Hetty J. Bontkes

Low Levels of Circulating Invariant Natural Killer T Cells Predict Poor Clinical Outcome in Patients With Head and Neck Squamous Cell Carcinoma

PURPOSE Evading antitumor immune responses is an important aspect of the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Invariant CD1d-restricted natural killer T (iNKT) cells play an allegedly pivotal role in such responses via transactivation of immune effector cells. It has been reported that iNKT cells are reduced in peripheral blood of cancer patients compared with healthy controls. Here, we investigated whether the extent of this deficiency affected disease outcome in HNSCC patients. PATIENTS AND METHODS In a prospective study, circulating iNKT cell numbers were evaluated in 47 patients before radiotherapy. Patients were stratified in three groups based on iNKT cell levels, and clinical data were obtained during a median follow-up period of 31 months. RESULTS A small, compared with an intermediate or large, circulating iNKT cell fraction was significantly associated with decreased 3-year overall survival rate (39% v 75% and 92%, respectively), disease-specific survival rate (43% v 87% and 92%, respectively), and locoregional control rate (31% v 74% and 92%, respectively) in HNSCC patients. Cox regression revealed that the iNKT cell level, as well as clinical T stage, was an independent prognostic parameter even after correction for the confounding effect of age. CONCLUSION A severe circulating iNKT cell deficiency was related to poor clinical outcome in HNSCC patients, suggesting their critical contribution to antitumor immune responses. Furthermore, screening for iNKT cell levels may be useful for determining which patients can benefit from immunotherapeutic adjuvant therapies aimed at reconstitution of the circulating iNKT cell pool.

HPV 16 infection and progression of cervical intra‐epithelial neoplasia: Analysis of HLA polymorphism and HPV 16 E6 sequence variants

High‐risk human papillomavirus (HPV) infection plays an important role in cervical intra‐epithelial neoplasia (CIN), but HPV infection alone is not sufficient for progression to cervical cancer. Several lines of evidence suggest that cellular immune surveillance is important in the control of HPV infection and the development of CIN. The presentation to T cells of target viral peptides in the context of HLA molecules is influenced by the genetic polymorphisms of both HPV and HLA and thereby influences the host immune response and clinical outcome of HPV infection. HLA class I and II polymorphism in susceptibility for HPV 16 infection, development and progression of CIN was analyzed in a group of 118 patients participating in a prospective study of women with initial abnormal cytology. Patients were stratified according to HPV status and course of the disease. HLA‐B*44 frequency was increased in the small group of patients with a lesion that showed clinical progression during follow‐up [OR = 9.0 (4.6–17.5),p= 0.007]. HLA‐DRB1*07 frequency was increased among HPV 16‐positive patients compared with patients who were negative for all HPV types [OR = 5.9 (3.0–11.3), p= 0.02]. Our results are consistent with the immunogenetic factors associated with disease progression being different from those associated with susceptibility to HPV 16 infection. Sequencing of the HPV 16 E6 and E7 open reading frames of a subset of these patients (n = 40) showed the frequency of HPV 16 variants to be similar to other studies. However, there was no significant correlation between variant incidence and disease progression or viral persistence and no significant correlation with any HLA allele. It appears that multiple HLA types can influence HPV 16‐associated cervical dysplasia but the role of HPV 16 variants in disease progression and susceptibility in relation to HLA polymorphism remains unclear. Int. J. Cancer 78:166–171, 1998.© 1998 Wiley‐Liss, Inc.

CD4+CD25hi regulatory T‐cell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia

CD4+CD25hiCTLA4+FoxP3+ regulatory T cells (Treg) have been shown to maintain immune tolerance against self antigens and increased circulating frequencies have been reported in various types of cancers. Circulating invariant natural killer T‐cells (iNKT) are reduced in cancer patients and low iNKT frequency is related to poor prognosis. It is not yet clear whether high Treg numbers and low iNKT cell numbers pose an increased risk for the progression of premalignant lesions or whether Treg and iNKT cell numbers are influenced by dysplasia. We therefore studied prospectively the relation between iNKT cell and Treg frequencies and the natural course of human papillomavirus type 16 (HPV16) induced pre‐malignant cervical dysplasia in 82 patients who participated in a nonintervention cohort study of women with abnormal cytology. Treg frequencies were significantly increased in women who had persistent HPV16 infection. Within the HPV16 persistence group there was no difference in Treg frequencies among patients who developed a CIN3 lesion and patients who did not progress to CIN3. Furthermore, Treg frequencies were increased in patients who had detectable HPV16 E7 specific IL‐2 producing T‐helper cells, which suggests a causal role of HPV infection in Treg development in parallel with HPV16 specific T helper cells. No evidence was found for a role for iNKT cells in persistence of HPV16 and progression of HPV16 induced CIN lesions. However, HPV‐persistence‐associated Tregs may explain the inefficacy of concomitant persistence associated immunity and may contribute to subsequent progression to neoplasia.

Immune responses against human papillomavirus (HPV) type 16 virus-like particles in a cohort study of women with cervical intraepithelial neoplasia II. Systemic but not local IgA responses correlate with clearance of HPV-16

To investigate whether there is an association between local or systemic IgG and IgA responses against human papillomavirus (HPV) type 16 virus-like particles (VLP) containing L1 and L2 and the possible influence of these responses on clearance of HPV-16 and its associated lesions, cervical mucus samples from 125 patients and plasma samples from 100 patients, all participating in a non-intervention cohort study of women with abnormal cytology, were analysed. The results show that local IgG and IgA HPV-16 VLP-specific antibodies do not correlate with virus clearance. However, systemic IgG responses were more frequently detected in patients with a persistent infection (11/24) compared with patients with cleared HPV-16 infections (3/28, P = 0.006). Furthermore, the ultimate development of high-grade lesions was associated with systemic VLP-specific IgG reactivity (P = 0.026). By contrast, systemic IgA responses were correlated with virus clearance (7/28 clearance compared with 1/24 persistence patients, P = 0.06). This correlation was statistically significant when only those clearance patients who tested HPV-16 DNA-positive at more than one visit were included in the analysis (5/11 compared with 1/24, P = 0.007). As these systemic IgA responses were not accompanied by local IgA responses, the systemic IgA responses in HPV-16 clearance patients are suggested to be a by-product of a successful cellular immune response induced at the local lymph nodes, mediated by cytokines.

Human papillomavirus type 16 E6/E7-specific cytotoxic T lymphocytes in women with cervical neoplasia.

Infection with oncogenic human papillomavirus (HPV) types is associated with the development of cervical neoplasia (CIN). The E6 and E7 oncoproteins are constitutively expressed in these lesions and are therefore putative targets for the immune response against HPV. The relation between HPV 16‐specific memory cytotoxic T‐cell precursor (mCTLp) activity to both oncoproteins and the natural course of cervical dysplasia was analyzed in 38 patients participating in a nonintervention cohort study of women with CIN and 11 HPV 16‐positive cervical carcinoma patients. In a cross‐sectional study at the end of follow‐up prior to biopsy, 8 of 20 patients with a persistent HPV 16 infection had specific mCTLp against at least one of the two oncoproteins. By contrast, no specific mCTLp activity was detected in 11 HPV‐negative patients or in 7 patients who had cleared an HPV 16 infection at the end of follow‐up. However, 5 of 11 cervical carcinoma patients showed mCTLp activity against the E7 protein only. This study demonstrates that HPV 16 oncogene‐specific mCTLp are present in women with HPV 16‐positive CIN prior to any intervention. Since HPV‐specific mCTLp were detected predominantly in women with high‐grade lesions or invasive cervical carcinoma and not in women who cleared the virus, the role of naturally occurring mCTLp in the protection against HPV‐associated cervical neoplasia remains to be established. Int. J. Cancer 88:92–98, 2000.

T cell proliferative responses against human papillomavirus type 16 E7 oncoprotein are most prominent in cervical intraepithelial neoplasia patients with a persistent viral infection

T cell proliferative responses against human papillomavirus type 16 (HPV-16) E7 protein were studied in relation to HPV status over time in 51 women originally diagnosed with abnormal cervical cytology and participating in a follow-up study. HPV-16-positive patients were grouped as having either a persistent, a cleared or a fluctuating HPV-16 infection as determined by PCR in consecutive cervical smears up until the moment of testing. Positive proliferative responses against HPV-16 E7 were found in 15/26 patients with a persistent, cleared or fluctuating HPV-16 infection (57.7%). In contrast, 0/15 patients who had been typed HPV-negative during follow-up showed positive responses (P = 0.0005). Further analysis showed positive responses to be more frequent in patients with persistent HPV-16 infections and stable or progressing cervical lesions (8/9 patients reactive, 88.9%) as compared to patients with cleared or fluctuating HPV-16 infections and stable or regressing cervical lesions (7/17, 41.1%, P = 0.04). The relatively strong T cell proliferative responses against HPV-16 E7 observed in patients with a persistent HPV-16 infection and progressive cervical lesions indicate that the effectivity of such responses cannot be predicted and apparently depends on additional factors.

Immune responses against human papillomavirus (HPV) type 16 virus-like particles in a cohort study of women with cervical intraepithelial neoplasia. I. Differential T-helper and IgG responses in relation to HPV infection and disease outcome.

T-helper (Th) cell-dependent IL-2 production and plasma IgG responses to virus-like particles consisting of the human papillomavirus type 16 (HPV-16) major capsid protein L1 (L1-VLP) were determined in patients with cytological evidence of cervical intraepithelial neoplasia (CIN) participating in a non-intervention prospective cohort study. IgG responses were associated with HPV-16 persistence and high-grade CIN lesions, while high frequencies of Th responses were observed in patients with both virus clearance and virus persistence, irrespective of CIN grade. The IgG response was found in conjunction with an IL-2 response to L1-VLP in 87% of the patients. Recognition of the HPV-16 L1 Th epitope (amino acids 311-335) was found to be more closely associated than recognition of L1-VLP as a whole to HPV exposure and CIN development. Among the HPV-16+ patients included in this study, those showing a Th response to amino acids 311-335 were more likely to carry the HLA DRB1*11/DQB1*0301 haplotype, while those showing an IgG response to L1-VLP were more likely to carry DRB1*0101/DQB1*0501. However, neither cell-mediated nor humoral immune responses against HPV-16 L1 appear to be sufficient for the natural control of HPV infection and CIN development.

Transition metal sensing by Toll‐like receptor‐4: next to nickel, cobalt and palladium are potent human dendritic cell stimulators

Background Nickel was recently identified as a potent activator of dendritic cells through ligating with human Toll‐like receptor (TLR)‐4.

Role of immune responses in the pathogenesis of low-risk MDS and high-risk MDS: implications for immunotherapy

The myelodysplastic syndromes (MDS) constitute a group of heterogeneous clonal haemopoietic stem cell disorders, characterized by ineffective and dysplastic haematopoiesis with varying degrees of peripheral cytopenia. Low‐risk MDS is characterized by increased apoptosis in the bone marrow (BM) with autoimmune characteristics whereas the advanced or high‐risk stages involve immune evasion and secondary DNA damage, giving cells growth potential to progress into acute myeloid leukaemia (AML). Nevertheless, the causes of MDS remain poorly defined and it is not clear how the disease progresses from an early stage to advanced MDS and AML. Although there are clear indications for a role of the immune system, the exact mechanism by which the immune response contributes to the progression is not yet clear. New insights into the pathophysiology of MDS with regard to the immune system will be instrumental for the development of novel patient‐oriented therapies. This review is focused on the role of immune responses in MDS and the implications for the development of novel immune therapies.

Expansion of dendritic cell precursors from human CD34+ progenitor cells isolated from healthy donor blood; growth factor combination determines proliferation rate and functional outcome

CD34+ haematopoietic progenitor cells, which circulate at extremely low frequencies in peripheral blood, are used to generate dendritic cells (DC) in vitro. Here, we describe a method to grow large numbers of DC precursors from these low frequent cells. Different combinations of early acting haematopoietic growth factors supported expansion of CD34+ cells. CD1a+ DC derived from precursors, expanded in fms‐like tyrosine kinase‐3 ligand (Flt3‐L), stem‐cell factor (SCF), interleukin (IL)‐3, and IL‐6, were less potent antigen‐presenting cells (APC) compared to CD1a+ DC derived from precursors expanded in Flt3‐L, trombopoietine (TPO), and SCF. Furthermore, the latter produced high levels of IL‐12 and low levels of IL‐10, a cytokine profile favorable for the priming cytotoxic T cells. In contrast, a mean increase of total cell number of 453‐fold was obtained with Flt3‐L, SCF, IL‐3, and IL‐6, and this increase was only 38‐fold with Flt3‐L, TPO, and SCF. Sequential cultures of both cocktails resulted in high numbers of potent APC, which can be useful DC‐based cancer vaccines.