of CD8 T_EFF into cytokine producer cells evaluated by ex vivo restimulation with different LCMV peptides at day 9 p.i. Results are displayed as the ratio between the frequency of IFNγ⁺ TNFα⁺ double-positive cells and the sum of IFNγ⁺ and TNFα⁺ single positive cells among CD8+ cells isolated from the blood of infected mice. (c) In vivo cytotoxicity capacity of GP33-specific T_EFF was evaluated by the differential killing of GP33 peptide-loaded splenocytes in comparison to control non-loaded splenocytes upon i.v. injection into LCMV-infected mice at day 6 p.i. (d) Surface expression of PD-1, LAG-3, 2B4, and KLRG1 in LCMV GP33-specific CD8 T_EFF isolated day 9 p.i. from the blood of LCMV-CL13 infected mice. (e-f) The capacity of LCMV-infected mice to control viral replication upon treatment with anti-LFA-1 Mab. Results show blood viremia. Detection limit 100pfu. See also Figure S2. * p < 0.05, ** p < 0.001, *** p < 0.0001. Error bars represent mean±SEM. Three or more independent experiments were performed including three mice per group with similar results.

Figure 3. Impact of LFA-1 on CD8 T_EFF differentiation and function after LCMV-CL13 infection

Differentially labeled WT and LFA-1^−/- P14 T_N were adoptively transferred to C57BL/6 mice and homed cells in lymphoid tissues were compared at steady-state (a) and WT and LFA-1^−/- P14 T_N expansion and differentiation and viremia was followed after i.v. infection with 5 × 10⁴ pfu LCMV-CL13 (b-f). (a) Homing indices (i.e. the ratio of WT:LFA-1^−/- T_N in a recipient organ relative to input ratio 24 hours after T_N transfer. (b) A small number (10,000 cells) of naïve LFA-1^+/+ CD45.1⁺ and LFA-1^−/- CD45.2⁺ P14 T_N cells were enriched by magnetic negative selection (>95% purity) and transferred into the same WT CD45.2⁺ recipient, thus generating P14 chimeric mice. (c) 24 hours later, recipient mice were i.v. challenged with LCMV-CL13 (5x10⁴ pfu) that were able to clear infection with kinetics similar to non-chimeric mice. See also Figure 2c. (d) The surface expression of CD45.1 and lack of LFA1 expression was used to identify adoptively transferred P14 T_EFF cells among CD8+ cells in recipient mice. (e) On day 9 p.i. the expression of cytokines (IFNγ and TNFα) by individuals LFA1^+/+ and LFA1^−/- P14 T_EFF cells was determined after ex vivo restimulation with gp33-41 or soluble CD3 antibody of splenocytes from LCMV-CL13 infected mice (d) At different times after infection the T_EFF burst size generated by adoptively transferred T_N P14 was monitored in lymphoid and non-lymphoid organs of P14 chimeric mice by flow cytometry. (e) During the expansion phase (day 6 p.i.) and at the beginning of the contraction phase (day 9 p.i.) the rate of proliferation of LFA1^+/+ and LFA1^−/- T_EFF was quantified by the incorporation of BrdU into dividing cells. BrdU was injected i.p. in LCMV-CL13 infected mice and 12 h later its incorporation into LFA1^+/+ and LFA1^−/- T_EFF was quantified by flow cytometry. (f) At day 9 p.i. splenocytes and lymph node cells of LCMV-CL13 infected mice were harvested and the binding to Annexin-V and incorporation of 7AAD into LFA1^+/+ and LFA1^−/- T_EFF was quantified by flow cytometry. See also Fig. S3&4. * p < 0.05, ** p < 0.001, *** p < 0.0001. Error bars represent mean±SEM.Three or more independent experiments were performed including three mice per group with similar results.

Figure 4. Role of LFA-1 usage and high viremia in T_EFF dysfunction after LCMV-CL13 infection (a) 10,000 cells of purified LFA-1^+/+ CD45.1⁺ and LFA-1^−/- CD45.2⁺ P14 T_N cells were transferred into the same C57BL/6 CD45.2⁺ recipients, thus generating P14 chimeric mice. 24 hours later, recipient mice were i.v. challenged with LCMV-CL13 (5x10⁴ or 2 × 10⁶ pfu). The surface expression of CD45.1 and lack of LFA1 expression was used to identify these cells among CD8+ cells in recipient mice. At day 9 p.i. the frequency (b) and (c) the expression of PD-1 and LAG-3 was quantified in P14 T_EFF from the blood of infected P14 chimeric mice. Results shown as mean fluorescence intensity (MFI). (d) In parallel, P14 T_EFF from the blood of infected mice were restimulated ex vivo with LCMV GP33 peptide to quantify their production of IFNγ and TNFα. Two independent experiments were performed including three mice per group with similar results

Figure 5. Global transcriptomics of T_EFF at the peak of anti-viral effector response (a) Genes differentially expressed in GP33-specific T_N and T_EFF from anti-LFA-1 Mab antibody and control treated mice. Each column represents an individual sample and each row a gene, and cells were colored to indicate relative expression. Top 200 genes upregulated or downregulated in each specific population are shown. (b) Principal Component Analysis of transcriptomic profiles of T_N and T_EFF. (c) Gene set enrichment analysis was performed between endogenous Dex-GP33 T_EFF from anti-LFA-1 Mab treated versus control mice and LFA-1^+/+ versus LFA-1^−/- P14 T_EFF. Overlap of enriched pathways are shown as a Venn diagram. (e,f) At day 9 p.i. splenocytes and blood cells of LCMV-CL13 infected mice were harvested and the binding to Annexin-V and incorporation of 7AAD into T_EFF was quantified by flow cytometry. Three independent experiments including three mice per group were performed with similar results

Figure 6. Transcriptional signatures of exhaustion, anergy and deletion tolerance in T_EFF in the absence of LFA-1 function. Geneset enrichment analysis (GSEA) on T cells exhaustion [41], anergy [40] and deletion tolerance [39] signatures from MSigDB v7.1 (Broad institute) was performed on day 9 Dex-GP33 (a,c) or P14 T_EFF (b,c) from LCMV-Cl13 infected mice. (*) represents signatures with a false discovery rate (FDR) of <0.05

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