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Purpose: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients.Experimental design: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures.Results: Comparable numbers of vaccine-induced CD8+ and/or CD4+ TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8+ T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8+ T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively.Conclusion: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8+ and CD4+ T-cell responses, particularly in stage III melanoma patients. Clin Cancer Res; 18(19); 5460–70. ©2012 AACR.

PDF file - 55K, Consort flow chart.

PDF file, 149K, CONSORT flow chart.

PDF file - 62K, This table describes patient details, including disease state and prior treatments.

Supplementary Figure 3. KLH-specific immune responses before and after DC vaccination. (A) KLH-specific T cell proliferation was analyzed before the first vaccination and after each DC vaccination during the first vaccination cycle in peripheral blood of vaccinated metastatic melanoma patients. Per time point each dot represents 1 patient. Proliferative response to KLH is given as proliferation index (proliferation with KLH/proliferation without KLH). The two patients not showing a KLH-specific T cell response are shown in grey. (B) KLH-specific IgG, IgA, and IgM antibodies were quantitatively measured after each vaccination cycle in sera of vaccinated patients. Per isotype each dot represents one patient. Best KLH responses per patient are shown.

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine.Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells.Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies.Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro–generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c+ myeloid DCs, naturally circulating in the blood.Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100.Results: Our results show that therapeutic vaccination against melanoma with small amounts (3–10 × 106) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12–35 months), which directly correlated with the development of multifunctional CD8+ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth.Conclusions: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155–66. ©2015 AACR.

Supplementary Figure legend

Purpose: Anticancer dendritic cell (DC) vaccines require the DCs to relocate to lymph nodes (LN) to trigger immune responses. However, these migration rates are typically very poor. Improving the targeting of ex vivo generated DCs to LNs might increase vaccine efficacy and reduce costs. We investigated DC migration in vivo in humans under different conditions.Experimental Design:HLA-A*02:01 patients with melanoma were vaccinated with mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (NCT00243594). For this study, patients received an additional intradermal vaccination with 111In-labeled mature DCs. The injection site was pretreated with nonloaded, activated DCs, TNFα, or Imiquimod; granulocyte macrophage colony-stimulating factor was coinjected or smaller numbers of DCs were injected. Migration was measured by scintigraphy and compared with an intrapatient control vaccination. In an ex vivo tissue model, we measured CCL21-directed migration of 19F-labeled DCs over a period of up to 12 hours using 19F MRI to supplement our patient data.Results: Pretreatment of the injection site induced local inflammatory reactions but did not improve migration rates. Both in vitro and in vivo, reduction of cell numbers to 5 × 106 or less cells per injection improved migration. Furthermore, scintigraphy is insufficient to study migration of such small numbers of 111In-labeled DCs in vivo.Conclusion: Reduction of cell density, not pretreatment of the injection site, is crucial for improved migration of DCs to LNs in vivo. Clin Cancer Res; 19(6); 1525–33. ©2013 AACR.

Supplementary Figure 1. Schematic overview of the 44 h mDC vaccine preparation cycle and vaccination strategy.

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3+ T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496–506. ©2016 AACR.

Multispectral imaging and melanin unmixing using automated quantitative analysis of tumor infiltrating lymphocytes by Vectra/InForm platform.

Supplementary Table 2 PDF file - 30K, Patient characteristics and historical controls

The gating strategy applied to isolate and determine the frequency of BDCA1+ DCs, BDCA1+ CD14+ cells, monocytes and MDSCs from PBMCS of melanoma patients or healthy donors by FACS (S1); The distribution of potentially "missed genes" over the RPKM value spectrum in all samples that underwent RNA sequencing (S2); A Venn diagram displaying the overlap between the four gene sets used in GSEA analysis: Mono high, infDC high, MoDC sig and BDCA1+ DC high (S3); The T cell suppressive capacity of BDCA1+ DCs, BDCA1+ CD14+ cells, monocytes and MDSCs (S4); Quantification of immunofluorescence stainings performed on sections of paraffin-embedded resection specimens of healthy and melanoma metastasized lymph nodes or healthy and melanoma metastasized colon (S5); Large view, showing multiple cells, of May-Grunwald/Giemsa-stained healthy donor-derived, FACS-sorted BDCA1+ DCs, BDCA1+CD14+ cells and monocytes (S6); GSEA enrichment plots (S7); BDCA1+ DCs, BDCA1+CD14+ cells and monocytes respond to TLR2 and TLR8 stimulation (S8); IL-12 levels determined in overnight supernatants of BDCA1+ DCs, BDCA1+ CD14+ cells and monocytes cultures following stimulation with pIC and R848 (S9); The T cell polarizing capacity of BDCA1+ DCs, BDCA1+ CD14+ cells and monocytes (S10); The effect of IL-10 on BDCA1+ CD14+ cell-induced T cell proliferation was determined by performing an MLR with allogenic naïve CD4+ T cells in the presence or absence of anit-IL-10 and anti-IL10 receptor or the matching isotype controls (S11); The suppressive capacity of BDCA1+ CD14+ cells is not reversed by PD-L1 blocking (S12).

Supplementary Figure 2 PDF file - 46K, Supplementary Figure 2 Plasmacytoid DCs acquire CCL21-driven chemotactic ability upon activation

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Supplementary Figure 1 PDF file - 34K, Supplementary Figure 1 CliniMACS-based pDC isolation is feasible

PDF file - 34K, KLH specific responses

The detailed composition of all gene sets used in GSEA.

PDF file - 233K, Definition of criteria for SKIL culture evaluation: Three examples are shown to illustrate the definition of SKIL culture criteria. (A) Criterion III, one patient (IV-B-16-St) with a clear population of TAA-specific CD8+ T cells in SKIL cultures, which produce high levels of IFNg upon encounter of both the defined gp100 peptides and naturally processed gp100 presented by a HLA-A*02:01 positive melanoma cell line (Mel624). Moderate amounts of IL-2 are produced and minimal production of IL-5, representing a clear IFNg dominant cytokine profile. (B) Criterion II, a patient (IV-B-11-Ro) with TAA-specific CD8+ T cells in SKIL cultures which produce high levels of IFNg and IL-2 upon encounter of the gp100 peptide, but fail to recognize the naturally processed gp100. (C) Criterion I, a patient (IV-A-07-Th) with a population of TAA-specific CD8+ T cells in SKIL cultures, recognizing the g100-154 peptide, but not the naturally processed gp100. Furthermore, those TAA-specific CD8+ T cells do not produce IFNg but produce elevated levels of IL-5 instead, indicating that the immune system is skewed towards tumor tolerance

The tumor microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1+CD14+ population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell–based vaccines, where they suppress CD4+ T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1+CD14+ cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1+CD14+ cell population expressed markers of both BDCA1+ dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumors for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy. Cancer Res; 76(15); 4332–46. ©2016 AACR.

PDF file - 40K, Vaccine characteristics

GSEA raw data generated by Bubble GUM software.

PDF file - 73K, Patient selection based on SKIL criteria: SKIL criteria are applied to select patients with immune responses meeting increasingly stringent criteria

PDF file - 52K, Characteristics matched controls

PDF file - 51K, CONSORT flow chart

PDF file - 71K, KLH-specific T cell responses according to SKIL classification: Regardless of the number of epitopes or the increasingly stringent criteria for TAA-specific immune responses; the levels of KLH-specific T cell responses were similar and highly variable between individuals. One-way ANOVA was used for comparison of KLH-specific T cell responses in groups of patients with increasing breadth of TAA-specific CD8+ T cell responses. Student t-test was used for comparison of KLH-specific T cell responses in groups of patients as defined by different SKIL criteria

Supplementary Table 1 PDF file - 29K, Induration (mm) of the different injection sites in the DTH skin test

PDF file - 30K, Levels of induration according to SKIL classification: Regardless of the number of epitopes or the increasingly stringent criteria for TAA-specific immune responses; the maximum induration at the injection site was similar in all groups. One-way ANOVA was used for comparison of induration in groups of patients with increasing breadth of TAA-specific CD8+ T cell responses. Student t-test was used for comparison of induration in groups of patients as defined by different SKIL criteria

Legends for Supplementary Tables S1-S2 and Supplementary Figures S1-S12.

PDF file - 34K, Schedule of treatment and monitoring:Patients received 3 vaccinations a biweekly interval, followed a delayed type hypersensitivity (DTH) skin test. After completing one cycle and if no progression of disease was confirmed on CT-scan, patients were eligible to receive a maximum of two more cycles and were followed up to 5 years. Abbreviations: Vx = visit x; M = month, D = day, W = week, Y = year, vacx = vaccination x; FU = follow-up; * follow-up visits were planned every 3 months; # CT-scans were planned every 6 months

Contains fulltext : 283535.pdf (Publisher’s version ) (Open Access) Dendritic cells (DCs) bridge the connection between innate and adaptive immunity. DCs present antigens to T cells and stimulate potent cytotoxic T-cell responses. Metabolic reprogramming is critical for DC development and activation; however, metabolic adaptations and regulation in DC subsets remains largely uncharacterized. Here, we mapped metabolomic and lipidomic signatures associated with the activation phenotype of human conventional DC type 1, a DC subset specialized in cross-presentation and therefore of major importance for the stimulation of CD8(+) T cells. Our metabolomics and lipidomic analyses showed that Toll-like receptor (TLR) stimulation altered glycerolipids and amino acids in cDC1. Poly I:C or pRNA stimulation reduced triglycerides and cholesterol esters, as well as various amino acids. Moreover, TLR stimulation reduced expression of glycolysis-regulating genes and did not induce glycolysis. Conversely, cDC1 exhibited increased mitochondrial content and oxidative phosphorylation (OXPHOS) upon TLR3 or TLR7/8 stimulation. Our findings highlight the metabolic adaptations required for cDC1 maturation.

Background Whereas neoadjuvant cisplatin-based chemotherapy (NAC) followed by a radical cystectomy remains the standard treatment for patients with muscle-invasive bladder cancer (MIBC), increasing evidence suggests that checkpoint inhibitors, either alone or in combination with chemotherapy, are effective in the (neo)adjuvant setting. The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in MIBC. Methods Tumor tissue of 81 patients was used, including 60 patients treated with NAC and 21 patients undergoing upfront cystectomy. Multiplex immunohistochemistry was performed to assess CD3+, CD3+CD8+, CD3+CD8−FoxP3−, CD3+FoxP3+, and CD20+ cells. Patients were classified into a favorable or unfavorable outcome group based on the development of a recurrence within a year. Results The density of intratumoral CD3+ T cells decreased following NAC in patients with a recurrence at one year, while it remained stable in patients without a recurrence (median fold change 0.6 [95CI 0.3; 1.0] versus 1.0 [95CI 0.6; 2.2]). This decrease was mainly attributable to a decrease in CD3+CD8−FoxP3− and CD3+FoxP3+ T cells and was not observed in patients with an early recurrence after upfront cystectomy. Additionally, in cystectomy tissue of patients treated with NAC, median CD3+ and CD3+CD8+ T cell densities were significantly lower in patients with versus patients without a recurrence (CD3: 261. cells/mm2 [95CI 22.4; 467.2]; CD8: 189.6 cells/mm2 [95CI 2.0;462.0]). Conclusion T cell density decreases following NAC in MIBC patients with poor clinical outcome. Further research is needed to investigate whether this decrease in T cell density affects the efficacy of subsequent checkpoint inhibitors. Précis The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in patients with MIBC. We reveal a decline in intratumoral CD3+ T cell density following NAC in patients with an early recurrence.

Prostaglandin E2 (PGE2) is an important maturation mediator for dendritic cells (DCs). However, increased PGE2 levels in the tumor exert immunosuppressive effects on DCs by signaling through two E-Prostanoid (EP) receptors: EP2 and EP4. Blocking EP-receptor signaling of PGE2 with antagonists is currently being investigated for clinical applications to enhance anti-tumor immunity. In this study, we investigated a new delivery approach by encapsulating EP2/EP4 antagonists in polymeric nanoparticles. The nanoparticles were characterized for size, antagonist loading, and release. The efficacy of the encapsulated antagonists to block PGE2 signaling was analyzed using monocyte-derived DCs (moDCs). The obtained nanoparticles were sized between 210 and 260 nm. The encapsulation efficacy of the EP2/EP4 antagonists was 20% and 17%, respectively, and was further increased with the co-encapsulation of both antagonists. The treatment of moDCs with co-encapsulation EP2/EP4 antagonists prevented PGE2-induced co-stimulatory marker expression. Even though both antagonists showed a burst release within 15 min at 37 °C, the nanoparticles executed the immunomodulatory effects on moDCs. In summary, we demonstrate the functionality of EP2/EP4 antagonist-loaded nanoparticles to overcome PGE2 modulation of moDCs.

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3‐dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN‐γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR‐stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC‐T cell cell‐cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross‐present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC‐based cancer vaccines with IDO inhibition., Among human blood dendritic cell subsets, cDC1s uniquely express biologically active IDO at steady‐state, whereas cDC2s express IDO upon IFN‐γ‐ or TLR agonist‐mediated signaling. The enzyme converts tryptophan into kynurenine, thereby limiting T cell proliferation. Selective inhibition of IDO by epacadostat impedes IDO‐mediated immunosuppressive effects in conventional DCs.

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.

BackgroundImmunotherapy is currently part of the standard of care for patients with advanced-stage non-small cell lung cancer (NSCLC). However, many patients do not respond to this treatment, therefore combination strategies are being explored to increase clinical benefit. The PEMBRO-RT trial combined the therapeutic programmed cell death 1 (PD-1) antibody pembrolizumab with stereotactic body radiation therapy (SBRT) to increase the overall response rate and study the effects on the tumor microenvironment (TME).MethodsHere, immune infiltrates in the TME of patients included in the PEMBRO-RT trial were investigated. Tumor biopsies of patients treated with pembrolizumab alone or combined with SBRT (a biopsy of the non-irradiated site) at baseline and during treatment were stained with multiplex immunofluorescence for CD3, CD8, CD20, CD103 and FoxP3 for lymphocytes, pan-cytokeratin for tumors, and HLA-ABC expression was determined.ResultsThe total number of lymphocytes increased significantly after 6 weeks of treatment in the anti-PD-1 group (fold change: 1.87, 95% CI: 1.06 to 3.29) and the anti-PD-1+SBRT group (fold change: 2.29, 95% CI: 1.46 to 3.60). The combination of SBRT and anti-PD-1 induced a 4.87-fold increase (95% CI: 2.45 to 9.68) in CD103+cytotoxic T-cells 6 weeks on treatment and a 2.56-fold increase (95% CI: 1.03 to 6.36) after anti-PD-1 therapy alone. Responders had a significantly higher number of lymphocytes at baseline than non-responders (fold difference 1.85, 95% CI: 1.04 to 3.29 for anti-PD-1 and fold change 1.93, 95% CI: 1.08 to 3.44 for anti-PD-1+SBRT).ConclusionThis explorative study shows that that lymphocyte infiltration in general, instead of the infiltration of a specific lymphocyte subset, is associated with response to therapy in patients with NSCLC.Furthermore, anti-PD-1+SBRT combination therapy induces an immunological abscopal effect in the TME represented by a superior infiltration of cytotoxic T cells as compared with anti-PD-1 monotherapy.

Whereas neoadjuvant cisplatin-based chemotherapy (NAC) followed by a radical cystectomy remains the standard treatment for patients with muscle-invasive bladder cancer (MIBC), increasing evidence suggests that checkpoint inhibitors, either alone or in combination with chemotherapy, are effective in the (neo)adjuvant setting. The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in MIBC.Tumor tissue of 81 patients was used, including 60 patients treated with NAC and 21 patients undergoing upfront cystectomy. Multiplex immunohistochemistry was performed to assess CD3The density of intratumoral CD3T cell density decreases following NAC in MIBC patients with poor clinical outcome. Further research is needed to investigate whether this decrease in T cell density affects the efficacy of subsequent checkpoint inhibitors. PRéCIS: The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in patients with MIBC. We reveal a decline in intratumoral CD3

Tissue specimens taken from primary tumors or metastases contain important information for diagnosis and treatment of cancer patients. Multispectral imaging allows in situ visualization of heterogeneous cell subsets, such as lymphocytes, in tissue samples. Many image processing pipelines first segment cell boundaries and then measure marker expression to assign cell phenotypes. In dense tissue environments such as solid tumors, segmentation-based phenotyping can be inaccurate due to segmentation errors or overlapping cell boundaries. Here we introduce a machine learning pipeline design called ImmuNet that directly identifies the positions and phenotypes of immune cells without determining their exact boundaries. ImmuNet is easy to train: human annotators only need to click on immune cells and rank their expression of each marker; full annotation of tissue regions is not necessary. We demonstrate that ImmuNet is a suitable approach for immune cell detection and phenotyping in multiplex im-munohistochemistry: it compares favourably to segmentation-based methods, especially in dense tissues, and we externally validate ImmuNet results by comparing them to flow cytometric measurements from the same tissue. In summary, ImmuNet performs well on diverse tissue specimens, takes relatively little effort to train and implement, and is a simpler alternative to segmentation-based approaches when only cell positions and phenotypes, but not their shapes are required for downstream analyses. We hope that ImmuNet will help cancer researchers to analyze multichannel tissue images more easily and accurately.