IL-2 treatment promotes CD8+ T effector expansion during chronic infection.

(A) Schematic showing C57BL/6 mice chronically infected with LCMV Docile and receiving intraperitoneal treatment of 30,000 IU recombinant human IL-2 daily from day 10 to 14 post infection, followed by the analyses on day 15. (B) Quantification showing CD4/CD8 ratio and the numbers of CD8+ T cells in the spleen. Cells were gated on 7AAD- B220- TCRβ+CD8+ or CD4+ cells using flow cytometry. (C,D) Representative FACS plots and statistics showing CD44highCD62Llow effector (C) and the expression of Granzyme B (D) in total CD8+ T cells in mice treated with PBS or IL-2. (E, F) Representative FACS plots and quantification showing the frequencies and numbers of H-2Db LCMV GP33 tetramer-specific CD8+ T cells (E) and the frequencies and numbers of CXCR5-TCF1- TEX cells and CXCR5+TCF1+ TPEX cells. Results are representative of two or three independent experiments (n = 4-5/group). Each dot represents one individual mouse. Student’s unpaired two-tailed t tests.

IL-2 alters tissue localization of CD8+ T cells and viral reservoirs in chronic infection.

(A) C57BL/6 mice were chronically infected with LCMV Docile and received intraperitoneal treatment with IL-2 as in Figure 1A. Representative confocal images (left: low magnification, right: high magnification) and quantification of LCMV infected cells in the spleen. (B) C57BL/6 mice chronically infected with LCMV Docile and receiving intraperitoneal treatment of gradient doses of recombinant human IL-2 (10,000 – 100,000 IU) daily from day 10 to 14 post infection, followed by the analyses on day 15. FACS plot showing the gating of TFH and non-TFH cells in CD4+ T cells (left), and quantification of virus-infected (VL4+) TFH and non-TFH cells in each group (right). (C, D) In the same experiment as (A), histograms showing the expression of CXCR5 on GP33 tetramer-specific CXCR5+ CD8+ T cells (C) and representative images (left) and quantification (right) of CD8+ T cells in B-cell follicles (D). (E) Representative FACS plots (left) and quantification (right) showing the expression of CXCR5+ on OT-I CD8+ T cells, co-cultured with splenocytes pulsed with the SINFEKL peptide (1 µM) and indicated cytokines: TGFβ (10 ng/mL), IL-6 (100 ng/mL) and IL-2 (500 I.U./mL). Results are representative of at least two independent experiments. Each dot represents one image count (A, D), one individual mouse (B), or one culture (E). Student’s unpaired two-tailed t tests.

An IL-2-STAT5-BLIMP1 axis regulates the balance of effector and TPEX generation.

(A-C) C57BL/6 mice were chronically infected with LCMV Docile and received intraperitoneal treatment with IL-2 as in Figure 1A. Quantification of the expression of CD122 and CD25 between CXCR5+ TPEX cells and CXCR5- TEX cells (A). The comparison of the expression of CD122 and CD25 on CXCR5+ TPEX cells or CXCR5- TEX cells between PBS treated and IL-2 treated mice (B). The comparison of the expression of pSTAT5 in CXCR5+ TPEX cells or CXCR5- TEX cells between PBS treated and IL-2 treated mice (C). (D-F) Schematic (D) showing the overexpression of constitutive active STAT5b (STAT5b-CA) in P14 cells, followed by the adoptive transfer in congenically marked recipient mice with chronical LCMV Docile infection. Representative FACS plots (left) and quantifications (right) of the frequencies of P14 cells in total CD8+ T cells (E) and the frequencies of CXCR5+ TPEX cells in P14 cells (F). (G, H) Schematic of the generation of bone marrow chimera mice carrying T cell specific deletion of BLIMP1 (encoded by Prdm1), followed with chronic LCMV Docile infection and IL-2 treatment (i.p. 30,000 I.U. daily from day 10 to 14 post-infection) (G). Representative FACS plots and quantifications showing the frequencies of CXCR5+ TPEX cells in CD44+ CD8+ T cells (H). Results are representative of two or three independent experiments (n = 4-5/group). Each dot represents one individual mouse. Student’s unpaired two-tailed t tests. N.S., statistically not significant.

Comparison of IL-2-STAT5 signaling and IL-2 receptors between CXCR5+ TPEX cells and CXCR5- TEX cells.

(A-C) Purified CXCR5+ TPEX and CXCR5− TEX P14 cells from host C57BL/6 mice at day 8 post LCMV (DOCILE) infection underwent RNA-seq to generate transcriptomes as described previously (Leong et al., 2016). Gene set enrichment analyses showing the downregulation of IL-2-STAT5 signalling in CXCR5+ TPEX cells (A). Heatmaps (B) and quantification (C) showing the expression of indicated cytokine receptor genes.

(D) C57BL/6 mice were chronically infected with LCMV Docile and receiving intraperitoneal treatment of IL-2 as in Figure 1A. FACS plots showing the gating of CXCR5+ TPEX cells and CXCR5− TEX populations in LCMV-specific GP33 tetramer+ CD8+ T cells (left) and histograms showing the expression of CD122 and CD25 on indicated populations.

An IL-2 fusion protein regulates the balance of effector and TPEX cell generation.

(A) Schematic showing P14 T cells being transferred into congenically marked mice, which were subsequently infected with LCMV Docile and treated with ANV410, a CD122-specific IL-2 fusion protein, three times every other day from day 8 to 12 post infection and analyzed on day 14 post infection (regimen 1, panels B-E) or from day 1 to 5 of infection and analyzed on day 7 post infection (regimen 2, panels F-H). (B) Quantifications of total CD8+ T cells (left) or P14 cells (right) on day 14 post infection (regimen 1). (C) FACS plot showing the gating strategy for CX3CR1+ TEX cells (left) and quantifications of percentages and numbers of CX3CR1+ TEX cells in P14 cells on day 14 post infection (regimen 1). (D) Quantification showing percentages and expression levels of GzmB+ P14 cells on day 14 post infection (regimen 1). (E) FACS plots showing the gating strategy for TCF1+ TPEX cells (left) and quantification showing the percentages and numbers of TCF1+ TPEX cells in P14 cells on day 14 post infection (regimen 1). (F) Quantification showing the percentages and numbers of P14 cells on day 7 post infection (regimen 2). (G) FACS plots showing the gating strategy for TCF1+ TPEX cells (left) and quantification showing the percentages and numbers of TCF1+ TPEX cells in P14 cells on day 7 post infection (regimen 2). (H) Quantification showing the percentages and numbers of TCF1+ TPEX cells in endogenous PD-1+ CD8+ T cells on day 7 post infection (regimen 2). (I) Schematic of low-dose IL-2 therapy in patients with systemic lupus erythematosus (SLE). (J) Quantification showing the frequencies of CXCR5+ TPEX in peripheral blood CD8+ T cells in consecutive time points (left) or before and after the therapy (right). Each dot represents one individual mouse (B-H) or one individual patient (J). Student’s unpaired two-tailed t tests (B-H) and paired two-tailed t test (J).

Treatment with a CD122-specific IL-2 fusion protein results in CD8 T cell and NK cell but not TREG cell expansion.

C57BL/6 mice were chronically infected with LCMV Docile and receiving intraperitoneal treatment of ANV410 as in Figure 4A (Regimen A).

(A) FACS plots (left) and quantifications (right) for total CD4+ or CD8+ T cells.

(B) Quantification for CD3-NK1.1+ NK cells.

(C) Quantification for Foxp3+ CD4+ TREG cells.