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Background Increasing evidence suggests that immune regulated pathways influence both breast cancer (BrC) development and response to (neo) adjuvant chemotherapeutic treatment. The sentinel lymph node (SLN) is the first site of BrC induced suppression of immune effector subsets, rendering them unable to mount an effective anti-tumor response. Since detailed knowledge of the functional status of these immune effector subsets is lacking, we compared the immune status of BrC SLN with healthy lymph nodes (HLN) using viable cell samples and correlated our findings to several clinico-pathological characteristics. Additionally we investigated if ex vivo conditioning with the Toll-like receptor-9 ligand CpG-B with or without simultaneous inhibition of JAK2/STAT3 signaling (a downstream signaling pathway implicated as the ‘master switch’ in tumor induced immune suppression) could overcome the observed immune suppression in BrC SLN. Methods Viable SLN cells were obtained from 40 clinically node negative BrC patients. Axillary HLN were obtained from 17 BRCA-1 or -2 patients undergoing a prophylactic mastectomy. Frequencies and activation state of dendritic cell (DC) subsets and regulatory T cells (Treg) were determined by extensive multi-color flowcytometric analyses. Additionally, SLN cells from 12 BrC patients were cultured in absence and presence of CpG-B (PF-3512676) and the combination of CpG-B with a JAK2-STAT3 inhibitor (AG-490). Results Of the 40 BrC SLN 11 contained metastasis. We found clear evidence of BrC-related immune suppression, as significantly higher Treg frequencies were observed in metastasis negative BrC SLN as compared to HLN. These frequencies further increased in metastasis positive BrC SLN. Activation state (by expression of the co-stimulatory molecules CD40, CD83 and CD86) of lymph node resident (LNR)-DC subsets (both CD11c+ myeloid DC and CD123+BDCA2+ plasmacytoid DC), but not of CD1a+ migratory subsets, was significantly lower in BrC SLN as compared to HLN. Additionally, in BrC SLN, activation state of these LNR-DC subsets was further reduced upon metastatic involvement and in non-luminal BrC subtypes (triple negative and HER-2+) as compared to luminal BrC subtypes (ER and/or PR positive). We previously showed in early-stage melanoma patients that local treatment with CpG-B led to the preferential activation of LNR-DC subsets. Similarly, ex vivo targeting of BrC SLN with CpG and CpG + JAK2/STAT3 inhibition resulted primarily in significantly enhanced activation of LNR-DC subsets, with superior effects of combined CpG and JAK2/STAT3 targeting evidenced by significantly higher CD83 expression. Conclusion BrC induced immune suppression in SLN seems to be primarily mediated by hampered activation of LNR-DC subsets, which was most effectively overcome (ex-vivo) with the immune activating compound CpG-B in combination with a small-molecule inhibitor of the immune suppressive JAK2/STAT3 signaling pathway (AG490). This combined immune targeting strategy might be of clinical benefit in enhancing effectiveness of conventional (neo)adjuvant chemotherapeutic treatment, especially in -more immune suppressed- non-luminal BrC subtypes with known poor prognosis. Citation Format: Kim M van Pul, Ronald JCLM Vuylsteke, Sinéad M Lougheed, Lisette EA te Velde, Petrousjka van den Tol, Emiel J Th Rutgers, Hein BAC Stockmann, Tanja D de Gruijl. Breast cancer related immune suppression in the sentinel lymph node can be effectively countered by combined CpG-B administration and JAK2/STAT3 inhibition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-01.