Anti cd8α

The following antibodies were given i.p. at 200µg per injection: anti-CD8α (Clone: 2.43; BioXcell), anti- CD8β (Clone: 53-5.8; BioXcell), anti-CD25 (Clone: PC61; eBioscience), as well as rat IgG2B (Clone: LTF-2; BioXcell) and rat IgG1 (Clone: HRPN; BioXcell) isotype controls. For in vivo T_Reg cell depletion with Diphteria Toxin (DT; Sigma-Aldrich), Foxp3^DTR mice were given 50µg/kg of DT i.p. every other day as previously described (28 (link),29 (link)).

Cellular subsets and cytokines were depleted by intraperitoneally administering depleting antibodies (BioXCell) beginning 1 d before therapy as we previously reported (Moynihan et al., 2016 (link)): CD8 T cells with anti-CD8-α (clone 2.43, 400 μg every 3 days), CD4 T cells with anti-CD4 (clone GK1.5, 400 μg every 3 days), NK cells with anti-NK1.1 (clone PK136, 400 μg every 3 days), neutrophils with anti-Gr-1 (clone RB6–8C5, 400 μg every 2 days), macrophages with anti-F4/80 (clone CI:A3–1, 200 μg every day) (Lin et al., 2017 (link)), IFN-γ with anti- IFN-γ (clone XT3.11, 200 μg every 3 days), TNF-α with anti-TNF-α (clone XMG1.2, 500 μg every 2 days) and CXCL9 with anti-CXCL9 (clone MIG-2F5.5, 300 μg every 2 days). VEGFR2 was blocked by anti-VEGFR2 (clone DC101, 500 μg every 3 days). Apoptosis of intratumoral T cells were induced with anti-CD3ε F(ab’)2 (clone 145–2C11, 50 μg for intratumoral injection or 100 μg for systematic i.p. injection every day) (Besançon et al., 2017 (link)) to avoid toxicity associated with full anti-CD3 antibodies in treated mice (data not shown). Cellular depletions of CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, neutrophils, macrophages and NK cells were confirmed by flow cytometry of PBMCs (Figures S3A and S4A).

C57BL/6J mice (stock #000664; Jackson labs, Bar Harbor, ME) were challenged subcutaneously with 500,000 MC38 colon cancer cells on their flanks and enrolled on-study when tumors reached 50 mm³. Mice were treated with vehicle plus IgG2a isotype control (10 mg/kg; Bio X Cell, West Lebanon, NH), anti–PD-1 (10 mg/kg; clone RMP1–14; Bio X Cell, West Lebanon, NH), afatinib (10 mg/kg; Selleck, Houston, TX), combination anti–PD-1 (10 mg/kg) and afatinib (10 mg/kg), or combination anti–PD-1 (10 mg/kg), afatinib (10 mg/kg), and anti-CD8α (200 μg; clone 53–6.7; Bio X Cell, West Lebanon, NH). Animals received intraperitoneal (IP) injections of anti–PD-1 on days 5, 8, and 12 and afatinib on days 6, 7, 8, 9, and 10 (as indicated). Depleting anti-CD8α was administered two days prior to first anti–PD-1 treatment. Mice used in experiments were 7–8 weeks of age at time of tumor challenge. Endpoint was considered to be when tumors reached a size of 2000 mm³ or as mandated by institutional guidelines due to development of necrotic lesions. Mice were monitored every 2–3 days and tumors measured with digital calipers. All animal studies were conducted in accordance with, as well as with the approval of, the Institutional Animal Care and Use Committee of Dana-Farber/Harvard Cancer Center.

Mice were treated with six doses of depleting antibodies or isotype
control delivered by intraperitoneal injection on days-1 (300 vg), 1 (200
μg), 4 (200μg), 8 (200μg), 12 (200 μg), and 16
(200 μg) relative to tumor injection (day 0). Depletion efficiency
was checked by flow cytometry on cheek bleeds on days 3, 10, and 18 using
antibodies targeting non-competing CD8 epitopes. For depletion experiments
with MC38 cell lines, the following antibodies were used: rat IgG2b isotype
control (BioXCell, Clone #LTF-2) or anti-CD8α (BioXCell, Clone
#2.43). For depletion experiments with PHD3-OE cell lines, the following
antibodies were used: rat IgG1 isotype control (BioXCell, Clone #TNP6A7) or
anti-CD8β (BioXCell, Clone #53-5.8)