Rolf Kiessling

Regulatory T Cells in Cancer

At the present time, regulatory T cells (Tregs) are an integral part of immunology but the route from discovery of suppressive lymphocytes in the 1980s to the current established concept of Tregs almost 20 years later has been a rollercoaster ride. Tregs are essential for maintaining self-tolerance as defects in their compartment lead to severe autoimmune diseases. This vitally important function exists alongside the detrimental effects on tumor immunosurveillance and antitumor immunity. Beginning with the identification of CD4(+)CD25(+) Tregs in 1995, the list of Treg subsets, suppressive mechanisms, and knowledge about their various origins is steadily growing. Increase in Tregs within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Several mechanisms, ranging from proliferation to specific trafficking networks, have been identified to account for their systemic and/or local accumulation. Since various immunotherapeutic approaches are being utilized for cancer therapy, various strategies to overcome the antagonistic effects exerted by Tregs are being currently explored. An overview on the biology of Tregs present in cancer patients, their clinical impact, and methods for modulating them is given in this review. Despite the extensive studies on Tregs in cancer many questions still remain unanswered. Even the paradigm that Tregs generally are disadvantageous for the control of malignancies is now under scrutiny. Insight into the specific role of Tregs in different types of neoplasias is the key for targeting them in a way that is beneficial for the clinical outcome.

Camouflage and sabotage: tumor escape from the immune system

The field of tumor immunology has made great progress in understanding tumor immune interactions. As a consequence a number of immuno-therapeutic approaches have been successfully introduced into the clinic and a large number of promising therapeutic strategies are investigated in ongoing clinical trials. Evaluation of anti-tumor immunity in such trials as well as in animal models has shown that tumor escape from immune recognition and tumor-mediated suppression of anti-tumor immunity can pose a significant obstacle to successful cancer therapy. Here, we review mechanisms of tumor immune escape and immune-subversion with a focus on the research interests in our laboratory: loss of MHC class I on tumor cells, increased oxidative stress, recruitment of myeloid-derived suppressor cells, and regulatory T cells.

Prognostic significance of tumor iNOS and COX-2 in stage III malignant cutaneous melanoma

PurposeNew prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stagexa0III malignant cutaneous melanoma.Experimental designThe expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease ≤14xa0months) and 17 slowly progressing (OS ≥60xa0months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. χ2 exact trend test and logistic regression analysis were used for statistical analysis.ResultsBoth iNOS (Pxa0=xa00.002) and COX-2 (Pxa0=xa00.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (Pxa0=xa00.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (ORxa0=xa010.4) was higher than that of COX-2 (ORxa0=xa05.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness.ConclusionOur data show that iNOS is an independent and stronger prognostic factor for OS in stagexa0III malignant cutaneous melanoma than COX-2.

IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells

Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.

HLA-dependent tumour development: a role for tumour associate macrophages?

HLA abnormalities on tumour cells for immune escape have been widely described. In addition, cellular components of the tumour microenvironment, in particular myeloid derived suppressor cells (MDSC) and alternatively activated M2 tumour-associated macrophages (TAMs), are involved in tumour promotion, progression, angiogenesis and suppression of anti-tumour immunity. However, the role of HLA in these activities is poorly understood. This review details MHC class I characteristics and describes MHC class I receptors functions. This analysis established the basis for a reflection about the crosstalk among the tumour cells, the TAMs and the cells mediating an immune response.The tumour cells and TAMs exploit MHC class I molecules to modulate the surrounding immune cells. HLA A, B, C and G molecules down-regulate the macrophage myeloid activation through the interaction with the inhibitory LILRB receptors. HLA A, B, C are able to engage inhibitory KIR receptors negatively regulating the Natural Killer and cytotoxic T lymphocytes function while HLA-G induces the secretion of pro-angiogenic cytokines and chemokine thanks to an activator KIR receptor expressed by a minority of peripheral NK cells. The open conformer of classical MHC-I is able to interact with LILRA receptors described as being associated to the Th2-type cytokine response, triggering a condition for the M2 like TAM polarization. In addition, HLA-E antigens on the surface of the TAMs bind the inhibitory receptor CD94/NKG2A expressed by a subset of NK cells and activated cytotoxic T lymphocytes protecting from the cytolysis.Furthermore MHC class II expression by antigen presenting cells is finely regulated by factors provided with immunological capacities. Tumour-associated macrophages show an epigenetically controlled down-regulation of the MHC class II expression induced by the decoy receptor DcR3, a member of the TNFR, which further enhances the M2-like polarization. BAT3, a positive regulator of MHC class II expression in normal macrophages, seems to be secreted by TAMs, consequently lacking its intracellular function, it looks like acting as an immunosuppressive factor.In conclusion HLA could cover a considerable role in tumour-development orchestrated by tumour-associated macrophages.

Correlation of HLA-A02* genotype and HLA class I antigen down-regulation with the prognosis of epithelial ovarian cancer

BackgroundIn recent years, evidence is accumulating that cancer cells develop strategies to escape immune recognition. HLA class I HC down-regulation is one of the most investigated. In addition, different HLA haplotypes are known to correlate to both risk of acquiring diseases and also prognosis in survival of disease or cancer. We have previously shown that patients with serous adenocarcinoma of the ovary in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A02* genotype. We aimed to study the relationship between HLA-A02* genotype in these patients and the subsequent HLA class I HC protein product defects in the tumour tissue.Materials and methodsOne hundred and sixty-two paraffin-embedded tumour lesions obtained from Swedish women with epithelial ovarian cancer were stained with HLA class I heavy chain (HC) and β2-microglobulin (β2-m)-specific monoclonal antibodies (mAb). Healthy ovary and tonsil tissue served as a control. The HLA genotype of these patients was determined by PCR/sequence-specific primer method. The probability of survival was calculated using the Kaplan–Meier method, and the hazard ratio (HR) was estimated using proportional hazard regression.ResultsImmunohistochemical staining of ovarian cancer lesions with mAb showed a significantly higher frequency of HLA class I HC and β2-m down-regulation in patients with worse prognosis (WP) than in those with better prognosis. In univariate analysis, both HLA class I HC down-regulation in ovarian cancer lesions and WP were associated with poor survival. In multivariate Cox-analysis, the WP group (all with an HLA-A02* genotype) had a significant higher HR to HLA class I HC down-regulation.ConclusionsHLA-A02* is a valuable prognostic biomarker in epithelial ovarian cancer. HLA class I HC loss and/or down-regulation was significantly more frequent in tumour tissues from HLA-A02* positive patients with serous adenocarcinoma surgical stage III–IV. In multivariate analysis, we show that the prognostic impact is reasonably correlated to the HLA genetic rather than to the expression of its protein products.

The two sides of HER2/neu: immune escape versus surveillance.

The oncogene HER2 is one of the prototypes for targeted immunotherapy of cancer using both monoclonal antibodies as well as T cell based immunotherapies. Effective humoral and cellular immune responses against HER2 can be induced, but these responses can be influenced by the effects of this oncogene on the target tumor cells. The processes involved in HER2-mediated adaptive and innate immunity and the molecular mechanisms underlying the escape of HER2-expressing tumor cells from immune surveillance, particularly from cytotoxic T cells, are discussed. Implementing this knowledge in clinical trials to revert immune evasion may help optimize immunotherapies directed against HER2-expressing tumors.

Stereotactic Ablative Radio Therapy (SABR) followed by immunotherapy a challenge for individualized treatment of metastatic solid tumours

Combination strategies surely play a crucial role in treatment of cancer. Stereotactic ablative radiotherapy (SABR) has been described to induce abscopal effects particularly in renal cell cancer metastases. This effect is a reaction induced following irradiation of tumour tissue and occurring in another metastatic location outside the treatment field. However, this effect is limited and occurs sparsely in about 1-5% of patient. We are planning to improve the clinical outcome of this treatment in metastatic solid tumours by combining SABR with sequential immunotherapeutic treatments including vaccination strategies, adoptive cell therapy, cytokine therapy, or anti-CTLA-4 therapy.

Non-classical HLA-class I expression in serous ovarian carcinoma: Correlation with the HLA-genotype, tumor infiltrating immune cells and prognosis.

In our previous studies, we have shown that patients with serous ovarian carcinoma in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A*02 genotype. This represent a stronger prognostic factor than loss or downregulation of the MHC class I heavy chain (HC) on tumor cells. In this study, we investigated the expression of the non-classical, immune tolerogenic HLA -G and -E on the tumor cells along with the infiltration of immune cells in the tumor microenvironment. FFPE primary tumors from 72 patients with advanced stages of serous adenocarcinoma and metastatic cells present in ascites fluid from 8 additional patients were included in this study. Both expression of HLA-G and aberrant expression of HLA-E were correlated to a significant worse prognosis in patients with HLA-A*02, but not with different HLA genotypes. Focal cell expression of HLA-G correlated to a site-specific downregulation of classical MHC class I HC products and aberrant HLA-E expression, showing a poor survival. HLA-G was more frequently expressed in metastatic cells than in primary tumor lesions and the expression of HLA-G inversely correlated with the frequency of tumor infiltrating immune cells. All these parameters can contribute together to identify and discriminate subpopulations of patients with extremely poor prognosis and can give them the opportunity to receive, and benefit of individually tailored treatments.

Differential tumor infiltration by T-cells characterizes intrinsic molecular subtypes in breast cancer.

BackgroundMolecular subtypes of breast cancer and presence of tumor-infiltrating immune cells have both been implicated as important predictive and prognostic factors for improved risk stratification and treatment individualization of breast cancer patients. Their association, however, has not been studied in detail. The aim of this study was to evaluate the expression of the T cell markers CD8, FoxP3, CD3 and ζ-chain in molecular subtypes of the invasive margin and tumor center of breast cancer and corresponding sentinel nodes and to deduct prognostic information from these findings.MethodsTumor and sentinel node sections from 177 patients with primary, invasive, unilateral early-stage breast cancer were stained by immunohistochemistry and T-cell phenotypes quantified manually. Clinical data were collected from medical records.ResultsThe degree of T-cell infiltration and expression of all markers differed significantly among the molecular subtypes, being highest in non-luminal, more aggressive tumors: more T-cell infiltration and higher expression of all markers were associated with hormone receptor negativity, higher proliferation and higher histological grades, but also with larger tumor size. Basal-like tumors, and most remarkably their tumor centers, hosted the highest number of FoxP3+xa0T-cells with an unfavorable ratio to cytotoxic CD8+xa0T-cells. T-cell infiltration was generally higher in the invasive margin than the tumor center. A scoring system based on densities of CD3 and CD8 could significantly separate molecular subtypes (pxa0<xa00.001).ConclusionsThus, immunological patterns with functional implications within each subtype are associated with prognostic factors. These findings should be further validated in studies using larger patient populations and longer follow-up.