Unique features and clinical importance of acute alloreactive immune responses.

Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.

Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

Identification of distinct human invariant natural killer T-cell response phenotypes to alpha-galactosylceramide.

BACKGROUND: Human CD1d-restricted, invariant natural killer T cells (iNKT) are a unique class of T lymphocytes that recognise glycolipid antigens such as alpha-galactosylceramide (alphaGalCer) and upon T cell receptor (TCR) activation produce both Th1 and Th2 cytokines. iNKT cells expand when cultured in-vitro with alphaGalCer and interleukin 2 (IL-2) in a CD1d-restricted manner. However, the expansion ratio of human iNKT cells varies between individuals and this has implications for attempts to manipulate this pathway therapeutically. We have studied a panel of twenty five healthy human donors to assess the variability in their in-vitro iNKT cell expansion responses to stimulation with CD1d ligands and investigated some of the factors that may influence this phenomenon. RESULTS: Although all donors had comparable numbers of circulating iNKT cells their growth rates in-vitro over 14 days in response to a range of CD1d ligands and IL-2 were highly donor-dependent. Two reproducible donor response patterns of iNKT expansion were seen which we have called 'strong' or 'poor' iNKT responders. Donor response phenotype did not correlate with age, gender, frequency of circulating iNKT, or with the CD1d ligand utilised. Addition of exogenous recombinant human interleukin 4 (IL-4) to 'poor' responder donor cultures significantly increased their iNKT proliferative capacity, but not to levels equivalent to that of 'strong' responder donors. However in 'strong' responder donors, addition of IL-4 to their cultures did not significantly alter the frequency of iNKT cells in the expanded CD3+ population. CONCLUSION: (i) in-vitro expansion of human iNKT cells in response to CD1d ligand activation is highly donor variable, (ii) two reproducible patterns of donor iNKT expansion were observed, which could be classified into 'strong' and 'poor' responder phenotypes, (iii) donor iNKT response phenotypes did not correlate with age, gender, frequency of circulating iNKT cells, or with the CD1d ligand utilised, (iv) addition of IL-4 to 'poor' but not 'strong' responder donor cultures significantly increased their in-vitro iNKT cell expansion to alphaGalCer.

ATP-induced autophagy is associated with rapid killing of intracellular mycobacteria within human monocytes/macrophages.

BACKGROUND: We have previously reported that ATP treatment of M bovis-BCG infected human macrophages induces P2X7 receptor-dependent killing of intracellular mycobacteria. The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes. In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy. RESULTS: We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocyte-derived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP). CONCLUSION: We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections.

Direct cell-to-cell spread of a pathogenic yeast.

BACKGROUND: Cryptococcosis, a fatal fungal infection of the central nervous system, is one of the major killers of AIDS patients and other immunocompromised hosts. The causative agent, Cryptococcus neoformans, has a remarkable ability to 'hide' and proliferate within phagocytic cells of the human immune system. This intracellular phase is thought to underlie the ability of the pathogen to remain latent for long periods of time within infected individuals. RESULTS: We now report that Cryptococcus is able to undergo 'lateral transfer' between phagocytes, moving directly from infected to uninfected macrophages. This novel process was observed in both C. neoformans serotypes (A and D) and occurs in both immortalised cell lines and in primary human macrophages. Lateral transfer is independent of the initial route of uptake, since both serum-opsonised and antibody-opsonised C. neoformans are able to undergo direct cell-to-cell transfer. CONCLUSION: We provide the first evidence for lateral transfer of a human fungal pathogen. This rare event may occur repeatedly during latent cryptococcal infections, thereby allowing the pathogen to remain concealed from the immune system and protecting it from exposure to antifungal agents.

Expulsion of live pathogenic yeast by macrophages.

Phagocytic cells, such as neutrophils and macrophages, perform a critical role in protecting organisms from infection by engulfing and destroying invading microbes . Although some bacteria and fungi have evolved strategies to survive within a phagocyte after uptake, most of these pathogens must eventually kill the host cell if they are to escape and infect other tissues . However, we now demonstrate that the human fungal pathogen Cryptococcus neoformans is able to escape from within macrophages without killing the host cell by a novel expulsive mechanism. This process occurs in both murine J774 cells and primary human macrophages. It is extremely rapid and yet can occur many hours after phagocytosis of the pathogen. Expulsion occurs independently of the initial route of phagocytic uptake and does not require phagosome maturation . After the expulsive event, both the host macrophage and the expelled C. neoformans appear morphologically normal and continue to proliferate, suggesting that this process may represent an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation .

The 5-deoxy-5-methylthio-xylofuranose residue in mycobacterial lipoarabinomannan. absolute stereochemistry, linkage position, conformation, and immunomodulatory activity.

Mycobacteria produce a cell-surface glycoconjugate, lipoarabinomannan (LAM), which has been shown to be a potent modulator of the immune response that arises from infection by these organisms. Recently, LAM from the human pathogens Mycobacterium tuberculosis and M. kansasii has been shown to contain an unusual 5-deoxy-5-methylthio-xylofuranose (MTX) residue as well as its corresponding oxidized counterpart, 5-deoxy-5-methylsulfoxy-xylofuranose (MSX). To date, the absolute configuration of these residues and their linkage position to the polysaccharide are unknown, as is their biological role. Through the combined use of chemical synthesis and NMR spectroscopy, we have established that the MTX/MSX residues in these glycoconjugates are of the d-configuration and that they are linked alpha-(1-->4) to a mannopyranose residue in the mannan portion of the glycan. Conformational analysis of the MTX/MSX residue using NMR spectroscopy showed differences in ring conformation and as well as in the rotamer populations about the C-4-C-5 bond, as compared to the parent compound, methyl alpha-d-xylofuranoside. Two of the synthesized disaccharides, 3 and 34, were tested in cytokine induction assays, and neither led to the production of TNF-alpha or IL-12p70. In contrast, both demonstrated modest inhibitory properties when these same cytokines were induced using a preparation of Interferon-gamma and Staphylococcus aureus Cowan strain (SAC/IFN-gamma). These latter observations suggest that this motif may play a role in the immune response arising from mycobacterial infection.