Sharon Louise Coleman

Recovery of CD8+ T-Cell Function During Systemic Chemotherapy in Advanced Ovarian Cancer

Immunologic approaches are emerging as new treatment options in several types of cancer. However, whereas the ability of patients to develop potent CD8+ T-cell responses is crucial for efficient antitumor responses, immunocompetence and T-cell function are not tested routinely in patients entering immunotherapy. The objective of our study was to monitor T-cell function in advanced cancer and during chemotherapy. CD8+ T-cell function of 21 patients with advanced ovarian cancer (stages III-IV) was assessed by cytokine flow cytometry following stimulation of 42 PBMC samples with a panel of synthetic viral peptides in vitro, consisting of pan-Caucasian epitopes. CD8+ T-cell responses were significantly lower in patients with high levels (>200 units/mL) of Ca125 (marker of tumor load and progression) than in those with low Ca125 levels (P = 0.0013). In longitudinal studies of nine patients, chemotherapy was associated with decreasing Ca125 levels in seven cases and also with improvement or maintenance of CD8+ T-cell function in seven cases. After the full course of chemotherapy, five of nine patients in remission displayed potent CD8+ T-cell responses, whereas four of nine patients in progression displayed low or decreasing T-cell responses, pointing toward a correlation between T-cell function and clinical response. Our results show for the first time that CD8+ T-cell function is not permanently suppressed in advanced cancer and successful chemotherapy is associated with improved antigen-specific T-cell reactivity. We suggest that functional assays determining T-cell immunocompetence can be valuable tools for optimizing cancer immunotherapy for improved clinical success.

Promoter polymorphisms in glutathione-S-transferase genes affect transcription.

The glutathione-S-transferases are a group of enzymes that play a major role in detoxification and defence against toxic, carcinogenic and other compounds. We analysed the proximal promoters of 14 genes encoding glutathione-S-transferase for polymorphism. Ten of the promoters contained sequence variants, nine of which we were able to clone into a reporter gene vector, pGL3. The relative ability of each haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671) using a cotransfected CMV-SEAP plasmid as a control. Four genes (GSTA1, GSTA2, GSTM4 and GSTT2) showed activity differences greater than 1.5-fold between haplotypes, and a fifth gene (MGST1) showed a 1.4-fold difference. The promoter sequence variants in these genes may therefore play a role in human variation, susceptibility to diseases and the effects of drugs.

Characterisation, mutation detection, and association analysis of alternative promoters and 5’UTRs of the human dopamine D3 receptor gene in schizophrenia

The dopamine D3 receptor gene (DRD3) is a candidate for a number of psychiatric conditions including schizophrenia, bipolar disorder and alcohol and drug abuse. Previous studies have reported associations between polymorphisms in DRD3 and these disorders, but these findings may have reflected linkage disequilibrium with pathogenic variants that are further upstream. We have isolated and sequenced approximately 9 kb of genomic sequence upstream of the human DRD3 translational start site. Using 5′ RACE, we have identified within this region three additional exons and two putative promoter regions which show promoter activity in three different cell lines. A 5′ UTR identified only in lymphoblasts is spread over three exons and is 353 bp long. A second 5′ UTR, found in adult and fetal brain, lymphocytes, kidney and placenta is spread over two exons and is 516 bp long. A 260-bp sequence within this 9 kb corresponds to a previously reported EST, but corresponding mRNA could not be found in the tissues above. The EST, 5′ UTRs and putative promoter regions have been analysed for polymorphisms, revealing 10 single nucleotide polymorphisms, seven of which were tested for association in a large sample of unrelated patients with schizophrenia and matched controls. No associations were observed with schizophrenia. In addition we failed to replicate previous findings of association with homozygosity of the Ser9Gly variant. The results from this study imply that neither the coding nor the regulatory region of DRD3 plays a major role in predisposition to schizophrenia.

Chronic myeloid leukemia as an immunological target

Various clinical observations have implicated T cells in the control of chronic myeloid leukemia (CML). These observations have in recent years been supported by laboratory results indicating the presence of CML‐specific T cells in the lymphocyte repertoire of both normal healthy individuals and disease‐bearing patients. Both MHC‐unrestricted and MHC‐restricted immune effector mechanisms are involved. Donor lymphocyte infusion has produced encouraging GvL effects. However, future adoptive immunotherapy may depend on the isolation and generation of leukemia‐specific T cells. Although many proteins may potentially act as leukemia antigens in CML for MHC‐restricted cytotoxicity, the bcr‐abl fusion protein has been most extensively investigated. There is now much evidence to suggest that the bcr‐abl junctional peptides are capable of eliciting both CD4 and CD8 responses in normal healthy donors and CML patients. Furthermore, the T‐cell lines generated react with autologous or HLA‐matched fresh CML cells, suggesting that the bcr‐abl fusion protein can be processed in vivo so that the joining segment is bound to HLA molecules in a configuration and concentration similar to those of the immunizing peptide for antigen recognition by the antigen‐specific T‐cell receptor. These results also indicate that the bcr‐abl junctional peptides may be used for immunotherapy of CML. Other strategies available for immunotherapy of CML include immunologically or genetically manipulated donor T‐cell infusion, the use of cytokines, adoptive immunotherapy with leukemia‐reactive T‐cells expanded ex vivo, and immune gene therapy. Novel and rational immunotherapy may therefore play an important adjuvant role in future in the management of patients with CML. Am. J. Hematol. 54:61–67, 1997

The human hyaluronan synthase genes: genomic structures, proximal promoters and polymorphic microsatellite markers.

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5 cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3 end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5-untranslated region (5-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.

Molecular analysis of T cell receptor beta variability in a patient with orofacial granulomatosis.

BACKGROUND: Orofacial granulomatosis (OFG) is a rare chronic inflammatory disorder of unknown causation and is characterised histologically by non-caseating granulomas and aggregates of small lymphocytes. The molecular nature of these T cells is, however, unclear. AIMS: To determine the T cell receptor (TCR) V beta gene usage of the T cell infiltrate associated with the primary lesions in a patient with OFG. METHODS: A molecular method involving reverse transcriptase (RT)-polymerase chain reaction (PCR), DNA cloning, single strand conformation polymorphism (SSCP), length analysis, and nucleotide sequencing was used. RESULTS: Compared with peripheral blood lymphocytes from the same patient, notably restricted TCRV beta gene usage was observed in the T cell infiltrate. Only three of the 24 major TCRV beta gene families were represented in the repertoire. There was preferential usage of the V beta 6 gene. In addition, more than 20% of the V beta 6 TCR transcripts exhibited an identical unique V-D-J junctional sequence, suggesting a local antigen driven V beta 6 T cell clonal expansion in vivo, a phenomenon not observed in normal oral mucosa. CONCLUSIONS: The TCRV beta repertoire of T cells associated with OFG is restricted. It is also associated with a local T cell clonal expansion. The results, therefore, provide a new perspective on the immunopathology of OFG.

A high proportion of chromosome 21 promoter polymorphisms influence transcriptional activity

We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes.

Cytokine enhancement of immunogenicity in chronic myeloid leukaemia

Various clinical and laboratory observations suggest that the leukaemia cells in chronic myeloid leukaemia (CML) are potentially immunogenic. Whilst the ability of the leukaemia cells to elicit an anti-leukaemic immune response in the allogeneic setting is established, it remains unclear why such anti-leukaemic response does not occur in vivo in the autologous setting. We previously demonstrated the presence of leukaemia-reactive T cells in a patient with CML. However, we found that the T cells were normally anergic unless pre-incubated in vitro in high-dose recombinant interleukin-2. We speculated that the T cell anergy was the result of a lack of the appropriate immune costimulatory molecules on the leukaemia cell surface. In this study, we confirm the absence of immune costimulatory molecules, CD80 (B7-1) and CD86 (B7-2), on leukaemia cells and demonstrated that these costimulatory molecules on the leukaemia cells can be upregulated by a combination of GM-CSF and IL-4. There was an associated restoration of leukaemia cell immunogenicity to autologous T cells in mixed lymphocyte leukaemia reactions, suggesting a possible enhancement of anti-leukaemic reaction. More importantly, T cells primed with ‘activated’ leukaemia cells were able to recognise fresh cytokine-naive leukaemia cells. Furthermore, leukaemia cells expressing the dendritic cell marker, CD1a, were also generated. Our findings therefore suggest the opportunity in future to use these combination cytokines in vivo or these leukaemia cells which have been activated in vitro for leukaemia immunotherapy.

Abnormal TCR V/3 repertoire in patients with chronic myeloid leukaemia

Summary. We have used 25 sets of oligonucleotide primers specific for the 24 known major human T‐cell receptor (TCR) V/? families in polymerase chain reactions to analyse the T‐cell repertoire of the peripheral blood in seven patients with chronic myeloid leukaemia (CML). In contrast to normal healthy individuals, all seven patients exhibited variable degrees of TCR V/3‐specific T‐cell deletion, ranging from two to eight of the 24 major families. T cells bearing V/3 17 and 8 were most commonly deleted. These results suggest a superantigen effect associated with CML. The patterns of deletion did not appear to correlate with either of the two bcr‐abl transcripts. The reason and aetiological agent responsible for the T‐cell deletion remain speculative. Further work is ongoing to characterize this phenomenon in animal models and patients with CML.

Resistance of CD45RA− T Cells to Apoptosis and Functional Impairment, and Activation of Tumor-Antigen Specific T Cells during Radiation Therapy of Prostate Cancer

The effect of radiation therapy (RT) to the pelvis on circulating T cells was studied in prostate cancer (PCa) patients to provide a baseline for a more informed design of combination radioimmunotherapy. Peripheral blood samples taken from 12 PCa patients with locally advanced tumor before, during, and after hypofractionated RT were analyzed for T cell phenotype and function. There was significantly more loss of naive and early memory compared with more differentiated T cells during RT. The proportions of annexin-V+ and Fas-expressing T cells were elevated in patients during RT and in PBMC irradiated in vitro (≤5.0 Gy), with preferential increases in CD45RA+ T cells. The baseline level of apoptosis of CD45RA− T cells increased >2-fold in the presence of an IκB-kinase inhibitor, indicating a protective effect via this pathway. T cell proliferation was impaired during RT with IL-2–dependent recovery post-RT. Recall T cell responses to common viral Ags, measured by IFN-γ production, were little affected by RT. In vitro irradiation of healthy donor PBMCs resulted in a significantly increased frequency of responding T cells, due at least partly to the preferential elimination of CD45RA+ T cells. Most importantly, antitumor CD4+ and CD8+ T cell responses were detectable after, but not before or during RT. The results indicate that generating tumor-specific T cell responses before RT and boosting their activity post-RT are ways likely to amplify the frequency and function of antitumor T cells, with implications for scheduling immunotherapy in PCa.