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Introduction Treatment with the immune checkpoint inhibitor anti-programmed cell death protein-1 (PD-1) often causes immune-related adverse events (irAEs). Since irAEs resemble autoimmune diseases, autoantibodies might play a role and could potentially be used to identify patients at risk. Therefore, we investigated the association between autoantibody-positivity and toxicity as well as clinical response in patients with melanoma treated with anti-PD-1.Materials and methods This two-center, retrospective study included 143 patients with melanoma treated with anti-PD-1. Toxicities grade ≥2 and recurrences/responses were captured until 6 months after treatment initiation. Autoantibody measurements were performed at baseline and 3 months after treatment initiation, including IgM-rheumatoid factor (RF), antinuclear antibodies (ANA), extractable nuclear antigen, anti-cyclic citrullinated peptide antibodies (anti-CCP2) and anti-thyroid antibodies.Results 169 irAEs were experienced by 86/143 patients (137 grades 1–2, 32 grades 3–4), the most common being thyroiditis (n=25), dermatitis (n=24), and sicca problems (n=19). Patients with autoantibodies at baseline experienced more irAEs (p=0.001), predominantly associated with anti-thyroid antibodies and thyroid dysfunction. No association was observed between any irAE and anti-CCP2, RF or ANA. In women, baseline and on-treatment anti-thyroid antibody-positivity as well as seroconversion during treatment was associated with thyroid dysfunction. In men, this association was only observed on-treatment. The presence of autoantibodies was not associated with melanoma recurrence (p=0.776) or response (p=0.597).Conclusion The presence of autoantibodies prior to anti-PD-1 therapy is associated with irAEs in patients with melanoma. Both baseline positivity and seroconversion of anti-thyroid antibodies were strongly associated with thyroid dysfunction. This association was stronger in women, with all women who were baseline positive developing thyroid dysfunction.

Background The presence of T cells and suppressive myeloid cells in epithelial ovarian cancer (EOC) correlate with good and bad clinical outcome, respectively. This suggests that EOC may be sensitive to adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL), provided that immunosuppression by myeloid-derived suppressor cells and M2 macrophages is reduced. Platinum-based chemotherapy can alleviate such immunosuppression, potentially creating a window of opportunity for T cell-based immunotherapy.Methods We initiated a phase I/II trial (NCT04072263) in patients with recurrent platinum-sensitive EOC receiving TIL during platinum-based chemotherapy. TILs were administered 2 weeks after the second, third and fourth chemotherapy course. Patients were treated in two cohorts with or without interferon-α (IFNa), as conditioning and TIL support regimen. The primary endpoint was to evaluate the feasibility and safety according to CTCAE V.4.03 criteria and the clinical response and immune modulatory effects of this treatment were evaluated as secondary endpoints.Results Sixteen patients were enrolled. TIL could be successfully expanded for all patients. TIL treatment during chemotherapy without IFNa (n=13) was safe but the combination with IFNa added to the chemotherapy-induced toxicity with 2 out of 3 patients developing thrombocytopenia as dose-limiting toxicity. Fourteen patients completed treatment with a full TIL cycle and were further evaluated for clinical and immunological response. Platinum-based chemotherapy resulted in reduction of circulating myeloid cell numbers and IL-6 plasma levels, confirming its immunosuppression-alleviating effect. Three complete (CR), nine partial responses and two stable diseases were recorded, resulting in an objective response rate of 86% (Response Evaluation Criteria In Solid Tumors V.1.1). Interestingly, progression free survival that exceeded the previous platinum-free interval was detected in two patients, including an exceptionally long and ongoing CR in one patient that coincided with sustained alleviation of immune suppression.Conclusion TIL therapy can be safely combined with platinum-based chemotherapy but not in combination with IFNa. The chemotherapy-mediated reduction in immunosuppression and the increase in platinum-free interval for two patients warrants further exploration of properly-timed TIL infusions during platinum-based chemotherapy, possibly further benefiting from IL-2 support, as a novel treatment option for EOC patients.

Background The survival of patients with cervical cancer who are treated with cisplatin in conjunction with the topoisomerase I inhibitor topotecan is enhanced when compared with patients treated with only one of these chemotherapeutics. Moreover, cisplatin-based and T cell-based immunotherapy have been shown to synergize, resulting in stronger antitumor responses. Here, we interrogated whether topotecan could further enhance the synergy of cisplatin with T cell-based cancer immunotherapy.Methods Mice bearing human papilloma virus 16 (HPV16) E6/E7-expressing TC-1 tumors were vaccinated with HPV16 E7 long peptides and additionally received chemotherapy consisting of cisplatin and topotecan. We performed an in-depth study of this combinatorial chemoimmunotherapy on the effector function and expansion/contraction kinetics of vaccine-induced CD8+ T cells in the peripheral blood and tumor microenvironment (TME). In addition, we interrogated the particular role of chemotherapy-induced upregulation of costimulatory ligands by tumor-infiltrated myeloid cells on T cell proliferation and survival.Results We show that E7 long peptide vaccination combined with cisplatin and topotecan, results in CD8+ T cell-dependent durable rejection of established tumors and 94% long-term survival. Although topotecan initially repressed the expansion of vaccine-induced CD8+ T cells, these cells eventually expanded vigorously, which was followed by delayed contraction. These effects associated with the induction of the proliferation marker Ki-67 and the antiapoptosis molecule Bcl-2 by intratumoral tumor-specific CD8+ T cells, which was regulated by topotecan-mediated upregulation of the costimulatory ligand CD70 on myeloid cells in the TME.Conclusions Taken together, our data show that although treatment with cisplatin, topotecan and vaccination initially delays T cell expansion, this combinatorial therapy results eventually in a more robust T cell-mediated tumor eradication due to enhancement of costimulatory molecules in the TME.

Background Primary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of pivotal importance to improve therapy outcome.Method Here, two mouse models with resistance against therapeutic vaccine-induced tumor regression were studied. Exploration of the tumor microenvironment by high dimensional flow cytometry in combination with therapeutic in vivo settings allowed for the identification of immunological factors driving immunotherapy resistance.Results Comparison of the tumor immune infiltrate during early and late regression revealed a change from tumor-rejecting toward tumor-promoting macrophages. In concert, a rapid exhaustion of tumor-infiltrating T cells was observed. Perturbation studies identified a small but discernible CD163hi macrophage population, with high expression of several tumor-promoting macrophage markers and a functional anti-inflammatory transcriptome profile, but not other macrophages, to be responsible. In-depth analyses revealed that they localize at the tumor invasive margins and are more resistant to Csf1r inhibition when compared with other macrophages. In vivo studies validated the activity of heme oxygenase-1 as an underlying mechanism of immunotherapy resistance. The transcriptomic profile of CD163hi macrophages is highly similar to a human monocyte/macrophage population, indicating that they represent a target to improve immunotherapy efficacy.Conclusions In this study, a small population of CD163hi tissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163hi M2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance.

Background Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.Experimental design Whole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+ T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+ cytotoxic T cells in tumors.Results Neoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+ (double positive, DP) CD8+ T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+ T cells could be attributed to CD4+ T cells. CD8+ T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.Conclusion Coexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+ T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients.

Background The complete response rate of cervical high-grade squamous intraepithelial lesion (cHSIL) patients to imiquimod immunotherapy is approximately 60%. Consequently, many patients are exposed to unnecessary adverse effects of imiquimod. On the other hand, conventional surgical large loop excision therapy is associated with increased risk of premature births in subsequent pregnancies. An in-depth analysis of the cHSIL immune microenvironment was performed in order to identify and develop a predictive biomarker for response to imiquimod, to maximize therapy efficacy and to avoid adverse effects in patients unlikely to respond.Methods Biopsies of 35 cHSIL patients, before and 10 weeks on imiquimod treatment, were analyzed by two multispectral seven-color immunofluorescence panels for T cell and myeloid cell composition in relation to treatment response. Based on these results a simplified immunohistochemical detection protocol was developed. Samples were scanned with the Vectra multispectral imaging system and cells were automatically identified using machine learning.Results The immune microenvironment of complete responders (CR) is characterized by a strong and coordinated infiltration by T helper cells (activated PD1+/type 1 Tbet+), M1-like macrophages (CD68+CD163-) and dendritic cells (CD11c+) prior to imiquimod. The lesions of non-responders (NRs) displayed a high infiltration by CD3+FOXP3+ regulatory T cells. At 10 weeks on imiquimod, a strong influx of intraepithelial and stromal CD4+ T cells was observed in CR but not NR patients. A steep decrease in macrophages occurred both in CR and NR patients, leveling the pre-existing differences in myeloid cell composition between the two groups. Based on the pre-existing immune composition differences, the sum of intraepithelial CD4 T cell, macrophage and dendritic cell counts was used to develop a quantitative simplified one color immunohistochemical biomarker, the CHSIL immune biomarker for imiquimod (CIBI), which can be automatically and unbiasedly quantified and has an excellent predictive capacity (receiver operating characteristic area under the curve 0.95, p

Background Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP.Methods A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-)malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 µg per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays.Results Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial.Conclusions Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity.Trial registration numbers NCT02821494 and 2014-000658-12.

Background Many solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells.Methods We performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells.Results Reovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth.Conclusions These findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.

Myeloid immune cells are frequently present in the tumor environment, and although they can positively contribute to tumor control they often negatively impact anticancer immune responses. One way of inhibiting the positive contributions of myeloid cells is by signaling through the cluster of differentiation 47 (CD47)/signal regulatory protein alpha (SIRPα) axis. The SIRPα receptor is expressed on myeloid cells and is an inhibitory immune receptor that, upon binding to CD47 protein, delivers a ‘don’t eat me’ signal. As CD47 is often overexpressed on cancer cells, treatments targeting CD47/SIRPα have been under active investigation and are currently being tested in clinical settings. Interestingly, the CD47/SIRPα axis is also involved in T cell-mediated antitumor responses. In this perspective we provide an overview of recent studies showing how therapeutic blockade of the CD47/SIRPα axis improves the adaptive immune response. Furthermore, we discuss the interconnection between the myeloid CD47/SIRPα axis and adaptive T cell responses as well as the potential therapeutic role of the CD47/SIRPα axis in tumors with acquired resistance to the classic immunotherapy through major histocompatibility complex downregulation. Altogether this review provides a profound insight for the optimal exploitation of CD47/SIRPα immune checkpoint therapy.

Background Cross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown.Methods In this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function.Results Here, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression.Conclusion These data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

Background High serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective.Methods IL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1.control. Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6rafl/fl×LysMcre+ mice.Results Our therapeutic vaccination protocol elicits a strong tumor-specific CD8+ T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8+ T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6rafl/fl×LysMcre+ but not cre-negative control mice, while no differences in the vaccine-induced CD8+ T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages.Conclusion IL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections.

Resistance to antitumor immunity can be promoted by the oncogenic pathways operational in human cancers, including the epidermal growth factor receptor (EGFR) pathway. Here we studied if and how EGFR downstream signaling in head and neck squamous cell carcinoma (HNSCC) can affect the attraction of immune cells. HPV-negative and HPV-positive HNSCC cell lines were analyzed in vitro for CCL2, CCL5, CXCL9, CXCL10, IL-6 and IL-1β expression and the attraction of T cells under different conditions, including cetuximab treatment and stimulation with IFNγ and TNFα using qPCR, ELISA and migration experiments. Biochemical analyses with chemical inhibitors and siRNA transfection were used to pinpoint the underlying mechanisms. Stimulation of HNSCC cells with IFNγ and TNFα triggered the production of T-cell attracting chemokines and required c-RAF activation. Blocking of the EGFR with cetuximab during this stimulation increased chemokine production in vitro, and augmented the attraction of T cells. Mechanistically, cetuximab decreased the phosphorylation of MEK1, ERK1/2, AKT, mTOR, JNK, p38 and ERK5. Chemical inhibition of EGFR signaling showed a consistent and pronounced chemokine production with MEK1/2 inhibitor PD98059 and JNK inhibitor SP600125, but not with inhibitors of p38, PI3K or mTOR. Combination treatment with cetuximab and a MEK1/2 or JNK inhibitor induced the highest chemokine expression. In conclusion, overexpression of EGFR results in the activation of multiple downstream signaling pathways that act simultaneously to suppress type 1 cytokine stimulated production of chemokines required to amplify the attraction of T cells.

The clearance of a high-risk human papillomavirus (hrHPV) infection takes time and requires the local presence of a strong type 1 cytokine T cell response, suggesting that hrHPV has evolved mechanisms to resist this immune attack. Using an unique system for non, newly and persistent hrHPV infection, we show that hrHPV infection renders keratinocytes (KCs) resistant to the anti-proliferative and necroptosis inducing effects of IFN and TNFα. HrHPV-impaired necroptosis was associated with the upregulation of several methyltransferases, including EZH2 and the downregulation of RIPK3 expression. Restoration of RIPK3 expression using the global histone methyltransferase inhibitor 3-deazaneplanocin increased necroptosis in hrHPV-positive KCs. Simultaneously, hrHPV effectively inhibited IFN/TNF-mediated arrest of cell growth at the S-phase by downregulating IFITM1 already at 48 hours after hrHPV infection, followed by an impaired increase in the expression of the anti-proliferative gene RARRES1 and a decrease of the proliferative gene PCNA. Knockdown of IFITM1 in uninfected KCs confirmed its role on RARRES1 and its anti-proliferativeory effects. Thus, our study reveals how hrHPV deregulates two pathways involved in cell death and growth regulation to withstand immune mediated control of hrHPV-infected cells.

There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.

Tumor associated macrophages (TAM) can promote angiogenesis, invasiveness and immunosuppression. The cytokine CSF-1 (or M-CSF) is an important factor of TAM recruitment and differentiation and several pharmacological agents targeting the CSF-1 receptor (CSF-1R) have been developed to regulate TAM in solid cancers. We show that the kinase inhibitor PLX3397 strongly dampened the systemic and local accumulation of macrophages driven by B16F10 melanomas, without affecting Gr-1(+) myeloid derived suppressor cells. Removal of intratumoral macrophages was remarkably efficient and a modest, but statistically significant, delay in melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly enhanced tumor control by immunotherapy using tumor-specific CD8 T cells. Elevated IFNγ production by T cells was observed in mice treated with the combination of PLX3397 and immunotherapy. These results support the combined use of CSF-1R inhibition with CD8 T cell immunotherapy, especially for macrophage-stimulating tumors.

Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing enrichment, and the ability to align cell subsets across multiple data samples for comparative analysis. In this manuscript, we develop hierarchical modeling extensions to the Dirichlet Process Gaussian Mixture Model (DPGMM) approach we have previously described for cell subset identification, and show that the hierarchical DPGMM (HDPGMM) naturally generates an aligned data model that captures both commonalities and variations across multiple samples. HDPGMM also increases the sensitivity to extremely low frequency events by sharing information across multiple samples analyzed simultaneously. We validate the accuracy and reproducibility of HDPGMM estimates of antigen-specific T cells on clinically relevant reference peripheral blood mononuclear cell (PBMC) samples with known frequencies of antigen-specific T cells. These cell samples take advantage of retrovirally TCR-transduced T cells spiked into autologous PBMC samples to give a defined number of antigen-specific T cells detectable by HLA-peptide multimer binding. We provide open source software that can take advantage of both multiple processors and GPU-acceleration to perform the numerically-demanding computations. We show that hierarchical modeling is a useful probabilistic approach that can provide a consistent labeling of cell subsets and increase the sensitivity of rare event detection in the context of quantifying antigen-specific immune responses.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Despite the presence of intracellular pathogen recognition receptors that allow infected cells to attract the immune system, undifferentiated keratinocytes (KCs) are the main targets for latent infection with high-risk human papilloma viruses (hrHPVs). HPV infections are transient but on average last for more than one year suggesting that HPV has developed means to evade host immunity. To understand how HPV persists, we studied the innate immune response of undifferentiated human KCs harboring episomal copies of HPV16 and 18 by genome-wide expression profiling. Our data showed that the expression of the different virus-sensing receptors was not affected by the presence of HPV. Poly(I:C) stimulation of the viral RNA receptors TLR3, PKR, MDA5 and RIG-I, the latter of which indirectly senses viral DNA through non-self RNA polymerase III transcripts, showed dampening in downstream signalling of these receptors by HPVs. Many of the genes downregulated in HPV-positive KCs involved components of the antigen presenting pathway, the inflammasome, the production of antivirals, pro-inflammatory and chemotactic cytokines, and components downstream of activated pathogen receptors. Notably, gene and/or protein interaction analysis revealed the downregulation of a network of genes that was strongly interconnected by IL-1β, a crucial cytokine to activate adaptive immunity. In summary, our comprehensive expression profiling approach revealed that HPV16 and 18 coordinate a broad deregulation of the keratinocyte's inflammatory response, and contributes to the understanding of virus persistence.