Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death and reduced immunoglobulin release.

Streptococcal pyrogenic exotoxin (Spe) A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage to Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leucocyte profile of tonsil is unique, the impact of SpeA production on human tonsil cell function was investigated. Human tonsil cells from routine tonsillectomy were co-incubated with purified streptococcal superantigens or culture supernatants from isogenic streptococcal isolates, differing only in superantigen production. Tonsil cell proliferation was quantified by tritiated thymidine incorporation, and cell surface characteristics assessed by flow cytometry. Soluble mediators including immunoglobulin were measured using enzyme-linked immunosorbent assay. Tonsil T cells proliferated in response to SpeA and demonstrated typical release of proinflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7 days. In contrast, marked B cell apoptosis and abrogation of total immunoglobulin (Ig)A, IgM, and IgG production occurred in the presence of SpeA and other superantigens. In SpeA-stimulated cultures, T follicular helper (Tfh) cells showed a reduction in C-X-C chemokine receptor (CXCR)5 (CD185) expression, but up-regulation of OX40 (CD134) and inducible T cell co-stimulator (ICOS) (CD278) expression. The phenotypical change in the Tfh population was associated with impaired chemotactic response to CXCL13. SpeA and other superantigens cause dysregulated tonsil immune function, driving T cells from Tfh to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a probable survival advantage.

Frequency of acute myeloid leukaemia-associated mouse chromosome 2 deletions in X-ray exposed immature haematopoietic progenitors and stem cells.

Exposure to ionising radiation can lead to an increased risk of cancer, particularly leukaemia. In radiation-induced acute myeloid leukaemia (rAML), a partial hemizygous deletion of mouse chromosome 2 is a common feature in several susceptible strains. The deletion is an early event detectable 24h after exposure in bone marrow cells using cytogenetic techniques. Expanding clones of bone marrow cells with chromosome 2 deletions can be detected less than a year after exposure to ionising radiation in around half of the irradiated mice. Ultimately, 15-25% of exposed animals develop AML. It is generally assumed that leukaemia originates in an early progenitor cell or haematopoietic stem cell, but it is unknown whether the original chromosome damage occurs at a similar frequency in committed progenitors and stem cells. In this study, we monitored the frequency of chromosome 2 deletions in immature bone marrow cells (Lin(-)) and haematopoietic stem cells/multipotent progenitor cells (LSK) by several techniques, fluorescent in situ hybridisation (FISH) and through use of a reporter gene model, flow cytometry and colony forming units in spleen (CFU-S) following ex vivo or in vivo exposure. We showed that partial chromosome 2 deletions are present in the LSK subpopulation, but cannot be detected in Lin(-) cells and CFU-S12 cells. Furthermore, we transplanted irradiated Lin(-) or LSK cells into host animals to determine whether specific irradiated cell populations acquire an increased proliferative advantage compared to unirradiated cells. Interestingly, the irradiated LSK subpopulation containing cells carrying chromosome 2 deletions does not appear to repopulate as well as the unirradiated population, suggesting that the chromosomal deletion does not provide an advantage for growth and in vivo repopulation, at least at early stages following occurrence.

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia.

The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.