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In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFβ, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.

A large number of non histone chromosomal proteins (NHCPs), many of which remain poorly characterized, are known to be associated with DNA. The tight binding group of NHCPs, which is thought to contain both structural and sequence-specific DNA binding proteins, has proved particularly difficult to study due to low solubility, lack of functional assays and, in many cases, low abundance. An immunological approach has proved popular and a number of studies, in which dehistonized chromatin (extracted with 2 M salt/5 M urea) has been used to immunize experimental animals, have indicated the presence of antigens which are both cell-specific and apparently dependent on DNA for immunological activity. Rabbit antiserum H27, prepared by immunization with Hela dehistonized chromatin (Dc), is such an example. Microcomplement fixation (C'F) analysis indicated the presence of the antigenic fraction in metaphase chromosomal scaffolds and also demonstrated that the antigen(s) could be reconstituted with human DNA, but not rat or calf DNA, to restore immunological activity lost on extensive nuclease digestion (Campbell . et al. , 1979; Dunn et al. , 1980). The apparent sequence-specific requirement of H27 antigens has been analysed further by screening a genomic library with a Hela chromosomal scaffold DNA fraction. In Chapter 4, 2 clones (Cl and C12), which apparently contain sequences suitable for reconstitution, are studied. Southern blotting techniques do not find any homology between the 2 cloned DNAs, which do not appear to be enriched in scaffold DNA sequences, although both clones contain repetitive sequences. It is suggested that either the H27 antigens recognize a small sequence not detectable by standard hybridization techniques, or that reconstitution could occur with 2 non-identical sequences. In the absence of additional H27 antiserum, it was not possible to determine the nature of the potential antigenic binding site or establish whether it represented a genuine scaffold associated sequence involved in chromosomal domain organisation. In Chapter 5, 5 rabbit antisera (R1-R5) and mouse monoclonal antibody (MCA), all elicited against Hela Dc, are examined. Analysis of the 5 antisera by C'F, an ELISA assay and immunoblotting indicated each anti serum reacted with different chromosomal determinants to different extents. This is attributed to the genetic variability of the 5 rabbits which were not inbred. R1-R5 were relatively cross reactive with human and non human chromatins in both C'F and ELISA assays and whilst total DNA dependence was observed in the C'F assay, the reactive antigens did not reconstitute with DNA, after extensive nuclease digestion had abolished activity. The antisera reacted strongly with isolated chromosomal scaffolds, and both immunoblotting and immunofluorescence suggested some reactivity with intermediate filament proteins which are common insoluble contaminants of both Dc and scaffold preparations. Interestingly, whilst C'F analysis suggested R1-R5 recognized different determinants to H27, immunoblotting with all 6 antisera indicated similarities. It is therefore suggested that the major reconstituting antigens reactive with H27 cannot be detected by immunoblotting in the absence of DNA to maintain antigenicity. DNA dependence studies of R1-R5 using the ELISA assay gave different results to those obtained by C'F. R1-R3 recognized only DNA independent determinants, whilst R4 and R5 were partially DNA dependent. Results are explained in terms of the different detection mechanisms employed by each assay system and may suggest that the DNA dependence of some determinants in the C'F assay reflects an assay dependent phenomenon. It is concluded that DNA dependence does not necessarily reflect a DNA-protein interaction of biological significance within the nucleus. Unlike polyclonal antisera, MCAs to tight binding NHCP-DNA complexes have the potential to characterize individual components. However, as demonstrated by M8D5, MCAs do have limitations. M8D5 recognizes a single protein of Mr 64,000 which is apparently a minor component of Dc and chromosomal scaffolds. ELISA analysis shows M8D5 to be cross reactive with a variety of chromatin types and to recognize a DNA independent antigen. M8D5 was not amenable to analysis by C'F or immunofluorescence. Limitations of M8D5 are discussed in relation to the general failure of MCAs to analyse the many different components of the tight binding NHCP group. In Chapter 6, Hela DNA binding proteins are assessed by DNA protein blotting (Bowen et al, 1980). Protein blots of chromatin and several subfractions were probed with [32P] labelled Hela genomic DNA and many proteins were shown to have DNA binding potential. Very similar DNA binding patterns were obtained using Dc, scaffold and crude cytokeratin preparations, indicating considerable similarities between the 3 fractions. Many of the antigenic proteins detected by immunoblotting also had DNA binding potential, including some intermediate filament proteins. This technique is limited by the inability of some proteins to renature after electrophoresis and blotting and the highly non-specific nature of the interaction of genomic DNA with immobilized protein. However, it is demonstrated that proteins may be distinguished by their relative abilities to bind single stranded or double stranded DNA and strategies are considered which may allow detection of higher affinity DNA. binding proteins, within a complex mixture.