T cells in colorectal cancer (CRC) are associated with improved survival. However, checkpoint immunotherapies antagonizing the suppression of these cells are ineffective in the great majority of patients. To better understand the immune cell regulation in CRC, we compared tumor-associated T lymphocytes and macrophages to the immune cell infiltrate of normal mucosa. Human colorectal tumor specimen and tumor-distant normal mucosa tissues of the same patients were collected. Phenotypes and functionality of tissue-derived T cells and macrophages were characterized using immunohistochemistry, RNA in situ hybridization, and multiparameter flow cytometry. CRC contained significantly higher numbers of potentially immunosuppressive CD39 and Helios-expressing regulatory T cells in comparison to normal mucosa. Surprisingly, we found a concomitant increase of pro-inflammatory IFNγ -producing T cells. PD-L1+ stromal cells were decreased in the tumor tissue. Macrophages in the tumor compared to tumor-distant normal tissue appear to have an altered phenotype, identified by HLA-DR, CD14, CX3CR1, and CD64, and tolerogenic CD206+ macrophages are quantitatively reduced. The prognostic effect of these observed differences between distant mucosa and tumor tissue on the overall survival was examined using gene expression data of 298 CRC patients. The combined gene expression of increased FOXP3, IFNγ, CD14, and decreased CD206 correlated with a poor prognosis in CRC patients. These data reveal that the CRC microenvironment promotes the coexistence of seemingly antagonistic suppressive and pro-inflammatory immune responses and might provide an explanation why a blockade of the PD1/PD-L1 axis is ineffective in CRC. This should be taken into account when designing novel treatment strategies.

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain m/z values have the same spatial distribution as CD3 T lymphocytes in the lymphoid follicular structures or as CD206 macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M and anti-inflammatory M phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the m/z values found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells.

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588 Background: Neoadjuvant chemotherapy (NAC) with epirubicin/cyclophosphamid (EC) followed by docetaxel (D) is currently a standard of care therapy in women with early, high-risk breast cancer (BC). New approaches aim to improve the outcome by combining chemo- with immunotherapy. It is therefore of great interest if chemotherapeutics differ in their effect on the immune system and if some substances are superior combination partners than others. Methods: 79 BC patients, who participated in the ABCSG-34 trial, were included. 39 patients were treated with 6 cycles of EC followed by 6 cycles of D and 40 received the reverse sequence (D→EC). Blood was collected before and after 6 cycles. The plasma levels of a variety of immune mediators were determined by multiplex bead array assay. The response to therapy was measured by Residual Cancer Burden (RCB)-score. A score of ≤1.36 was determined as good response. Lymphocyte activation was assessed after stimulation with phytohaemagglutinin (PHA) by flow cytometric analysis of IFNgamma. Additionally, lymphocytes of 6 healthy probands were stimulated with PHA and treated with E or D. The stimulation was quantified by measuring cluster formation after 5 days. Further, a human BC cell line (SK-BR3) was treated with E or D. The induced cell death was determined morphologically as well as by flow cytometry after staining of phosphatidylserine, Sub-G1 DNA and active caspase-3. Results: The treatment of BC patients with 6 cycles of EC resulted in a decrease of lymphocyte stimulation whereas 6 cycles of D had no effect. The plasma levels of most immune mediators decreased significantly after six cycles EC when compared to baseline. Under the influence of D, the effect was much weaker. The changes of Eotaxin and OPG during the first 6 cycles of D correlated with the RCB-score in the reverse group. A decrease in Eotaxin (p = 0.0136) or in OPG (p = 0.0487) correlated with good response. The in vitro lymphocyte stimulation assay showed that E and D have similar inhibitory effects on lymphocytes.The annexin V/PI analysis confirmed that E more often leads to apoptotic cell death in SK-BR3 cells than D (E:53% vs. D:14%). SK-BR3 cells formed more often polyploid cells when treated with D. This suggests that D induces a regulated form of necrosis whereas E apoptosis. Conclusions: This study is the first to compare the immunomodulatory effect of E and D in BC patients. E inhibits lymphocyte activation in vitro and in vivo and suppresses many soluble immune mediators. This suggests that it is not suited for combination with immunotherapies. D showed a much weaker effect.