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

BACKGROUND: Molecular 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. METHODS: Tumor 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. RESULTS: The 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+ T-cells with an unfavorable ratio to cytotoxic CD8+ T-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 (p < 0.001). CONCLUSIONS: Thus, 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.

Cripto-1 vaccination elicits protective immunity against metastatic melanoma.

Metastatic melanoma is a fatal disease that responds poorly to classical treatments but can be targeted by T cell-based immunotherapy. Cancer vaccines have the potential to generate long-lasting cytotoxic CD8(+) T cell responses able to eradicate established and disseminated tumors. Vaccination against antigens expressed by tumor cells with enhanced metastatic potential represents a highly attractive strategy to efficiently target deadly metastatic disease. Cripto-1 is frequently over-expressed in human carcinomas and melanomas, but is expressed only at low levels on normal differentiated tissues. Cripto-1 is particularly upregulated in cancer-initiating cells and is involved in cellular processes such as cell migration, invasion and epithelial-mesenchymal transition, which are hallmarks of aggressive cancer cells able to initiate metastatic disease. Here, we explored the potential of Cripto-1 vaccination to target metastatic melanoma in a preclinical model. Cripto-1 was overexpressed in highly metastatic B16F10 cells as compared to poorly metastatic B16F1 cells. Moreover, B16F10 cells grown in sphere conditions to enrich for cancer stem cells (CSC) progressively upregulated cripto1 expression. Vaccination of C57Bl/6 mice with a DNA vaccine encoding mouse Cripto-1 elicited a readily detectable/strong cytotoxic CD8(+) T cell response specific for a H-2 Kb-restricted epitope identified based on its ability to bind H-2(b) molecules. Remarkably, Cripto-1 vaccination elicited a protective response against lung metastasis and subcutaneous challenges with highly metastatic B16F10 melanoma cells. Our data indicate that vaccination against Cripto-1 represents a novel strategy to be tested in the clinic.

Tumour-induced immune suppression: role of inflammatory mediators released by myelomonocytic cells.

Tumour-induced immune dysfunction is a serious challenge to immunotherapy for cancer, and intact adaptive and innate cellular immunity is key to its success. Myelomonocytic cells have a central role in this immune suppression, and tumour-associated macrophages, eosinophils, neutrophils and myeloid-derived suppressor cells have all been shown to be of major importance. These myelomonocytic cells secrete a broad repertoire of inflammatory mediators providing them with powerful tools to inhibit tumour-reactive T cells and natural killer cells; free oxygen radicals including reactive oxygen species and NO, arginase, indoleamine 2,3-dioxygenase, prostaglandins, the pro-inflammatory heterodimer S100A8/9 and cytokines, such as granulocyte-macrophage colony-stimulating factor and transforming growth factor-ß, have proven particularly potent in suppressing antitumour cellular immunity. Determining which of these factors prevail in individual cancer patients and designing methods aimed at neutralization or inhibition of their effects on target tissues have the potential to greatly enhance the clinical efficacy of immunotherapy.

Immunogenic tumor cell death induced by chemoradiotherapy: molecular mechanisms and a clinical translation.

Chemoradiotherapy can induce immunogenic cell death, triggering danger signals such as high-mobility group box 1 protein, and resulting in T-cell immunity. This concept can potentially be harnessed for clinical therapy to enhance tumor-specific immunity. There is however limited information to translate this theory directly in a clinical setting. In this review, we will discuss and summarize molecular and cellular mechanisms underlying immunogenic tumor cell death induced by chemoradiotherapy, with emphasis on a clinical translation.

Myeloid-derived suppressor cells impair the quality of dendritic cell vaccines.

Myeloid-derived suppressor cells (MDSC) are important regulators of the immune system and key players in tumor-induced suppression of T-cell responses. CD14+HLA-DR-/low MDSC have been detected in a great number of malignancies, including melanoma. MDSC are known to be impaired in their ability to differentiate along the myeloid lineage, e.g., into dendritic cells (DC). This is a concern for utilization of monocyte-derived DC for vaccination of patients with melanoma or other cancers exhibiting accumulation of CD14+ MDSC. When producing DC according to standard operating procedures of two currently ongoing clinical trials, we found that MDSC co-purified with monocytes isolated by elutriation. MDSC frequencies did not affect yield or viability of the produced DC, but induced a dose-dependent decrease in DC maturation, ability to take up antigen, migrate and induce T-cell IFNγ production. Changes in DC characteristics were most notable when 'pathological' frequencies of >50% CD14+HLA-DR- cells were present in the starting culture. The impaired DC quality could not be explained by altered cytokine production or increased oxidative stress in the cultures. Tracking of HLA-DR- cells throughout the culture period revealed that the observed changes were partially due to the impaired maturation and functionality of the originally HLA-DR- population, but also to their negative effects on HLA-DR+ cells. In conclusion, MDSC could be induced to differentiate into DC but, due to the impairment of overall DC vaccine quality when >50% HLA-DR- cells were present in the starting culture, their removal could be advisable.

Tumor-dependent down-regulation of the ζ-chain in T-cells is detectable in early breast cancer and correlates with immune cell function.

Suppressive factors produced by tumors or tumor-associated cells represent a major obstacle to immune-mediated tumor eradication and immunotherapy. We studied tumor-dependent changes in expression of the ζ-chain, a key molecule for the transduction of stimulatory signals through the T-cell receptor and activating receptors on natural killer (NK) cells, in patients with early (stages 1 and 2) breast cancer. Ex-vivo levels of ζ-chain expression, proliferation and cytokine expression in lymphocyte subpopulations were measured in breast tumors, their draining lymph nodes and in pre- and postoperative blood samples of cancer patients and healthy controls by multi-parametric flow cytometry. We found that T-cell ζ-chain expression in peripheral blood of breast cancer patients (n = 29) was down-regulated compared with healthy controls (n = 10) (p ≤ 0.033), which was most pronounced in stage 2 (n = 15, p ≤ 0.004). T- and NK-cell ζ-chain loss was most distinct in the tumor and decreased with increasing distance (p ≤ 0.015). After surgical tumor resection, peripheral blood ζ-chain levels normalized to those observed in healthy controls. ζ-chain expression in peripheral blood T-cells correlated with lymphocytic proliferative activity (p ≤ 0.035), and with the expression of proinflammatory cytokines in CD8+ T-cells (p ≤ 0.035). Breast cancer patients had lower numbers of circulating early memory CD8+ T-cells than healthy donors (p ≤ 0.000), correlating with lower T-cell ζ-chain levels (p ≤ 0.011). Our findings suggest that phenotypic and functional alterations in lymphocytes can be detected even in early stage breast cancer patients. The observed immunosuppression appears to be systemic and tumor dependent, as it is strongest in the tumor, correlates with tumor stage and normalizes after surgery.

Tumor-induced changes in the phenotype of blood-derived and tumor-associated T cells of early stage breast cancer patients.

Tumor-induced immune suppression has mainly been studied in patients with advanced cancer. Despite the fact that they are most likely to benefit from immunotherapy, patients with early stage cancers were under-represented in these studies. We analyzed blood and tumor-derived T cells from patients with stage 1 (n = 20), stage 2 (n = 23) or stage 3 (n = 1) breast cancer and found that, even early stage tumors induced T cell differentiation. Breast cancer patients had significantly more circulating CD8+ memory and fewer CD8+ naïve T cells than healthy controls (n = 10). Up-regulation of CD69 and PD1 on cancer patient T cells suggests previous activation, and increased expression of the chemokine receptors CCR5 and CXCR3 on CD8+ T cells indicates that their homing capacity differs from that of healthy individuals. Comparison of blood-derived and tumor-associated T cells from patients with different metastatic status and tumor grades revealed that tumor progression and aggressiveness seem to favor the expansion of memory T cells over naive T cells. We have previously shown that immunosuppression in this patient population is stronger in the tumor than in the blood. Here, we report signs of exhaustion, such as loss of CD28, on tumor-associated as compared to blood-derived CD8+ T cells, despite the fact that tumor-associated T cells are predominantly effector memory cells and express high levels of CD69. The finding that the presence of a tumor potentially induces immunosenescence early during tumorigenesis indicates that efficient immunotherapy might be difficult even in patients with early stage cancer due to T cell exhaustion and tolerance.

Improving the efficacy of cancer immunotherapy.

A series of cancer vaccines have been evaluated in clinical trials with encouraging results, but the demonstration of clinical benefit in confirmatory studies has so far proven to be difficult. The development of cancer vaccines is hampered by a range of issues particular to this field of research. On 12th March 2008, the Biotherapy Development Association convened a workshop to discuss issues faced by scientists and clinicians involved in the development of cancer vaccines. This paper is a review of the field, based on discussions held at the BDA workshop, and describes biological barriers encountered in generating effective immune responses to tumours, methodological obstacles encountered in the improvement of immunological monitoring which aims to improve inter-laboratory and inter-trial comparisons, challenges in clinical trial design and problems posed by the lack of specific regulation for cancer vaccines and the impact on their development. Ultimately, a number of general solutions are posed: (1) better patient selection, (2) use of multi-modal treatments that affect several aspects of the immune system at once, (3) a requirement for the development of good biomarkers to stratify patients for selection prior to trial and as surrogates for clinical response and (4) harmonisation of SOPs for immunological monitoring of clinical trials.

Analysis of HLA class I-II haplotype frequency and segregation in a cohort of patients with advanced stage ovarian cancer.

In solid tumors, human leucocyte antigen (HLA)-A2 has been suggested to be a risk factor and a negative prognostic factor. The HLA-A2 allele in Scandinavia has a high prevalence; it decreases with latitude and also with ovarian cancer mortality in Europe. Furthermore, an association of the HLA-A2 allele with severe prognosis in serous adenocarcinoma of the ovary in stages III-IV was found. Thirty-two unrelated Swedish women with relapsing or progressive ovarian cancer were analysed for the genotypes at the HLA-A, HLA-B, HLA-Cw, and HLA-DRB1 loci by the polymerase chain reaction/sequence-specific primer method. The frequencies of HLA alleles of healthy Swedish bone marrow donors provided by the coordinating centre of the Bone Marrow Donors Worldwide Registries, Leiden, the Netherlands were used as controls. When this cohort of epithelial ovarian cancer patients was compared with healthy Swedish donors, the frequency of HLA-A1 and HLA-A2 gene/phenotype appears, although not statistically significant, to be increased in patients with ovarian carcinoma, while HLA-A3 was decreased. HLA-A2 homozygotes were twofold higher in patients. The A2-B8 haplotype was significantly increased (corrected P value). A2-B5, A2-B15, A2-DRB1*03, A2-DRB1*04, A2-B15-Cw3, and A2-B8-DRB1*03 had odds ratio as well as the level of the lower confidence interval above 1 and significant P value only when considered as single, non-corrected analysis. HLA-B15 and HLA-Cw3 were only present in HLA-A2-positive patients showing that the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes were segregated. In this selected cohort with advanced disease, there are indications of an unusual overrepresentation of HLA class I and II genes/haplotypes as well as segregation for the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes. These findings are presented as a descriptive analysis and need further investigations on a larger series of ovarian cancer patients to establish prognostic associations.

A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer.

Prostate-specific antigen (PSA) is a serine protease secreted at low levels by normal luminal epithelial cells of the prostate and in significantly higher levels by prostate cancer cells. Therefore, PSA is a potential target for various immunotherapeutical approaches against prostate cancer. DNA vaccination has been investigated as immunotherapy for infectious diseases in patients and for specific treatment of cancer in certain animal models. In animal studies, we have demonstrated that vaccination with plasmid vector pVAX/PSA results in PSA-specific cellular response and protection against tumour challenge. The purpose of the trial was to evaluate the safety, feasibility and biological efficacy of pVAX/PSA vaccine in the clinic. A phase I trial of pVAX/PSA, together with cytokine granulocyte/macrophage-colony stimulating factor (GM-CSF) (Molgramostim) and IL-2 (Aldesleukin) as vaccine adjuvants, was carried out in patients with hormone-refractory prostate cancer. To evaluate the biologically active dose, the vaccine was administered during five cycles in doses of 100, 300 and 900 microg, with three patients in each cohort. Eight patients were evaluable. A PSA-specific cellular immune response, measured by IFN-gamma production against recombinant PSA protein, and a rise in anti-PSA IgG were detected in two of three patients after vaccination in the highest dose cohort. A decrease in the slope of PSA was observed in the two patients exhibiting IFN-gamma production to PSA. No adverse effects (WHO grade >2) were observed in any dose cohort. We demonstrate that DNA vaccination with a PSA-coding plasmid vector, given with GM-CSF and IL-2 to patients with prostate cancer, is safe and in doses of 900 microg the vaccine can induce cellular and humoral immune responses against PSA protein.

Her-2/neu as a paradigm of a tumor-specific target for therapy.

Her-2/neu (HER2) is a protooncogene that is known to be amplified in a proportion of many different types of carcinomas. Overexpression of HER2 is generally associated with poor prognosis and resistance to chemo and radiotherapy. There exist many approaches that may be potentially utilized for therapy of HER2 overexpressing tumors. Understanding the biochemical and physiological function of HER2 in oncogenesis as well as its role in facilitating immune escape of tumor cells is critical for the improvement of existing strategies as well as developing new therapeutic candidates targeting HER2.

Development of a technology platform for large-scale clinical grade production of DC.

BACKGROUND: Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DC are produced according to good manufacturing practice (GMP) guidelines. METHODS: In the present study, using leukapheresis products from 10 cancer patients, we validated an elutriation technology for large-scale clinical grade production of monocyte-derived DC. RESULTS: The elutriation method gave a very high purity (mean+/-SD) (86+/-5.3%) and recovery (66+/-10.4%) of monocytes. Specifically for the two monocyte-rich fractions (3 and 4,) the recovery was 42+/-13% of viable cells that could be further differentiated into immature DC in hydrophobic culture bags using GM-CSF and IL-4. The immature DC exhibited<1% CD83+ expression and >98% phagocytic activity. Maturation with TNF-alpha or poly I:C resulted in DC with expression of CD80+, CD86+ and HLA-DR+ (>99%) and CD83+ (80+/-11.9%), as well as producing IL-12p70 and lacking phagocytic activity (<5%). This cell product can be cryopreserved with cell viability >85% and cell recovery >80% after thawing. DISCUSSION: The elutriation procedure, when optimized and if the monocyte content of the starting material exceeds 5%, does not require further selection or depletion using affinity approaches.

Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker.

The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT-PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a 'therapeutic window' to be exploited in cancer immunotherapy.

Hyaluronic acid and chondroitin sulphate A rapidly promote differentiation of immature DC with upregulation of costimulatory and antigen-presenting molecules, and enhancement of NF-kappaB and protein kinase activity.

Dendritic cells (DCs) have been identified as effective antigen-presenting cells (APCs). We demonstrate that extracellular matrix (ECM), hyaluronic acid (HA) and chondroitin sulphate A (CSA), in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), can rapidly promote the differentiation of monocyte-derived immature DCs, as characterized by the remarkable upregulation of human leucocyte antigen (HLA-DR), CD40, CD54, CD80 and CD86 expression to levels higher than those in the DCs generated by culturing with GM-CSF and interleukin (IL)-4 for 7 days and aggregation of the cells within 48 h. The upregulation of expression of HLA-DR, CD40, CD54, CD80 and CD86 was dose-dependent. Further studies showed that HA and CSA were able to augment nuclear factor (NF)-kappaB activity, as determined by gel mobility shift assay and promote protein phosphorylation. Inhibition of NF-kappaB by pyrolidine dithiocarbamate and sodium salicylate, and serine-threonine and tyrosine kinase by starosporine as well as phosphatidylinositide-3-kinase (PI-3-K) by wortmannin could prevent the effects of HA and CSA on the expression of HLA-DR, CD40, CD80 and CD86 in various degrees. Thus, our data demonstrate that HA or CSA can effectively and rapidly promote the differentiation of immature DC, suggesting that HA and CSA may possess a potential capacity in regulating immune responses.

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.

DNA immunization of HLA transgenic mice with a plasmid expressing mycobacterial heat shock protein 65 results in HLA class I- and II-restricted T cell responses that can be augmented by cytokines.

Infection with Mycobacterium tuberculosis (MTB) remains a major cause of morbidity and mortality world-wide. An effective vaccination strategy is the immunization with plasmid DNA (pDNA), expressing an antigen (Ag) from a pathogen in vivo, which results in specific immune response against the encoded protein as well as the pathogen itself or cells infected with it. To test the ability to induce HLA-restricted T cell immune response against a mycobacterial antigen in humans by pDNA vaccination, we have used transgenic mice that express HLA class I (A*0201/Kb) or HLA class II (DRB1*0301) molecules. pDNA immunization with mycobacterial heat shock protein 65 (Mhsp65)-expressing plasmid (P3M.65) resulted in HLA-II-restricted, Ag-specific T cell-mediated immune responses characterized by proliferation and cytokine production. These T cell responses could be further augmented by the coinjection of P3M.65 and plasmid expressing murine GM-CSF. Furthermore, coimmunizing HLA-I transgenic mice with P3M.65 and a plasmid expressing murine IFN-gamma induced a specific cytotoxic T lymphocyte response restricted by HLA-A2. These results represent the first evidence of a concomitant in vivo induction of HLA class I- as well as class II-restricted T cell responses by pDNA immunization, which is induced or augmented by the codelivery of cytokine-expressing plasmids, supporting its potential use in clinical trials.

Association of HLA class I and class II antigen expression and mortality in uveal melanoma.

PURPOSE: Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. These abnormalities may play a role in the clinical course of the disease, because HLA antigens mediate interactions of tumor cells with T cells and NK cells. Uveal melanoma is a highly malignant tumor of the eye and is characterized by a hematogenic spread to the liver. Little is known about the role of HLA expression in progression of this malignant disease. METHODS: In the present study HLA class I antigen, beta(2)-microglobulin (beta(2)-m), and HLA class II antigen expression was analyzed in primary uveal melanoma lesions by immunoperoxidase staining with monoclonal antibodies of 65 archival clinical samples. The results were correlated with the clinical course of the disease. RESULTS: HLA class I antigen expression and beta(2)-m expression were downregulated in 40 and 35 lesions, respectively. HLA class II antigens were expressed in 30 lesions. Patients with high HLA class I, including beta(2)-m, and HLA class II antigen expression in their primary melanoma lesions had a significantly decreased survival (P = 0.009, P < 0.001, and P = 0.006, respectively). CONCLUSIONS: The findings argue against a major role of cytotoxic T-lymphocyte (CTL)-mediated control of tumor growth in the clinical course of uveal melanoma and are compatible with a potential role of NK-cell-mediated control of hematogenic metastatic spread.

Tumor necrosis factor-alpha induces coordinated changes in major histocompatibility class I presentation pathway, resulting in increased stability of class I complexes at the cell surface.

It is demonstrated that similar to interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) induces coordinated changes at different steps of the major histocompatibility complex (MHC) class I processing and presentation pathway in nonprofessional antigen-presenting cells (APCs). TNF-alpha up-regulates the expression of 3 catalytic immunoproteasome subunits--LMP2, LMP7, and MECL-1--the immunomodulatory proteasome activator PA28 alpha, the TAP1/TAP2 heterodimer, and the total pool of MHC class I heavy chain. It was also found that in TNF-alpha--treated cells, MHC class I molecules reconstitute more rapidly and have an increased average half-life at the cell surface. Biochemical changes induced by TNF-alpha in the MHC class I pathway were translated into increased sensitivity of TNF-alpha--treated targets to lysis by CD8(+) cytotoxic T cells, demonstrating improved presentation of at least certain endogenously processed MHC class I--restricted peptide epitopes. Significantly, it was demonstrated that the effects of TNF-alpha observed in this experimental system were not mediated through the induction of IFN-gamma. It appears to be likely that TNF-alpha--mediated effects on MHC class I processing and presentation do not involve any intermediate messengers. Collectively, these data demonstrate the existence of yet another biologic activity exerted by TNF-alpha, namely its capacity to act as a coordinated multi-step modulator of the MHC class I pathway of antigen processing and presentation. These results suggest that TNF-alpha may be useful when a concerted up-regulation of the MHC class I presentation machinery is required but cannot be achieved by IFN-gamma. (Blood. 2001;98:1108-1115)