SUMMARYCessation of milk removal causes mammary tissue involution, which in rodents is characterized by extensive tissue degeneration and loss of the majority of luminal epithelial cells by apoptosis. In contrast, bovine mammary tissue shows little histological evidence of tissue remodelling between lactations. In this study, we combined histology with molecular biology to examine the cellular and molecular changes in bovine mammary tissue on cessation of milking. Oligonucleosomal laddering of genomic DNA extracted from lactating tissue indicated that a proportion of cells were dying by apoptosis. This was confirmed by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labelling of apoptotic cells in lactating tissue sections (TUNEL). One week after cessation of milking, a-lactalbumin and a,5-casein messenger RNA (mRNA) abundance had decreased by 99 and 85 %, respectively, whereas lactoferrin mRNA had increased 20-fold. Drying off was also accompanied by an increase in oligonucleosomal laddering of genomic DNA, and by an increase in the proportion of TUNEL-positive cells, which were localized preferentially in regions where alveolar structure had deteriorated. Therefore, termination of lactation was associated with partial loss of the mammary cell population and dedifferentiation of the remainder.

Frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. Screening of goat's milk proteins in rabbit mammary explant cultures identified a single whey protein of M(r) 7600 able to inhibit synthesis of milk constituents. The active whey protein, which we term FIL (Feedback inhibitor of Lactation), also decreased milk secretion temporarily when introduced into a mammary gland of lactating goats. FIL was synthesized by primary cultures of goat mammary epithelial cells, and was secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by this milk protein.

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.

Minor or histocompatibility (H) antigens are recognized by CD4+ and CD8+ T lymphocytes as short polymorphic peptides associated with MHC molecules. They are the targets of graft versus host and graft versus leukemia responses following bone marrow transplantation between HLA-identical siblings. Several genes encoding class I-restricted minor H epitopes have been identified, but approaches used for these have proved difficult to adapt for cloning class II-restricted minor H genes. We have combined the unique antigen-presenting properties of dendritic cells and high levels of episomal expression following transfection of COS cells to identify a Y chromosome gene encoding two HY peptide epitopes, HYAb and HYEk.

Programmed cell death in mammary tissue was studied during natural weaning in lactating mice and after litter removal or milk stasis. All treatments stimulated mammary apoptosis, indicating that this process is an integral part of the tissue's involution after lactation. Induction of apoptosis was slower in natural weaning than after litter removal but occurred earlier when mice were concurrently pregnant during natural weaning. Ipsilateral induction of apoptosis by milk stasis in teat-sealed glands indicates that cell death is under local (i.e., intramammary) as well as endocrine regulation. Apoptosis detected by DNA laddering was associated with changes in expression of p53 and bax, two genes implicated in the regulation of cell death, and was accompanied by structural degeneration characteristic of mammary involution. Reciprocal changes in stromelysin mRNA, and that of its inhibitor TIMP-2, suggested that this structural reorganisation was the result of coordinated changes in gene expression favouring proteolysis of the extracellular matrix.

This study provides the first evidence that Foxp3-transduced T cells can control the rejection of an allogeneic transplant and suggests that T-cell Foxp3 gene transfer may have therapeutic value in clinical transplantation.

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty246–254 epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7−/−) skin grafts by female LMP7−/− mice. Second, we ruled out an altered Uty246–254-specific T cell repertoire in LMP7−/− female mice and demonstrated efficient Uty246–254 presentation by re-expressing LMP7 in male LMP7−/− cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty246–254-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7−/− female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.

Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.