Max Petersson

Tumor-induced immune dysfunction.

Abstract Immune system-based approaches for the treatment of malignant disease over the past decades have often focused on cytolytic effector cells such as cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL and NK cell activity. However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from CTL- or NK cell-based antitumor approaches. The underlying causes of tumor-associated immune suppression of CTL and NK cell activity are discussed, and features of interest shared with HIV infection, leprosy, and rheumatoid arthritis are also be mentioned. Remarkable and very recent observations have shed more light upon the causes of dysfunctional alterations in CTL and NK cells often associated with these diseases, that in turn have suggested new immunotherapeutic approaches for cancer and infectious disease.

Inhibition of Activated/Memory (CD45RO+) T Cells by Oxidative Stress Associated with Block of NF-κB Activation

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-γ release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO+) T cells and correlated with a block in NF-κB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-γ, TNF-α, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO+, T cells found in the tumor milieu are functionally suppressed.

T Cell Tolerance Based on Avidity Thresholds Rather Than Complete Deletion Allows Maintenance of Maximal Repertoire Diversity

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1α (EF1α). EF1α occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1α peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1α peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1α isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.

Immunosuppression in human tumor-host interaction: role of cytokines and alterations in signal-transducing molecules

Cancer is a disease in which flank systemic immunosuppression, which typically occurs only in the advavced stages, may cause considerable problems, manifested by recurrent infections and possibly also progression of malignant disease. The possibility of finding subtle alterations in the immune system of cancer patients before these are clinically manifest is increasing as more refined and quantitative methods of measuring immune functions are developed. These methods are shedding light upon mechanisms underlying escape from immune surveillance of tumors. The mystery behind the frequent observation of tumor growth in the face of a seemingly normal systemic immune response is slowly being elucidated. Recently there has been a shift in the ;ocus of research interest from systemic to local events during tumor establishment and subsequent growth. Tumor-infiltrating lymphocytes (TIL) are consistently devoid of cellular effector functions unless cultured in vitro in the presence of non-physiological concentrations of cytokines such as interleukin-2 (IL-2) [64]. The low activity of freshly isolated TIL has been ascribed to suppressor lymphocytes or macrophages [22, 23, 42] or to the secretion of suppressor factors by tumor cells [16, 65, 77]. We will not review in detail immunodepression in malignancy [9, 85], but will rather focus on recent research into the mechanisms responsible for decreased immune responses in cancer patients. We submit that suppressed anti-tumor responses can arise from the normal regulation of T and natural killer (NK) cells as mediated by cells, cytokines and other regulatory molecules. In the first part of this review, we will discuss alterations in cytokine production by patient lymphocytes and cytokine expression within tumors, but are well aware of the plethora of other immunosuppressire factors which have been described in human cancer [11, 86]. The second major focus of this review will De on recently described alterations in signal transducing molecules in patient T and NK cells.

Mechanisms of escape from CD8+ T‐cell clones specific for the HER‐2/NEU proto‐oncogene expressed in ovarian carcinomas: Related and unrelated to decreased MHC class 1 expression

We have developed an in vitro model to study mechanisms by which ovarian tumor cells that over‐express the HER‐2/neu proto‐oncogene escape recognition by TCD8+. Nine tumor‐specific, HLA A2‐restricted TCD8+ clones were isolated from 2 ovarian tumor‐specific TCD8+ lines derived from tumor‐infiltrating or ‐associated lymphocytes. Of these, 2 clones recognized the previously defined HER‐2/neu epitope E75 (a.a. 369–377) and one recognized the C85 epitope (a.a. 971–979), whereas the specificity of the remaining 6 clones was unknown. Three different tumor escape variants (EVC8, EVC22 and EVC36) were produced by co‐culturing an ovarian tumor line over‐expressing HER‐2/neu with these autologous TCD8+ clones. Cell surface expression of HLA A2 was markedly decreased on all 3 escape variants, relative to the parental tumor line, while no significant decrease in their expression of the HER‐2/neu, ICAM‐1 or LFA‐3 molecules was found. There was a correlation between the level of tumor‐specific recognition and HLA A2 expression among the tumor clones isolated from 2 of the escape variants (EVC8 and EVC36). In contrast, high HLA A2‐expressing tumor clones isolated from the EVC22 variant, or EVC22 which had regained high HLA A2 expression through IFN‐γ treatment, were not recognized by the HER‐2/neu‐specific TCD8+ clone C‐22. No mutations were found in the cDNA or the genomic DNA derived from the PCR product corresponding to a 496 bp fragment including the region coding for the E75 epitope of the HER2/neu gene in the EVC22 variant. Collectively, this in vitro model underlines the importance of decreased expression of the HLA restriction element for escape from tumor‐specific TCD8+ but also demonstrates that additional mechanisms exist.

Immunization with heat shock protein 70 from methylcholanthrene-induced sarcomas induces tumor protection correlating with in vitro T cell responses

Abstract. The cytosolic members of the heat shock protein 70 (hsp70) family have in recent years been shown to elicit protective immunity mediated via binding to antigenic peptides in tumor and viral animal models. In this study we have used the methylcholanthrene (MC)-induced sarcomas MC57S and MC57X, previously shown to express individually distinct MHC-I associated peptides recognized by tumor necrosis factor-alpha (TNF-α) producing CD8+ T cells. With hsp70 purified from tumor or liver tissue, we were able to confirm that tumor-derived hsp70 elicited in vivo protection against a challenge with the same tumor as that used for hsp70 isolation. We also observed that immunizing with hsp70 isolated from tumor tissue resulted in a significantly better protection than immunizing with hsp70 isolated from the liver tissue of healthy mice. In two out of three experiments however, immunization with liver-derived hsp70 as well as tumor-derived hsp70 resulted in a significantly delayed tumor outgrowth as compared to saline-injected controls. In vitro, T cell lines from mice immunized with tumor-derived hsp70 could recognize tumor cells from the same MC57 tumor as that used for the hsp70 purification, resulting in TNF-α production. In sera from hsp70-immunized mice we observed undetectable or only very low levels of anti-hsp70 antibodies, suggesting that it is possible to repeatedly immunize the mice with no significant interference from neutralizing antibodies. We therefore conclude that hsp70 immunization can lead to protection against the tumor it was purified from, with a low risk of eliciting neutralizing antibodies that could affect further immunizations.

An active T-cell-independent mechanism enhances MHC class I transcription and expression on a mouse T-cell lymphoma in vivo

The present study focuses on the mechanism underlying changes in H-2 cell surface antigen expression after passage of the in vitro grown YAC-1 lymphoma as an ascites tumor. The increase in cell-surface expression correlated with elevated levels of class I transcripts as revealed by Northern blots. The enhanced H-2 expression was also seen with a cloned YAC-1 line, and not until 2 weeks after in vitro explantation had levels of H-2 decreased to those on the in vitro established YAC-1. Arguing for the necessity of a mature functioning immune system, suckling mice were unable to increase H-2 expression on inoculated lymphoma cells. Also pretreatment with cyclophosphamide or irradiation abolished the capacity of adult mice to increase cell surface H-2 on YAC-1 cells. A functioning T-cell system was not required for H-2 enhancement to occur since athymic nude mice were fully competent. The possible significance of an active T-cell-independent host mechanism which enhances tumor H-2 expression at the transcriptional level is discussed.

ENGAGEMENT OF MHC CLASS I PROTEINS ON NATURAL KILLER CELLS INHIBITS THEIR KILLING CAPACITY

We have studied whether engagement of MHC class I (MHC—I) molecules on natural killer (NK) cells can influence the NK killing activity. Human NK effector cells, enriched by nylon wool passage, were incubated with monoclonal antibodies (MoAb) to MHC—I followed by cross‐linking with secondary rabbit anti mouse Ig or streptavidin. Cross linking of MHC—I molecules on NK cells resulted in a clear inhibition of the NK activity against the target cells K562, Molt‐4 and U937. The inhibitory effect was selective for MHC—I and was not seen with MoAb to MHC—II or CD56 molecules. The inhibition was not mediated via Fc receptors since F(ab)2 fragments of the MHC—I MoAb W6/32 were as effective as the intact antibody. The best inhibition of NK activity was obtained using biotin‐labelled F(ab)2 fragments of W6/32 and streptavidin as a cross‐linker, where up to 70 % reduction in NK cell activity was observed. Antibody dependent cellular cytotoxicity (ADCC) was also inhibited by cross‐linking MHC—I molecules on the effector cells.