Fig 1.
T cells inhibit the growth of EBV-infected B cells in cord blood-humanized mice.
NSG mice were injected i.p. with EBV-infected cord blood cells and then treated with or without the OKT3 T-cell depleting ab (50 μg i.p. three times a week starting 11 days after injection of cells, 6 mice/group). Mice were euthanized 26 days post-injection. Grossly visible lymphomas were dissected and weighed. The weight of tumors is normalized to the average size of the untreated tumors (set as 1).
Fig 2.
EBV-infected lymphoma cells express the PD-L1 and PD-L2 ligands in cord blood-humanized mice.
A) B cells isolated from pancreatic tissue or spleens of mice injected with EBV-infected cord blood, or mock-treated cord blood, were stained with antibodies specific for human CD45, CD19, CD20, CD3, PD-L1, PD-L2 or isotype matched negative controls and analyzed by flow cytometry. Samples were gated on lymphocytic cells expressing human CD45, CD19, and CD20 (B cells). Filled histograms show staining for PD-L1 (left column) or PD-L2 (right column), in comparison to staining of the same population of cells by the isotype control (dashed histograms). B) EBV–infected lymphoma cells in a cord blood-humanized mouse were co-stained for EBNA2 (purple) and PD-L1 (brown) by IHC. Examples of co-staining cells are indicated with black arrows.
Fig 3.
T cells express the PD-1 and CTLA-4 receptors in EBV-infected cord blood-humanized mice.
T cells isolated from spleens of EBV-infected cord blood-humanized mice were stained with anti-PD-1 ab (A), CTLA-4 (B) or isotype control, and analyzed by flow cytometry. Filled histograms show staining for PD-1 (A), or CTLA-4 (B), in comparison to staining of the same population of cells by the isotype control (dashed histograms). Similar results were obtained in multiple mice and representative results are shown.
Fig 4.
Combined PD-1/CTLA-4 blockade inhibits the growth of EBV-induced lymphomas in cord blood-humanized mice.
A) M81 strain (2000 infectious units) EBV- infected cord blood-humanized NSG mice were treated with PBS (untreated), anti-PD-1 ab (anti-CD279 J116 mouse monoclonal), anti-CTLA-4 ab (ipilimumab), or anti-PD-1 and anti-CTLA-4 abs as indicated (starting 5 days post infection, 100 μg/animal i.p. 3x/week). Mice were euthanized at 4 weeks after cord blood injection and grossly visible tumors were weighed. The tumor weight is shown for each condition (normalized to the average tumor weight of untreated animals, 4 mice/group). B and C) Similar experiments were performed in NSG mice injected with M81 EBV strain-infected cord blood derived from two additional cord blood donors; mice were treated with PBS or the combination of both abs as described above (6 mice/group). D. Data from each of the three experiments shown in figure A-C was combined and analyzed. E. The survival curves of EBV- infected cord blood-humanized NSG mice treated with or without anti-PD-1/CTLA-4 abs (9 animals each group, infected with 500 infectious M81 EBV units) are shown.
Fig 5.
T cells are required for the therapeutic effect of PD-1/CTLA-4 blockade in EBV-infected humanized mice.
A. Human T cells were harvested at 4 weeks post-injection from spleens of EBV- infected mice that were PD-1/CTLA-4 Ab treated, or treated with istoype control Abs. The T cells were incubated for 72 hr in medium containing IL-2, then exposed to autologous umbilical cord mononuclear cells in the presence of vehicle control (shown as a peptide concentration of "0"), a mixture of synthetic EBV peptides (“EBV peptide”), or a mixture of CMV peptides (“CMV peptide”), and IFN-γ secreted into the culture supernatant was quantified by ELISA. The plot shows the means of 3–6 replicates for each condition with error bars indicating the standard deviations. The p value shown (calculated by a 2-tailed student's t test) is for the IFN-γ values produced by T cells against the EBV peptide. B. NSG mice were injected i.p. with EBV-infected cord blood cells and then treated with or without anti-PD-1/anti-CTLA-4 antibodies, in the presence of the OKT3 T-cell depleting ab (6 mice/group). Mice were euthanized 26 days post-injection. Grossly visible lymphomas were dissected and weighed. The weight of tumors is normalized to the average size of the tumors not treated with PD-1/CTLA-4-abs (set as 1).
Fig 6.
PD-1/CTLA-4 blockade increases T cell tumor infiltration and T cell activation in EBV-infected cord-blood humanized mice.
A) Lymphomas from euthanized untreated EBV-infected cord blood-humanized NSG mice were formalin-fixed and paraffin embedded. Tumors were stained with H&E (10x magnification and 100x magnification), or antibodies for CD20 (B cells), CD8 (cytotoxic T cells), CD4 (helper T cells) and NFATC1 (activated T cell) as indicated. No nuclear NFATC1 staining (indicative of T cell activation) was observed. At least 8 mice were analyzed from each condition and representative figures are shown. B) Lymphomas from EBV-infected cord blood-humanized NSG mice treated with PD-1 and CTLA-4 antibodies were formalin-fixed and paraffin embedded. Tumors were stained with H&E (10x magnification and 100x magnification), or antibodies for CD20 (B cells), CD8 (cytotoxic T cells), CD4 (helper T cells) and NFATC1as indicated. At least 8 mice were analyzed from each condition and representative figures are shown. C) The average number of infiltrating CD4 and CD8 T cells in PD-1/CTLA-4 treated versus untreated tumors was quantitated from 5 different views (60X), using three different tumors for each condition. D) The number of infiltrating FoxP3 positive cells, divided by the number of infiltrating CD4 T cells, was quantitated from 5 different views (60X), using three different tumors for each condition.
Fig 7.
PD-1/CTLA-4 blockade increases the number of intra-tumor high endothelial venules, and enhances RANTES production in tumors.
A). Lymphomas from EBV-infected cord blood-humanized NSG mice treated with or without PD-1 and CTLA-4 antibodies were formalin-fixed and paraffin embedded, and stained with antibodies for CD20 (B cells), CD8 (cytotoxic T cells), CD4 (helper T cells) and MECA-79 (a marker of high endothelial venules). Arrows are examples of positive staining HEVs. At least 8 mice were analyzed from each condition and representative figures are shown. B). The average number of Meca-79 positive venules in PD-1/CTLA-4 treated versus untreated tumors was quantitated from 5 different views (60X), using seven different tumors for each condition. C). Lymphomas from EBV-infected cord blood-humanized NSG mice treated with or without PD-1 and CTLA-4 antibodies were formalin-fixed and paraffin embedded, and co-stained with antibodies for CD20 (B cells) and RANTES. Arrows are examples of cells staining positive for RANTES and negative for CD20. At least 8 mice were analyzed from each condition and representative figures are shown.
Fig 8.
PD-1/CTLA-4 blockade decreases the number of latently, and lytically, EBV-infected B cells in cord-blood humanized mice.
A. Lymphomas from EBV-infected cord blood-humanized NSG mice treated with or without PD-1 and CTLA-4 antibodies were formalin-fixed and paraffin embedded. Tumors were examined for EBER expression using ISH, and for expression of EBV latent protein EBNA2, and lytic proteins BZLF1 and BMRF1, using IHC as indicated. Arrows are examples of positively BMRF1-staining cells. At least 8 mice were analyzed from each condition and representative figures are shown. B. Lymphomas from EBV-infected cord blood-humanized NSG mice treated with or without PD-1 and CTLA-4 antibodies were formalin-fixed and paraffin embedded. Tumors were examined for expression of EBV latent proteins EBNA2 and LMP1 using IHC as indicated. Arrows are examples of positively LMP1-staining cells. At least 8 mice were analyzed from each condition and representative figures are shown.
Table 1.
Antibodies used for immunohistochemistry.