Identification of promiscuous HPV16‐derived T helper cell epitopes for therapeutic HPV vaccine design

Cervical carcinoma and several other human papillomavirus (HPV)‐induced malignancies are a global public health problem, thus novel treatment modalities are urgently needed. Immunotherapy is an attractive option for treatment of HPV infection and HPV‐mediated premalignant and malignant lesions. However, previous approaches—focusing on the induction of cytotoxic CD8+ T cells (CTLs)—have as yet not yielded clinical successes. Since CD4+ T cells have been shown to be crucial for the induction and maintenance of CTL responses, and more recently to be also important for direct anti‐tumor immunity, human leukocyte antigen (HLA) class II‐restricted epitopes are intensively investigated to improve the efficacy of peptide‐based HPV immunotherapy. We here present an approach to identify promiscuous HPV16‐derived CD4+ T helper epitopes, which are capable of inducing T cell immunity in a large proportion of the population. To this end, we combined HLA class II epitope prediction servers with in vitro immunological evaluation to identify HPV16 E2‐, E5‐, E6‐, and E7‐derived CD4+ T cell epitopes. Candidate selected HPV16‐derived epitopes were found to be restricted by up to nine HLA‐DR molecules. Furthermore, they were found to induce frequent and robust HPV16 peptide‐specific Th1 responses in healthy donors, as monitored by interferon (IFN)‐γ ELISPOT and cytokine secretion assays. Moreover, these selected peptides also induced specific IFN‐γ T cell responses in blood from HPV16+ CIN2/3 and cervical carcinoma patients. We thus conclude that the identified T helper epitopes are valuable candidates for the development of a comprehensive therapeutic HPV vaccine.     

Experimental and clinical characteristics in myelodysplastic syndrome patients with or without HLA‐DR15 allele

We studied the effects of the presence of the HLA‐DR15 allele on the experimental and clinical features of myelodysplastic syndrome (MDS) by assessing the clinical data of 136 patients with MDS. We observed that the frequency of HLA‐DR15 expression in MDS patients (38.7%) was significantly higher than that in the healthy controls (p < 0.01). We noted the following observations with regard to disease progression: None of the 46 HLA‐DR15 positive patients with international prognostic scoring system (IPSS) scores ≤1 developed acute myeloid leukaemia (AML) during the follow‐up period, while six of the 63 DR15‐negative patients with the same IPSS score developed AML within a shorter follow‐up period (p = 0.039). Furthermore, the incidence of poor chromosomal abnormalities, the percentage of patients with IPSS scores ≥1.5 and the presence of ≥5% blasts in the bone marrow in the DR15‐positive patients were lower than the corresponding findings in the DR15‐negative patients. In addition, we also recorded the following observations with regard to bone marrow (BM) failure: The bicytopenia/pancytopenia ratio in the DR15‐positive patients was higher than that in the DR15‐negative patients (92.4 vs. 78.3%; p = 0.029). The peripheral–neutrophil count and the platelet count in the DR15‐positive patients were lower than those in the DR15‐negative patients (p = 0.028 and p = 0.011, respectively). Moreover, hypocellularity was more easily detectable in the DR15‐positive patients (26.4 vs. 16.9%). In addition, the BM CD4+ lymphocyte count and the CD4/CD8 ratio in the DR15‐positive patients were higher than the corresponding values in the DR15‐negative patients (p < 0.05 for both). However, there were no significant differences between the polarization of T‐helper (T_h) and T‐cytotoxic (T_c) cells and the cytokine levels in these two patient groups. We concluded that the presence of the HLA‐DR15 allele is indicative of a genetic susceptibility to MDS and, the presence of the HLA‐DR15 allele showed less association with disease progression and greater association with BM failure. Copyright © 2009 John Wiley & Sons, Ltd.     

CD8‐alpha T‐cell infiltration in human papillomavirus‐related oropharyngeal carcinoma correlates with improved patient prognosis

Patients with human papillomavirus (HPV)‐related oropharyngeal tumors display improved prognosis. The biological basis of this tumor phenotype is poorly understood. We investigated whether increased lymphocyte infiltrate in HPV‐positive oropharyngeal squamous cell carcinomas could account for better prognosis. We previously identified, in an Affymetrix GeneChip analysis of 83 HPV‐unrelated and 11 HPV‐related squamous cell carcinoma of the oropharynx, several candidate genes, including CD8α and CD3ζ. Their expression was validated in this study by qRT–PCR on an independent clinical series of 144 oropharyngeal tumors. Immunohistochemical staining of tumor specimens was performed to evaluate infiltration of tumor stroma by CD8+ and CD4+ lymphocytes. The prognostic value of CD8α and CD3ζ expression levels was measured by Kaplan–Meier and Cox regression model analyses. Immune response‐related signaling pathways were found to be deregulated in HPV‐positive oropharyngeal tumors. Expression of CD8α, CD3ζ, granzyme K, CD28 and integrin αL RNAs was upregulated in HPV‐positive lesions when compared with HPV‐unrelated tumors (p < 0.05). Stroma of HPV‐positive tumors was frequently and strongly infiltrated by CD8α‐ and CD3ζ‐positive T cells. CD8α RNA expression correlated with both improved global (Kaplan–Meier; p = 0.005; Cox regression: p = 0.003) and disease‐free (Cox regression: p = 0.04) survival. CD3ζ RNA expression correlated with improved overall survival (Cox regression: p = 0.024). These results suggest that an increased cytotoxic T‐cell‐based antitumor immune response is involved in improved prognosis of patients with HPV‐positive tumors.     

Tumor cell expression of immune inhibitory molecules and tumor‐infiltrating lymphocyte count predict cancer‐specific survival in pancreatic and ampullary cancer

Understanding the mechanisms of immune resistance in pancreatic and ampullary cancers is crucial for the development of suitable biomarkers and effective immunotherapeutics. Our aim was to examine the expression of the immune inhibiting molecules PD‐L1, Galectin‐9, HVEM, IDO and HLA‐G, as well as CD8+ and FoxP3+ tumor infiltrating lymphocytes (TIL), in pancreatic and ampullary cancers, and to relate their individual, as well as their combined expression, to cancer survival. Tumor tissue from 224 patients with resected pancreatic (n = 148) and ampullary (n = 76) cancer was used to construct tissue‐microarrays. Expression of immune inhibitory molecules and TIL was examined by immunohistochemistry. We show that immune inhibitory molecules are prevalently expressed. Moreover, high tumor expression of PD‐L1 (p = 0.002), Gal‐9 (p = 0.003), HVEM (p = 0.001), IDO (p = 0.049), HLA‐G (p = 0.004) and high CD8/FoxP3 TIL ratio (p = 0.006) were associated with improved cancer‐specific survival. All immune biomarkers, with the exception of IDO, were individually predictive of cancer‐specific survival when adjusted for clinicopathologic characteristics. For every additional immune biomarker present survival was almost two‐fold prolonged (HR 0.57 95%CI 0.47–0.69, p < 0.0001). When patients with pancreatic and ampullary cancer were analyzed separately the results were similar. We conclude that pancreas and ampullary cancers are rich in expression of immune‐inhibitory molecules. These molecules can be targets for future immunotherapeutics, as well as form powerful immunological biomarkers. We propose that such immune biomarker panels be included in future prospective immunotherapy trials.     

CD56‐positive lymphocyte infiltration in relation to human papillomavirus association and prognostic significance in oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV)‐related squamous cell carcinoma of the oropharynx (OSCC) are clinical and biological distinct from their HPV‐unrelated counterparts. Patients with HPV‐related OSCC display improved prognosis and therefore we investigated possible immune cell infiltrations associated with this tumor phenotype. We retrospectively analyzed a randomly selected cohort of 140 OSCC for presence of immune cells and HPV by immunohistochemistry and PCR followed by bead‐based hybridization (Luminex technology). HPV prevalence was 24.3% as determined by positive staining of p16INK4a and detection of high risk HPV‐DNA. We found significantly higher numbers of CD56 positive (CD56+) cells in tumor and surrounding microenvironment in HPV‐associated compared to HPV‐negative OSCC (t‐test: p = 0.004 and p = 0.002). For the entire cohort presence of CD56+ cells was associated with increased overall survival independent from HPV (Kaplan–Meier: p = 0.002; Cox regression: p = 0.042). Presence of CD56+ cells also correlated with a better outcome in HPV‐negative and especially in HPV‐negative OSCC with alcohol consumption ≤2 standard drinks per day (Kaplan–Meier: p = 0.05 and p = 0.003). Immunofluorescence localization of granular Granzyme B (GZMB) within CD56+ cells and coexpression of CD16 and CD56 suggests that detected CD56+ cells mainly represent cytotoxic Natural Killer (NK) cells. The fraction of potentially cytotoxic NK cells was significantly higher in HPV‐associated compared to HPV‐negative OSCC (Mann‐Whitney‐U‐Test: p = 0.011). The elevated abundance and activity of cytotoxic NK cells in OSCC with HPV driven carcinogenesis might contribute to favorable outcome in HPV‐related OSCC.     

Increased intratumoral regulatory T cells are related to intratumoral macrophages and poor prognosis in hepatocellular carcinoma patients

Immunosuppression mediated by regulatory T cells (Tregs) is a key facilitator of tumor immune evasion, but the source of these Tregs and their contribution to human cancer progression remains unclear. This study investigated the properties of FoxP3⁺ Tregs, their prognostic value in patients with hepatocellular carcinoma (HCC) and the underlying mechanisms of FoxP3⁺ Treg intratumoral accumulation. In addition to an increased number of circulating FoxP3⁺ Tregs, the results also showed that FoxP3⁺ Tregs gathered in the tumor site, where they suppressed tissue‐derived CD4⁺CD25^? T‐cell activation (p < 0.001), promoting disease progression and poor prognosis in HCC patients (< 0.01). The intratumoral prevalence of FoxP3⁺ Tregs was associated with a high density of macrophages (Mφ) (p < 0.001). Depletion of tissue Mφ thus attenuated the increase of liver FoxP3⁺ Treg frequency attributed to in vivo inoculation with hepatoma (p = 0.01), whereas Mφ exposed to tumor culture supernatants from hepatoma‐derived cell lines increased FoxP3⁺ Treg frequency in vitro (p < 0.001). This increase was partially blocked by antiinterleukin‐10 antibody (p < 0.01). In conclusion, tumor‐associated Mφ may trigger a rise of the intratumoral FoxP3⁺ Treg population, which in turn may promote HCC progression. © 2009 UICC     

A high proportion of bone marrow T cells with regulatory phenotype (CD4+CD25hiFoxP3+) in Ewing sarcoma patients is associated with metastatic disease

Immunosuppressive CD4+CD25^hiFoxP3+ T cells (T_reg cells) have been found at increased densities within the tumor microenvironment in many malignancies and interfere with protective antitumor immune responses. Osseous Ewing sarcomas (ESs) are thought to derive from a bone marrow (BM) mesenchymal cell of origin, and microscopic marrow involvement defines a subpopulation of patients at a high risk of relapse. We hypothesized that BM‐resident T cells may contribute to a permissive milieu for immune escape of ESs. Using 6‐color‐flow cytometry, we investigated the pattern of immune cell subset distribution including NK cells, γδ T cells, central and effector memory CD8+ and CD4+ T cells as well as T cells with regulatory phenotype (T_reg cells) in BM obtained at diagnosis from 45 primary or relapsed ES patients treated within standardized protocols. Although patients at relapse had an inverted CD4:CD8 T‐cell ratio, neither CD8+ effector/memory T‐cell subsets nor T_reg cells significantly differed from patients at diagnosis. No significant associations of innate and effector/memory T‐cell subpopulations with known risk factors were found, including age, gender, tumor site, primary metastases and histological tumor response. By contrast, T_reg cells were found at significantly higher frequencies in patients with primary metastatic disease compared with localized ESs (5.0 vs. 3.3%, p = 0.01). Thus, increased BM T_reg cells in patients with metastasized ES may reflect an immune escape mechanism that contributes to the development of metastatic disease. Immunotherapeutic strategies will have to adequately consider the regulatory milieu within areas of Ewing tumor‐immune interactions. © 2009 UICC     

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan–Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearman's rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6–0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7–0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3–0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64? HLA‐DR‐myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.     

MHC II lung cancer vaccines prime and boost tumor‐specific CD4+ T cells that cross‐react with multiple histologic subtypes of nonsmall cell lung cancer cells

Nonsmall cell lung cancer (NSCLC) is the major cause of lung cancer‐related deaths in the United States. We are developing cell‐based vaccines as a new approach for the treatment of NSCLC. NSCLC is broadly divided into 3 histologic subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Since these subtypes are derived from the same progenitor cells, we hypothesized that they share common tumor antigens, and vaccines that induce immune reactivity against 1 subtype may also induce immunity against other subtypes. Our vaccine strategy has focused on activating tumor‐specific CD4⁺ T cells, a population of lymphocytes that facilitates the optimal activation of effector and memory cytotoxic CD8⁺ T cells. We now report that our NSCLC MHC II vaccines prepared from adeno, squamous or large cell carcinomas each activate CD4⁺ T cells that cross‐react with the other NSCLC subtypes and do not react with HLA‐DR‐matched normal lung fibroblasts or other HLA‐DR‐matched nonlung tumor cells. Using MHC II NSCLC vaccines expressing the DR1, DR4, DR7 or DR15 alleles, we also demonstrate that antigens shared among the different subtypes are presented by multiple HLA‐DR alleles. Therefore, MHC II NSCLC vaccines expressing a single HLA‐DR allele activate NSCLC‐specific CD4⁺ T cells that react with the 3 major classes of NSCLC, and the antigens recognized by the activated T cells are presented by several common HLA‐DR alleles, suggesting that the MHC II NSCLC vaccines are potential immunotherapeutics for a range of NSCLC patients.     

The PD‐1/PD‐L1 axis and human papilloma virus in patients with head and neck cancer after adjuvant chemoradiotherapy: A multicentre study of the German Cancer Consortium Radiation Oncology Group (DKTK‐ROG)

We examined the prognostic role of PD‐1+ and CD8+ tumor infiltrating lymphocytes (TILs), and PD‐L1+ cells in patients with squamous cell carcinoma of the head and neck (SCCHN) treated with surgery and postoperative chemoradiotherapy (CRT). FFPE samples from 161 patients were immunohistochemically stained for PD‐1, CD8 and PD‐L1. The immune marker expression was correlated with clinicopathologic characteristics, and overall survival (OS), local progression‐free survival (LPFS) and distant metastases free‐survival (DMFS), also in the context of HPV16 DNA/p16 status. The median follow‐up was 48 months (range: 4–100). The 2‐year‐OS was 84.1% for the entire cohort. High PD‐1 and PD‐L1 expression were more common in patients with positive HPV16 DNA (p < 0.001 and p = 0.008, respectively) and high infiltration by CD8+ TILs (p < 0.001 for both markers). High PD‐L1 expression correlated with superior OS (p = 0.025), LPFS (p = 0.047) and DMFS (p = 0.048) in multivariable analysis, whereas no significance could be demonstrated for PD‐1. Patients with CD8^high/PD‐L1^high expression had favorable outcome (p < 0.001 for all endpoints) compared to other groups. We validated the superior OS data on CD8^high/PD‐L1^high using the Cancer Genome Atlas TCGA dataset (n = 518; p = 0.032). High PD‐L1 expression was a favorable prognostic marker in HPV16‐negative but not HPV16‐positive patients. In conclusion, HPV‐positive tumors showed higher expression of immune markers. PD‐L1 expression constitutes an independent prognostic marker in SCCHN patients post‐adjuvant CRT. In conjunction with CD8 status, these data provide an important insight on the immune contexture of SCCHN and are directly relevant for future treatment stratification with PD‐1/PD‐L1 immune checkpoint inhibitors to complement CRT.     

CD8+ tumour‐infiltrating lymphocytes in relation to HPV status and clinical outcome in patients with head and neck cancer after postoperative chemoradiotherapy: A multicentre study of the German cancer consortium radiation oncology group (DKTK‐ROG)

We examined the prognostic value of tumour‐infiltrating lymphocytes (TILs) in patients with squamous cell carcinoma of the head and neck (SCCHN) after surgery and postoperative cisplatin‐based chemoradiotherapy. FFPE‐tissue originating from the surgery of 161 patients treated in 8 DKTK partner sites was immunohistochemically stained for CD3 and CD8. Their expression was correlated with clinicopathological characteristics as well as overall survival (OS), local progression‐free survival (LPFS) and distant metastases free‐survival (DMFS), also in the context of the HPV16‐DNA/p16 status. After a median follow‐up of 48 months (range: 4100 months), OS at 4 years was 46.5% for the entire cohort. In multivariate analysis, high CD8 expression was confirmed as an independent prognostic parameter for OS (p = 0.002), LPFS (p = 0.004) and DMFS (p = 0.006), while CD3 expression lacked significance. In multivariate analysis HPV16 DNA positivity was associated with improved OS (p = 0.025) and LPFS (p = 0.013) and p16‐positive patients showed improved DMFS (p = 0.008). Interestingly, high CD8 expression was a prognostic parameter for the clinical outcome in both HPV16 DNA‐positive and HPV16 DNA‐negative patients. Similar findings were observed in the multivariate analysis for the combined HPV16 DNA/p16 status. Altogether, CD8+ TILs constitute an independent prognostic marker in SCCHN patients treated with adjuvant chemoradiotherapy. These data indicate that CD8‐positive TILs have antitumour activity and could be used for treatment stratification. Further validation of the prognostic value of CD8+ TILs as a biomarker and its role in the immune response in SCCHN patients after adjuvant chemoradiotherapy is warranted and will be performed in the prospective DKTK‐ROG study.     

Serum interferon gamma level predicts recurrence in hepatocellular carcinoma patients after curative treatments

Host immunity may have important role in the prognosis of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the correlation between circulating immune regulators and clinical outcome in patients with HCC. Sixty‐three HCC patients were prospectively enrolled. Serum levels of interleukin‐10 (IL‐10), transforming growth factor‐beta (TGF‐β), interferon‐gamma (IFN‐γ) and interferon gamma‐inducible protein 10 (IP‐10) were measured, as well as the prevalence of regulatory T cells (Treg), NK⁺ T cells, invariant natural killer T cells (iNKT), programmed cell death‐1 (PD‐1)⁺CD8⁺ T cells, T helper 17 cells (Th17), CD69⁺ and CD45RO⁺ T cells in peripheral blood mononuclear cells (PBMC). Correlation between these immune regulators and clinical outcome were analyzed. A low serum IFN‐γ level (<50 pg/mL) was significantly associated tumor stage (BCLC stage B: 61.25% vs. stage A: 25%, p = 0.010) and tumor size (>5 cm: 53.8% vs. <5 cm: 25%, p = 0.047). Recurrence‐free survival was evaluated in 48 patients receiving curative treatment of HCC. By multivariate analysis, BCLC stage [hazard ratio (HR) = 32.180, p < 0.001], tumor size (HR = 15.373, p = 0.005), AST (HR = 3.796, p = 0.011) and IFN‐γ (HR = 0.354, p = 0.018) levels were independent factors associated with recurrence‐free survival. In conclusion, serum IFN‐γ level correlates with tumor stage and tumor size in HCC patients. Patients with lower baseline IFN‐γ levels have a higher risk of tumor recurrence after curative treatment. IFN‐γ may reflect host anti‐tumor immunity and may be a potential marker of HCC recurrence after curative treatment.     

HLA‐DR expression is associated with better prognosis in sporadic Australian clinicopathological Stage C colorectal cancers

Predicting patient outcome for colorectal carcinoma (CRC) with lymph node but not distant metastases remains challenging. Various prognostic markers have been identified including microsatellite instability (MSI) and possibly expression of the MHC Class II protein, HLA‐DR. About 15% of sporadic CRC exhibits MSI associated with methylation of the DNA mismatch repair gene hMLH1 promoter. In addition, a significant proportion of unselected CRC demonstrates expression of HLA‐DR. We sought to examine the relationship between HLA‐DR expression, MSI status and prognosis in sporadic Australian Clinicopathological (ACP) Stage C CRC. Two hundred seventy consecutive patients with sporadic ACP Stage C CRC were treated at Concord Repatriation General Hospital between 1986 and 1992. None of these patients received adjuvant chemotherapy and all were followed for a minimum of 5 years or until death. DNA was extracted from paraffin sections and MSI status determined by PCR. HLA‐DR expression was determined immunohistochemically using an antibody against the HLA‐DR α chain. MSI status could be assigned in 235 cases: 176 CRCs (74.9%) were microsatellite stable, whereas 23 (9.8%) had high levels of MSI (MSI‐H) and 36 (15.3%) had low levels of MSI (MSI‐L). HLA‐DR expression by CRC cells was seen in 148 (60.1%) cases and correlated with the presence of tumor‐infiltrating lymphocytes (p = 0.0005) and peritumoral lymphocytes (p = 0.003), but not other clinicopathological features or MSI status. HLA‐DR‐positive CRCs were strongly associated with better patient outcome (p < 0.0001). © 2009 UICC     

Association of HLA‐DRB1, interleukin‐6 and cyclin D1 polymorphisms with cervical cancer in the Swedish population—A candidate gene approach

High‐risk human papillomavirus (hrHPV) infection is the major risk factor for cervical cancer (CxCa). The role of genetic susceptibility in the disease has been suggested, but the existing data lack consistency. We conducted a nested case‐control study on 973 CxCa cases and 1,763 matched controls, from two Swedish population‐based cohorts to examine the association of common genetic variants with CxCa risk. Human leukocyte antigen (HLA) alleles and 24 other polymorphisms in 14 genes were selected on the basis of reported association or mechanistic plausibility with an HPV infection or cervical cancer development. Genotyping was conducted using multiplex PCR and Luminex technology. A significant association of CxCa with various polymorphisms was observed: rs1800797 in the IL‐6 gene (odds ratio [OR] = 0.88, 95% confidence intervals [CI]: 0.79–0.99); rs1041981 in the LTA gene (OR = 0.87, 95% CI: 0.78–0.98), and rs9344 in the CCND1 gene (OR = 1.14, 95% CI: 1.02–1.27), for those individuals carrying the rare allele. Additionally, the alleles 0401 and 1501 of the HLA class II DRB1 locus were associated with an increased risk (OR = 1.23, 95% CI: 1.04–1.45 and OR = 1.29, 95% CI: 1.11–1.50, respectively), and allele 1301 was associated with decreased risk (OR = 0.59, 95% CI: 0.47–0.73). The effects of CCND1 and the HLA*DRB1 alleles were independent of the effect of smoking. We did not find any association of risk with polymorphisms in genes related to the innate immune system. In conclusion, our study provides evidence for genetic susceptibility to CxCa due to variations in genes involved in the immune system and in cell cycle. © 2009 UICC     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

Prognostic value of intra‐tumoral CD8+/FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis

Background and Objectives

Patients with isolated colorectal‐cancer‐liver‐metastases (CRCLM) frequently undergo metastatectomy. Tumor‐infiltrating‐lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor‐infiltrating CD8⁺ cytotoxic T‐cells and FoxP3⁺ regulatory T‐cells at the metastatic site of CRCLM patients.

Methods

TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin‐embedded tissue, from the same patients, was retrieved. CD8⁺ and FoxP3⁺ cells, both in the intra‐tumoral and the peri‐tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T‐cells (CD8⁺) and regulatory T‐cells (CD4⁺CD25⁺FoxP3⁺), within CD45⁺TILs, were measured by flow‐cytometry.

Results

By immunohistochemistry, individual densities of intra‐tumoral or peri‐tumoral CD8⁺ and FoxP3⁺ cells were not prognostic of survival. However, the intra‐tumoral, but not the peri‐tumoral, CD8⁺/FoxP3⁺ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19‐0.95, P = 0.032). By flow cytometry, the intra‐tumoral CD8⁺/regulatory T‐cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20‐0.99, P = 0.044).

Conclusions

The ratio of cytotoxic (CD8⁺) to regulatory (FoxP3⁺) T‐cells, in the intra‐tumoral compartment, but not in the peri‐tumoral compartment, can predict survival after resection of CRCLM.     

A prospective study on the natural course of low‐grade squamous intraepithelial lesions and the presence of HPV16 E2‐, E6‐ and E7‐specific T‐cell responses

This study investigates the clinical course of low grade squamous intraepithelial lesions (LSIL), HPV status and HPV16‐specific immune response in a large prospective study of 125 women with LSIL followed cytologically, virologically and histologically. Women with low‐grade abnormal smears were recruited and followed‐up for one year. Colposcopy, cervical biopsy for histology and brushings for HPV typing was performed at recruitment, 6 months (no biopsy) and upon completion of the study at one year. HPV16‐specific T‐cell responses were analysed by interferon‐γ ELISPOT at entry, 6 and 12 months. Infection with multiple HPV types was detected in 70% of all patients, HPV16 was found in 42% of the patients. LSIL lesions progressed to HSIL in 24%, persisted in 60% and regressed to normal in 16% of the patients. No difference was observed in the clearance rate of infections with single or multiple HPV types among the groups with a different histological outcome. HPV16‐specific type 1 T‐cell responses were detected in only half of the patients with an HPV16+ LSIL, and predominantly reactive to HPV16 E2 and E6. Interestingly, the presence of HPV16 E2‐specific T‐cell responses correlated with absence of progression of HPV16+ lesions (p = 0.005) while the detection of HPV16 E6 specific reactivity was associated with persistence (p = 0.05). This large prospective study showed that the majority of LSIL persisted or progressed within the first year.This was paralleled by immune failure as most of the patients with an HPV16+ LSIL failed to react to peptides of HPV16 E2, E6 or E7.     

Tumor‐reactive CD8+ T‐cell responses after vaccination with NY‐ESO‐1 peptide,CpG 7909 and Montanide® ISA‐51: association with survival

Peptide‐based vaccines have led to the induction of antigen‐specific CD8⁺ T‐cell responses in patients with NY‐ESO‐1 positive cancers. However, vaccine‐induced T‐cell responses did not generally correlate with improved survival. Therefore, we tested whether a synthetic CpG 7909 ODN (deoxycytidyl‐deoxyguanosin oligodeoxy‐nucleotides) mixed with NY‐ESO‐1 peptide p157‐165 and incomplete Freund's adjuvants (Montanide® ISA‐51) led to enhanced NY‐ESO‐1 antigen‐specific CD8⁺ immune responses in patients with NY‐ESO‐1 or LAGE‐1 expressing tumors. Of 14 HLA‐A2+ patients enrolled in the study, 5 patients withdrew prematurely because of progressive disease and 9 patients completed 1 cycle of immunization. Nine of 14 patients developed measurable and sustained antigen‐specific CD8⁺ T‐cell responses: Four had detectable CD8+ T‐cells against NY‐ESO‐1 after only 2 vaccinations, whereas 5 patients showed a late‐onset but durable induction of NY‐ESO‐1 p157‐165 specific T‐cell response during continued vaccination after 4 months. In 6 patients, vaccine‐induced antigen‐specific T‐cells became detectable ex vivo and reached frequencies of up to 0.16 % of all circulating CD8⁺ T‐cells. Postvaccine T‐cell clones were shown to recognize and lyse NY‐ESO‐1 expressing tumor cell lines in vitro. In 6 of 9 patients developing NY‐ESO‐1‐specific immune responses, a favorable clinical outcome with overall survival times of 43+, 42+, 42+, 39+, 36+ and 27+ months, respectively, was observed.     

Overexpression of hypoxia‐inducible factor 1 alpha impacts FoxP3 levels in mycosis fungoides—Cutaneous T‐cell lymphoma: Clinical implications

Mycosis fungoides (MF) is the most common variant of primary cutaneous T‐cell lymphoma, and decreased forkhead box P3 (FoxP3) expression has been reported in MF late stages. Hypoxia‐inducible factor 1 alpha (HIF‐1α) may regulate FoxP3 expression; however, it is unknown whether HIF‐1α is expressed in the CD4⁺ T cells of MF patients and how it could affect the expression of FoxP3. Therefore, we evaluated the expression of HIF‐1α and FoxP3 in CD4⁺ T cells obtained from the skin lesions of MF patients. We found increased cell proliferation and an increase in CD4⁺ T cells with an aberrant phenotype among early stage MF patients. HIF‐1α was overexpressed in these CD4⁺ T cells. In addition, we found a decrease in the percentage of FoxP3⁺ cells both in the skin of MF patients, when compared with control skin samples, and with disease progression. In addition, a negative correlation was established between HIF‐1α and FoxP3 expression. Skin HIF‐1α expression in MF patients correlated with the extent of the affected area and increased with the disease progression. Finally, we showed that ex vivo inhibition of HIF‐1α degradation increases the percentage of FoxP3⁺ T cells in skin lesions. Our results suggest that overexpression of HIF‐1α affects the levels of FoxP3 in MF patients, which could have relevant implications in terms of disease outcome.     

T cells raised against allogeneic HLA-A2/CD20 kill primary follicular lymphoma and acute lymphoblastic leukemia cells

T cells mediating a graft‐versus‐leukemia/lymphoma effects without causing graft‐versus‐host disease would greatly improve the safety and applicability of hematopoietic stem cell transplantation. We recently demonstrated that highly peptide‐ and HLA‐specific T cells can readily be generated against allogeneic HLA‐A*02:01 in complex with a peptide from the B cell‐restricted protein CD20. Here, we show that such CD20‐specific T cells can easily be induced from naïve precursors in cord blood, demonstrating that they do not represent cross‐reactive memory cells. The cells displayed high avidity and mediated potent cytotoxic effects on cells from patients with the CD20^pos B cell malignancies follicular lymphoma (FL) and acute lymphoblastic leukemia (ALL). However, the cytotoxicity was consistently lower for cells from two of the ALL patients. The ALL cells that were less efficiently killed did not display lower surface expression of CD20 or HLA‐A*02:01, or mutations in the CD20 sequence. Peptide pulsing fully restored the levels of cytotoxicity, indicating that they are indeed susceptible to T cell‐mediated killing. Adoptive transfer of CD20‐specific T cells to an HLA‐A*02:01^pos patient requires an HLA‐A*02:01^neg, but otherwise HLA identical, donor. A search clarified that donors meeting these criteria can be readily identified even for patients with rare haplotypes. The results bear further promise for the clinical utility of CD20‐specific T cells in B cell malignancies.