Tumor cell-intrinsic and tumor microenvironmental conditions co-determine signaling by the glycoimmune checkpoint receptor Siglec-7.

Tumors create an immunosuppressive tumor microenvironment by altering protein expression, but also by changing their glycosylation status, like altered expression of sialoglycans. Sialoglycans are capped with sialic acid sugar residues and are recognized by Siglec immune receptors. Siglec-7 is an inhibitory immune receptor similar to PD-1, and is emerging as glycoimmune checkpoint exploited by cancer cells to evade the immune system. However, the exact cellular and molecular conditions required for Siglec-7-mediated immune cell inhibition remain largely unknown. Here, we report on the development of a chimeric Siglec-7 cell system that enables dissection of Siglec-7 signaling, rather than Siglec-7 binding. Antibody-induced clustering, sialic acid-containing polymers, and highly sialylated erythrocytes effectively induced Siglec-7 signaling, thereby validating functionality of this reporter system. Moreover, the system reveals tumor cell-dependent Siglec-7 signaling. Tumor-associated conditions important for Siglec-7 signaling were defined, such as Siglec-7 ligand expression levels, presence of the known Siglec-7 ligand CD43, and sialic acid availability for sialylation of glycans. Importantly, therapeutic targeting of the Siglec-7/sialic acid axis using a sialyltransferase inhibitor resulted in strong reduction of Siglec-7 signaling. In conclusion, using a newly established cellular tool, we defined a set of tumor-associated conditions that influence Siglec-7 signaling. Moreover, the system allows to assess the efficacy of novel cancer drugs interfering with the Siglec-7/sialic acid axis as immunotherapy to treat cancer.

Comprehensive multiplexed immune profiling of the ductal carcinoma in situ immune microenvironment regarding subsequent ipsilateral invasive breast cancer risk.

BACKGROUND: Ductal carcinoma in situ (DCIS) is treated to prevent subsequent ipsilateral invasive breast cancer (iIBC). However, many DCIS lesions will never become invasive. To prevent overtreatment, we need to distinguish harmless from potentially hazardous DCIS. We investigated whether the immune microenvironment (IME) in DCIS correlates with transition to iIBC. METHODS: Patients were derived from a Dutch population-based cohort of 10,090 women with pure DCIS with a median follow-up time of 12 years. Density, composition and proximity to the closest DCIS cell of CD20+ B-cells, CD3+CD8+ T-cells, CD3+CD8- T-cells, CD3+FOXP3+ regulatory T-cells, CD68+ cells, and CD8+Ki67+ T-cells was assessed with multiplex immunofluorescence (mIF) with digital whole-slide analysis and compared between primary DCIS lesions of 77 women with subsequent iIBC (cases) and 64 without (controls). RESULTS: Higher stromal density of analysed immune cell subsets was significantly associated with higher grade, ER negativity, HER-2 positivity, Ki67 ≥ 14%, periductal fibrosis and comedonecrosis (P < 0.05). Density, composition and proximity to the closest DCIS cell of all analysed immune cell subsets did not differ between cases and controls. CONCLUSION: IME features analysed by mIF in 141 patients from a well-annotated cohort of pure DCIS with long-term follow-up are no predictors of subsequent iIBC, but do correlate with other factors (grade, ER, HER2 status, Ki-67) known to be associated with invasive recurrences.

Correction to: Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

In the original publication of the article the following abstract and keywords were inadvertently omitted.

Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

Background: In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC). Methods: A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.7­1.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week. Results: Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response. Conclusions: ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]

Highly specific and potently activating Vγ9Vδ2-T cell specific nanobodies for diagnostic and therapeutic applications.

Vγ9Vδ2-T cells constitute the predominant subset of γδ-T cells in human peripheral blood and have been shown to play an important role in antimicrobial and antitumor immune responses. Several efforts have been initiated to exploit these cells for cancer immunotherapy, e.g. by using phosphoantigens, adoptive cell transfer, and by a bispecific monoclonal antibody based approach. Here, we report the generation of a novel set of Vγ9Vδ2-T cell specific VHH (or nanobody). VHH have several advantages compared to conventional antibodies related to their small size, stability, ease of generating multispecific molecules and low immunogenicity. With high specificity and affinity, the anti-Vγ9Vδ2-T cell receptor VHHs are shown to be useful for FACS, MACS and immunocytochemistry. In addition, some VHH were found to specifically activate Vγ9Vδ2-T cells. Besides being of possible immunotherapeutic value, these single domain antibodies will be of great value in the further study of this important immune effector cell subset.

Topical rather than intradermal application of the TLR7 ligand imiquimod leads to human dermal dendritic cell maturation and CD8+ T-cell cross-priming.

Toll-like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra-)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara-treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro-inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara-stimulated DCs showed enhanced cross-presentation of the melanoma antigen MART-1, which resulted in increased priming and activation of MART-1-specific CD8(+) T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.

Differential capacity of human interleukin-4 and interferon-α monocyte-derived dendritic cells for cross-presentation of free versus cell-associated antigen.

Dendritic cells (DC) vaccination is a potent therapeutic approach for inducing tumor-directed immunity, but challenges remain. One of the particular interest is the induction of an immune response targeting multiple (unknown) tumor-associated antigens (TAA), which requires a polyvalent source of TAA. Previously, we described the preferred use of apoptotic cell-derived blebs over the larger apoptotic cell remnants, as a source of TAA for both in situ loading of skin-resident DC and in vitro loading of monocyte-derived DC (MoDC). Recent reports suggest that MoDC cultured in the presence of GM-CSF supplemented with IFNα (IFNα MoDC), as compared to IL-4 (IL-4 MoDC), have an increased capacity to cross-present antigen to CD8(+) T cells. As culture conditions, maturation methods and antigen sources differ between the conducted studies, we analyzed the functional differences between IL-4 MoDC and IFNα MoDC, loaded with blebs, in a head-to-head comparison using commonly used protocols. Our data show that both MoDC types are potent (cross-) primers of CD8(+) T cells. Whereas IFNα MoDC were more potent in their capacity to cross-present a 25-mer MART-1 synthetic long peptide (SLP) to a MART-1aa26-35 recognizing CD8(+) T cell line, IL-4 MoDC proved more potent cross-primers of antigen-specific CD8(+) T cells when loaded with blebs. The latter is likely due to the observed greater capacity of IL-4 MoDC to ingest apoptotic blebs. In conclusion, our data indicate the use of IFNα MoDC over IL-4 MoDC in the context of DC vaccination with SLP, whereas IL-4 MoDC are preferred for vaccination with bleb-derived antigens.

Notch controls generation and function of human effector CD8+ T cells.

The generation of effector CD8(+) T cells with lytic capacity is crucial for tumor control. Dendritic cells (DCs) provide important signals to promote naive CD8(+) T cell priming and activation of effector T cells. Here, we report that the Notch pathway has an important role in both these processes in human CD8(+) T cells. Activated monocyte-derived DCs express Notch ligands Jagged1 and Delta-like4, whereas naive CD8(+) T cells express Notch2. The role for Notch signaling in CD8(+) T cell priming was determined using an ex-vivo model system in which tumor antigen-specific primary CD8(+) T cell responses were measured. Inhibition of Notch using γ-secretase inhibitors or soluble Delta-like4-Fc during activation reduced expansion of antigen-specific CD8(+) T cells, which was mirrored by decreased frequencies of interferon (IFN)γ-, tumor necrosis factor-α-, and granzymeB-producing CD8(+) T cells. Moreover, T cells primed when Notch signaling was prevented are functionally low-avidity T cells. In addition, Notch partially regulates established effector T cell function. Activation-induced Notch signaling is needed for IFNγ release but not for cytolytic activity. These data indicate that Notch signaling controls human CD8(+) T cell priming and also influences effector T cell functions. This may provide important information for designing new immunotherapies for treatment of cancer.

Gene-dosage dependent overexpression at the 13q amplicon identifies DIS3 as candidate oncogene in colorectal cancer progression.

Colorectal cancer (CRC) development is in most cases marked by the accumulation of genomic alterations including gain of the entire q-arm of chromosome 13. This aberration occurs in 40%-60% of all CRC and is associated with progression from adenoma to carcinoma. To date, little is known about the effect of the 13q amplicon on the expression of the therein located genes and their functional relevance. We therefore aimed to identify candidate genes at the 13q amplicon that contribute to colorectal adenoma to carcinoma progression in a gene dosage-dependent manner. Integrative analysis of whole genome expression and DNA copy number signatures resulted in the identification of 36 genes on 13q of which significant overexpression in carcinomas compared with adenomas was linked to a copy number gain. Five genes showing high levels of overexpression in carcinomas versus adenomas were further tested by quantitative reverse transcription-PCR in two independent sample sets of colorectal tumors (n = 40 and n = 47). DIS3 and LRCH1 revealed significant overexpression in carcinomas compared with adenomas in a 13q gain dependent manner. Silencing of DIS3 affected important tumorigenic characteristics such as viability, migration, and invasion. In conclusion, significant overexpression of DIS3 and LRCH1 associated with adenoma to carcinoma progression is linked to the CRC specific gain of 13q. The functional relevance of this copy number aberration was corroborated for DIS3, thereby identifying this gene as novel candidate oncogene contributing to the 13q-driven adenoma to carcinoma progression.

Apoptotic blebs from leukemic cells as a preferred source of tumor-associated antigen for dendritic cell-based vaccines.

Since few leukemia-associated antigens (LAA) are characterized for acute myeloid leukemia (AML), apoptotic tumor cells constitute an attractive LAA source for DC-based vaccines, as they contain both characterized and unknown LAA. However, loading DC with apoptotic tumor cells may interfere with DC function. Previously, it was shown in mice that apoptotic blebs induce DC maturation, whereas apoptotic cell remnants (ACR) do not. Here, we analyzed human monocyte-derived DC (MoDC) functionality in vitro, after ingesting either allogeneic AML-derived ACR or blebs. We show that MoDC ingest blebs to a higher extent and are superior in migrating toward CCL19, as compared to ACR-loaded MoDC. Although MoDC cytokine production was unaffected, co-culturing bleb-loaded MoDC with T cells led to an increased T cell proliferation and IFNγ production. Moreover, antigen-specific CD8(+) T cells frequencies increased to 0.63 % by priming with bleb-loaded MoDC, compared to 0.16 % when primed with ACR-loaded MoDC. Importantly, CD8(+) T cells primed by bleb-loaded MoDC recognized their specific epitope at one to two orders of magnitude lower concentrations compared to ACR-loaded MoDC. In conclusion, superior ingestion efficiency and migration, combined with favorable T cell cytokine release and CD8(+) T cell priming ability and avidity, point to blebs as the preferred component of apoptotic leukemic cells for LAA loading of DC for the immunotherapy of AML.

Spatial relationships in the urothelial and head and neck tumor microenvironment predict response to combination immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICI) can achieve remarkable responses in urothelial cancer (UC), which may depend on tumor microenvironment (TME) characteristics. However, the relationship between the TME, usually characterized by immune cell density, and response to ICI is unclear. Here, we quantify the TME immune cell densities and spatial relationships (SRs) of 24 baseline UC samples, obtained before pre-operative combination ICI treatment, using multiplex immunofluorescence. We describe SRs by approximating the first nearest-neighbor distance distribution with a Weibull distribution and evaluate the association between TME metrics and ipilimumab+nivolumab response. Immune cell density does not discriminate between response groups. However, the Weibull SR metrics of CD8+ T cells or macrophages to their closest cancer cell positively associate with response. CD8+ T cells close to B cells are characteristic of non-response. We validate our SR response associations in a combination ICI cohort of head and neck tumors. Our data confirm that SRs, in contrast to density metrics, are strong biomarkers of response to pre-operative combination ICIs.

IL-21 promotes the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells.

BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+ CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT.

Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients.

BACKGROUND: New treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells. METHODS: Tetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells. RESULTS: Ex vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein. CONCLUSIONS: Here we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.

Atezolizumab With or Without Radiotherapy for Advanced Squamous Cell Carcinoma of the Penis (The PERICLES Study): A Phase II Trial.

PURPOSE: Patients with advanced penile squamous cell carcinoma have a poor prognosis (21% 2-year overall survival [OS] from diagnosis). We assessed the activity of atezolizumab (anti-PD-L1) in patients with advanced penile cancer, with or without radiotherapy (RT). PATIENTS AND METHODS: A single-center, nonrandomized phase II study with two treatment arms was conducted in 32 patients with histologically confirmed advanced penile cancer. All patients received atezolizumab (1,200 mg) once every 3 weeks. Twenty patients, who were expected to benefit from RT for locoregional disease control, received additional irradiation. The primary end point was 1-year progression-free survival (PFS) for the complete cohort and was reached if the actual 1-year PFS was at least 35%. Secondary end points included OS, objective response rate (ORR), and tolerability. Exploratory biomarker analyses were conducted in pretreatment specimens. RESULTS: Median follow-up was 29.1 months (IQR, 18.1-33.5). Grade 3-4 adverse events related to atezolizumab or RT were observed in 3/32 (9.4%) and 13/20 (65%) patients, respectively. One-year PFS was 12.5% (95% CI, 5.0 to 31.3), which did not meet the study's primary end point. Median OS was 11.3 months (95% CI, 5.5 to 18.7). In the objective response-evaluable population (n = 30; 93.8%), the ORR was 16.7% (95% CI, 6 to 35), including 2 (6.7%) complete responders and 3 (10%) partial responders. Improved PFS was observed in patients with high-risk human papillomavirus (hrHPV)-positive tumors (P = .003) and those with high infiltration of intratumoral CD3+CD8+ T cells (P = .037). CONCLUSION: Although the primary end point of 1-year PFS was not met, durable antitumor activity to atezolizumab was observed in a subset of patients. Biomarkers, such as hrHPV and intratumoral CD3+CD8+ T-cell infiltration, may help to better select responders.

Poor Outcome in Postpartum Breast Cancer Patients Is Associated with Distinct Molecular and Immunologic Features.

PURPOSE: Patients with postpartum breast cancer diagnosed after cessation of breastfeeding (postweaning, PP-BCPW) have a particularly poor prognosis compared with patients diagnosed during lactation (PP-BCDL), or to pregnant (Pr-BC) and nulliparous (NP-BC) patients, regardless of standard prognostic characteristics. Animal studies point to a role of the involution process in stimulation of tumor growth in the mammary gland. However, in women, the molecular mechanisms that underlie this poor prognosis of patients with PP-BCPW remain vastly underexplored, due to of lack of adequate patient numbers and outcome data. EXPERIMENTAL DESIGN: We explored whether distinct prognostic features, common to all breast cancer molecular subtypes, exist in postpartum tumor tissue. Using detailed breastfeeding data, we delineated the postweaning period in PP-BC as a surrogate for mammary gland involution and performed whole transcriptome sequencing, immunohistochemical, and (multiplex) immunofluorescent analyses on tumor tissue of patients with PP-BCPW, PP-BCDL, Pr-BC, and NP-BC. RESULTS: We found that patients with PP-BCPW having a low expression level of an immunoglobulin gene signature, but high infiltration of plasma B cells, have an increased risk for metastasis and death. Although PP-BCPW tumor tissue was also characterized by an increase in CD8+ cytotoxic T cells and reduced distance among these cell types, these parameters were not associated with differential clinical outcomes among groups. CONCLUSIONS: These data point to the importance of plasma B cells in the postweaning mammary tumor microenvironment regarding the poor prognosis of PP-BCPW patients. Future prospective and in-depth research needs to further explore the role of B-cell immunobiology in this specific group of young patients with breast cancer.

Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives induces rapid dendritic cell differentiation: implications for cancer immunotherapy.

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

Immune cells in mesothelioma microenvironment simplistic marker of response to nivolumab plus ipilimumab?

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a malignant disease of the pleura which recently can be treated with immune checkpoint inhibitors (ICI). To optimize this treatment, a better understanding of the tumor micro environment is needed. We investigated subgroups of immune cells in subsequent tumor biopsies of patients treated with ICI. METHODS: Biopsies from MPM patients included in two clinical ICI trials (nivolumab alone and an ipilimumab/nivolumab combination) were examined. At baseline and after 6 weeks of treatment, pleural biopsies were taken to examine the tumor microenvironment (CD20+, CD4+, CD8+, FoxP3+ and PD-1+ ). Cell density was defined as the number of marker positive cells per mm2. Radiological responses were evaluated as partial response, stable disease or progressive disease according to modified RECIST criteria. RESULTS: Thirty-four and 36 patients were included in the nivolumab and ipiliumumab/nivolumab trial respectively. In the nivolumab trial, no significant differences in cell densities were seen in baseline biopsies of patients with partial response versus progressive disease. In contrast, in the ipilimumab/nivolumab trial, a higher cell density of CD4+, CD8+, FoxP3+ and PD-1+ cells at baseline was significantly correlated with partial responses. On-treatment biopsies of both trials did not show significant changes when compared to baseline biopsies. CONCLUSION: Biopsies from patients responding to nivolumab plus ipilimumab treatment show a significant higher cell density of CD4+, CD8+, FoxP3+ and PD-1+ cells, without a change after 6 weeks of treatment. This observation is a first step in exploring the tumor microenvironment as predictor of response in ICI treatment in MPM.

High susceptibility of c-KIT+CD34+ precursors to prolonged doxorubicin exposure interferes with Langerhans cell differentiation in a human cell line model.

As neoadjuvant and adjuvant chemotherapy schedules often consist of multiple treatment cycles over relatively long periods of time, it is important to know what effects protracted drug administration can have on the immune system. Here, we studied the long-term effects of doxorubicin on the capacity of dendritic cell (DC) precursors to differentiate into a particular DC subset, the Langerhans cells (LC). In order to achieve high telomerase activity as detected in hematological stem cells, precursor cells from the acute-myeloid leukemia (AML)-derived cell line MUTZ3 were stably transduced with human telomerase reverse transcriptase (hTERT) to facilitate their growth potential, while preventing growth, and drug-induced senescence, and preserving their unique capacity for cytokine-dependent DC and LC differentiation. The hTERT-MUTZ3 cells were selected with increasing concentrations of the anthracyclin doxorubicin. After 1-2 months of selection with 30-90 nM doxorubicin, the cells completely lost their capacity to differentiate into LC. This inhibition turned out to be reversible, as the cells slowly regained their capacity to differentiate after a 3- to 4-month drug-free period and with this became capable again of priming allogeneic T cells. Of note, the loss and gain of this capacity to differentiate coincided with the loss and gain of a subpopulation within the CD34(+) proliferative compartment with surface expression of the stem cell factor receptor (SCF-R/CD117/c-Kit). These data are in favor of cytostatic drug-free intervals before applying autologous DC-based vaccination protocols, as specific DC precursors may need time to recover from protracted chemotherapy treatment and re-emerge among the circulating CD34(+) hematopoietic stem and precursor cells.

Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer.

BACKGROUND: Infection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. METHODS: HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. RESULTS: Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. CONCLUSION: Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies.

Breast adipocyte size associates with ipsilateral invasive breast cancer risk after ductal carcinoma in situ.

Although ductal carcinoma in situ (DCIS) is a non-obligate precursor to ipsilateral invasive breast cancer (iIBC), most DCIS lesions remain indolent. Hence, overdiagnosis and overtreatment of DCIS is a major concern. There is an urgent need for prognostic markers that can distinguish harmless from potentially hazardous DCIS. We hypothesised that features of the breast adipose tissue may be associated with risk of subsequent iIBC. We performed a case-control study nested in a population-based DCIS cohort, consisting of 2658 women diagnosed with primary DCIS between 1989 and 2005, uniformly treated with breast conserving surgery (BCS) alone. We assessed breast adipose features with digital pathology (HALO®, Indica Labs) and related these to iIBC risk in 108 women that developed subsequent iIBC (cases) and 168 women who did not (controls) by conditional logistic regression, accounting for clinicopathological and immunohistochemistry variables. Large breast adipocyte size was significantly associated with iIBC risk (odds ratio (OR) 2.75, 95% confidence interval (95% CI) = 1.25-6.05). High cyclooxygenase (COX)-2 protein expression in the DCIS cells was also associated with subsequent iIBC (OR 3.70 (95% CI = 1.59-8.64). DCIS with both high COX-2 expression and large breast adipocytes was associated with a 12-fold higher risk (OR 12.0, 95% CI = 3.10-46.3, P < 0.001) for subsequent iIBC compared with women with smaller adipocyte size and low COX-2 expression. Large breast adipocytes combined with high COX-2 expression in DCIS is associated with a high risk of subsequent iIBC. Besides COX-2, adipocyte size has the potential to improve clinical management in patients diagnosed with primary DCIS.