Alan M. Krensky

RANTES chemokine expression in cell-mediated transplant rejection of the kidney

RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemotactic cytokine (a chemokine) for memory T lymphocytes, monocytes, and eosinophils. RANTES expression was studied in renal allograft biopsy specimens. Although RANTES was not expressed in samples taken one hour after transplantation, or in native renal biopsy specimens from patients with cyclosporin nephrotoxicity, it was expressed during cell-mediated transplant rejection. RANTES mRNA was detected in infiltrating mononuclear cells and renal tubular epithelium, and RANTES protein was localised to mononuclear cells, tubular epithelium, and vascular endothelium. This suggests RANTES has a role in allograft rejection.

CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia.

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34+CD38− AML cells in many cases (74.0 ± 25.3% in 19 of 29 cases), whereas only a few (4.9 ± 1.6%) cells in the normal HSC-enriched population (Lin−CD34+CD38−CD90+) expressed CD96 weakly. To examine whether CD96+ AML cells are enriched for LSC activity, we separated AML cells into CD96+ and CD96− fractions and transplanted them into irradiated newborn Rag2−/− γc−/− mice. In four of five samples, only CD96+ cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

Chemokines, Chemokine Receptors, and Allograft Rejection

Chemokines are pivotal mediators in allograft rejection by virtue of their activity as regulators of leukocyte movement, adhesion, and effector function. Because the regulation of effector cell infiltration is complex, it is difficult to dissect the relative role of each chemokine in the inflammatory processes leading to allograft rejection, especially since many chemokines and chemokine receptors are seemingly redundant. Nevertheless, it is clear from knockout and inhibitor experiments that specific chemokine and/or chemokine receptor blockades may influence various aspects of the rejection process. Therefore, therapeutic interventions aimed at chemokines and/or their receptors may prove useful in treatment of transplant rejection and/or induction of immunologic tolerance. Therapeutic effects will likely differ, depending upon the stage of rejection and the other therapeutics administered. The marked enhancement of long-term kidney transplant survival in patients homozygous for a CCR5 null allele is very encouraging for future therapies directed at CCR5-positive mononuclear cells. Results with CCR1, CXCR3, and CX3CR1 blockades are also remarkable. Clearly, antagonists for chemokines and their receptors have the potential to become important therapeutics in treatment of acute and chronic allograft rejection.The authors apologize to all colleagues whose work could not be cited in this review due to space limitations.

Granulysin-Dependent Killing of Intracellular and Extracellular Mycobacterium tuberculosis by Vγ9/Vδ2 T Lymphocytes

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Cutting Edge: Decreased Accumulation and Regulatory Function of CD4+CD25high T Cells in Human STAT5b Deficiency

We show that STAT5b is important for the in vivo accumulation of CD4+CD25high T cells with regulatory cell function. A patient homozygous for a missense A630P STAT5b mutation displayed immune dysregulation and decreased numbers of CD4+CD25high T cells. STAT5bA630P/A630P CD4+CD25high T cells had low expression of forkhead box P3 and an impaired ability to suppress the proliferation of or to kill CD4+CD25− T cells. Expression of CD25, a component of the high-affinity IL-2R, was also reduced in response to IL-2 or after in vitro propagation. The impact of the STAT5b mutation was selective in that IL-2-mediated up-regulation of the common γ-chain cytokine receptor and perforin, and activation-induced expressions of CD154 and IFN-γ were normal. These results indicate that STAT5b propagates an important IL-2-mediated signal for the in vivo accumulation of functional regulatory T cells.

Human NKT Cells Express Granulysin and Exhibit Antimycobacterial Activity

Human NKT cells are a unique subset of T cells that express an invariant Vα24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid α-galactosylceramide (αGalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of αGalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-γ, proliferated, and exerted lytic activity in response to αGalCer-pulsed monocyte-derived cells. Importantly, αGalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that αGalCer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection.

T-cell release of granulysin contributes to host defense in leprosy

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Treatment of steroid-resistant focal segmental glomerulosclerosis with pulse methylprednisolone and alkylating agents

In children, steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis (FSGS) is frequently a progressive condition resulting in end-stage renal disease. There have been no reports of effective treatment for this condition. For the past several years, the Pediatric Nephrology services at the University of California, San Diego and Stanford University Schools of Medicine have treated these patients with a protocol involving infusions of high doses of methylprednisolone, often in combination with oral alkylating agents. Twenty-three children have been treated in this manner with a follow-up of 46±5 months. Twelve of these children are in complete remission. Six have minimal to moderate proteinuria. Four children remain nephrotic. Each of these children has a normal glomerular filtration rate. One child developed chronic renal failure and subsequently died while on dialysis. These results appear significantly better than previous series of children with FSGS. A controlled, multi-center trial of this protocol has been proposed.

Tubulo-Interstitial Nephritis Associated with Polyomavirus (BK Type) Infection

We studied viral injury to the kidney in a six-year-old boy with hyperimmunoglobulin M immunodeficiency who presented with irreversible acute renal failure and eventually died after five months of dialysis. Renal biopsy at the time of his presentation revealed a predominantly tubulo-interstitial process with numerous viral inclusions that were identified as polyomavirus. Urine cultures showed a massive viruria with BK-type, polyomavirus. The kidney disease was end stage, with persistence of BK virus identified by morphologic techniques and by culture. DNA hybridization analysis showed virus in low concentration in the lymph nodes, spleen, and lungs. The marked viruria, the high concentration of BK virus, and the extensive distribution of viral antigen throughout the kidney all suggest that infection with BK virus was the basis of the severe renal parenchymal injury.