Figure 1.
Nrp1 is highly expressed on tolerant CD8+ T cells primed by a self-antigen in healthy liver.
Naive Gag-specific CD8+ T cells (CD90.1+) were transferred into B6 mice (naive), B6 mice bearing an immunogenic FBL tumor (immune), Alb∶Gag mice (tolerant). (A) Two days after transfer, T cells were purified by cell sorting and RNA isolated for gene expression by microarray. Graphs displays nrp1 and pdcd1 relative gene expression pooled from biological triplicate samples, and error bars represent SD. (B) Nrp1 protein expression on T cells 3 days after adoptive transfer into the naive, immune or tolerant environment. Data are representative of 3 experiments, each with 3 recipient mice per group. (C) Nrp1 protein upregulation on T cells 2 and 4 days after transfer into Alb∶Gag recipients. Data are representative of 2 time course experiments.
Figure 2.
Nrp1 expression corresponds with a modest increase in CD8+ T cell proliferation in response to self-antigen.
Naive WT or Nrp1-deficient CD8+ T cells were labeled with efluor670 cytoplasmic dye and co-transferred into B6 (naive) or Alb∶Gag (tolerant) mice. (A) Nrp1 expression on WT and nrp1f/f T cells 3 days after transfer is compared in the overlaid histograms. (B) Dilution of efluor670 dye in transferred T cells was assessed 3 days after transfer and is displayed in overlayed histograms. (C) Geometric mean fluorescent intensity of efluor670 in T cells from either WT or Nrp1-deficient T cells (lower dye expression corresponds to more proliferation) is pooled from 4 separate experiments, each with 3 mice per group. Error bars are standard error of the mean (SEM) with P value indicated.
Figure 3.
Nrp1 expression does not contribute to the lack of effector function in tolerant CD8+ T cells.
Naive WT (CD90.1+) and Nrp1-deficient (CD90.1+/90.2+) Gag-specific CD8+ T cells were transferred into Alb∶Gag recipients with (lower) or without (upper) a Listeria infection. Three days later, production of IFNγ and TNFα by transferred T cells was measured after overnight restimulation with Gag peptide. (A) Plots display T cell frequency (left) and IFNγ and TNFα production (right). Inset numbers are the percent of total splenocytes within the inscribed square region (left). Numbers in each quadrant represent the percent of gated CD8+ CD90.1+ T cells (right). (B) The percent of gated CD8+ CD90.1+ or CD8+ CD90.1+/90.2+ T cells that express IFNγ in differentially treated Alb∶Gag recipients was graphed, with each circle representing individual mice pooled from 4 separate experiments. Horizontal bars represent the average for each group (ns = not significant).
Figure 4.
The cytotoxic potential of tolerant CD8+ T cells is not regulated by Nrp1.
WT or Nrp1-deficient CD90.1+ Gag-specific CD8+ T cells were transferred into Alb∶Gag recipients with or without Listeria infection. Three days after transfer, recipients were infused with a 1∶1 ratio of Gag (eFluor 670low) and control (eFluor 670high) peptide-pulsed target cells. Twenty hours later (day 4), recipient spleens were harvested and the frequency of transferred Gag-specific T cells was determined by flow cytometry (upper panels). Inset numbers are the percent of total splenocytes within the inscribed regions. Target cell frequency is displayed as histograms with the percentage of total eFluor 670-positive cells inset above the indicated regions (lower panels). (B) Graph displays relative target cell killing (% eFluor 670high/% eFluor 670low) pooled from 3 independent experiments. Error bars represent standard deviation (ns = not significant).
Figure 5.
Deletion of self-reactive CD8+ T cells is not mediated by expression of Nrp1.
Gag-specific CD8+ T cells from WT (CD90.1+) and Nrp1-deficient (CD90.1+/90.2+) donor mice were co-transferred into B6 or Alb∶Gag recipients. The frequency of transferred T cells in recipient tissues was assessed after 8 days. The total number of WT or Nrp1-deficinet T cells in the indicated tissue is displayed graphically and shows pooled data from 4 independent experiments, with each circle representing data from one mouse. Horizontal bars represent the average for each group and P values are indicated (ns = not significant).
Figure 6.
Nrp1 expression on adoptively transferred CD8+ T cells does not influence the efficacy of immunotherapy for leukemia.
B6 or Alb∶Gag recipient mice were inoculated with FBL leukemia. Seven days later, tumor-bearing recipients were infused with WT or Nrp1-deficient Gag-specific CD8+ T cells. Recipient survival was tracked for 40 days, and results pooled from 2 separate experiments are depicted in the graph showing percent survival (y-axis) over time in days (x-axis), with a total 10 mice in each treatment group. Data from B6 and Alb∶Gag groups were compared and P values are indicated (ns = not significant).
Figure 7.
Nrp1 expression is increased on human tumor infiltrating T cells and corresponds with antigen experience.
CD45+ lymphocytes were analyzed from the blood (PBL) and tumor (TIL) of patients undergoing surgical resection of metastatic melanoma, and compared to lymphocytes from the blood of healthy donors. (A) The frequency of CD4+ and CD8+ T cells from the indicated tissues was compared (left) and co-expression of CD45R0 and Nrp1 assessed on these gated T cell populations (right). (B) The frequency of Nrp1+ T cells among TIL, patient PBL, normal skin tissue adjacent to tumor, or healthy donor PBL is displayed graphically. Results are pooled from 8–20 independent samples, with each circle representing data from one patient/donor. Horizontal bars represent the average for each group and P values are indicated (ns = not significant). (C) Proliferation of CD8+ T cells from patient PBL or TIL was directly compared following 3 day in vitro stimulation with anti-CD3/CD28 beads. Histograms show relative dilution of efluor670 dye in stimulated (black line) versus non-stimulated (grey filled peaks) for 2 representative patients. Inset numbers are the percent of cells within the indicated region.