The role of CD4(+) T cells in BKV-specific T cell immunity.

Reactivation of polyomavirus BK (BKV) infection represents a severe complication in kidney transplant (KTX) patients. We previously reported an association between a declining BK viral load and the reconstitution of CD4(+) T cell BKV-specific immunity in patients following kidney transplantation. However, the specific contribution of CD4(+) T cells in the regulation of BKV-replication is unknown. Nevertheless, in vitro enrichment of BKV-specific T cells and subsequent adoptive T cell transfer may improve the restoration of immune competence in KTX patients with BKV infection. To date, strategies to capture human BKV-specific T cells with the ensuing expansion to clinically useful numbers are lacking. Here, we demonstrated a comprehensive flow cytometric analysis of the BKV-specific T cell response that permits access to the majority of T cells specific for immunodominant BKV antigens. A full-spectrum evaluation of the BKV-specific T cell response was performed by stimulating peripheral blood mononuclear cells (PBMC) with a mixture of BKV immunodominant peptide pools at varying concentrations and measuring activation marker expression and cytokine secretion. We also examined the effects of co-stimulation and PBMC resting time prior to activation. We defined the narrow range of stimulation conditions that permit the capture and expansion of functional BKV-specific T cell lines. The generated BKV-specific T cell lines showed the highest specificity and functionality when the T cells were captured according to IFNγ-secretion. This study highlights the multifunctional and cytolytic BKV-specific CD4(+) T cells as a dominant population within the generated T cell product. This method offers a novel approach for the generation of BKV-specific T cell lines for adoptive immunotherapy and underscores the critical role of CD4(+) T cells in the clearance of BKV.

Loss of DRAK2 signaling enhances allogeneic transplant survival by limiting effector and memory T cell responses.

Here, we demonstrate that loss of DRAK2 signaling significantly promotes the acceptance of allogeneic engraftment in two separate transplant models without promoting generalized immunosuppression. Drak2-/- T cells failed to reject allogeneic tumors, and were defective in rejecting Balb/C allogeneic skin grafts on C57BL6/J recipients. A significant fraction of alloreactive Drak2-/- T cells underwent apoptosis following activation, whereas enforced expression of Bcl-xL in Drak2-/- T cells restored allograft rejection. Formation of allogeneic memory was also greatly hampered in T cells lacking the Drak2 gene. Adoptive transfer of memory T cells from Drak2-/- mice failed to promote the rejection of allogeneic tumors, and such cells led to significantly delayed rejection of skin allografts in the Balb/C->C57BL/6J model. Costimulatory blockade by in vivo administration of Cytotoxic T-Lymphocyte Antigen 4 fusion protein (CTLA4-Ig) synergized with the DRAK2 deficiency and led to long-term allogeneic skin graft acceptance. Overall, these results demonstrate that DRAK2 plays an important role in primary and memory T cell responsiveness to allografts. Because previous studies have demonstrated that a loss of DRAK2 does not negatively impact antiviral immunity, the studies here underscore the potential utility of pharmacological blockade of DRAK2 to achieve transplant maintenance without the imposition of generalized immunosuppression.

Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry.

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.

A high risk phenotype of hypertrophic cardiomyopathy associated with a compound genotype of two mutated beta-myosin heavy chain genes.

Hypertrophic cardiomyopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that is in most cases familial and transmitted in a dominant fashion. The most frequently affected gene codes for the cardiac (ventricular) beta-myosin heavy chain. We have investigated the genetic cause of an isolated case of HCM, which was marked by an extremely severe phenotype and a very early age of onset. HCM is normally not a disease of small children. The proband was a boy who had suffered cardiac arrest at the age of 6.5 years (resuscitation by cardioconversion). Upon screening of the beta-myosin heavy chain gene as a candidate, two missense mutations, one in exon 19 (Arg719Trp) and a second in exon 12 (Met349Thr), were identified. The Arg719Trp mutation was de novo, as it was not found in the parents. In contrast, the Met349Thr mutation was inherited through the maternal grandmother. Six family members were carriers of this mutation but only the proband was clinically affected. Segregation and molecular analysis allowed us to assign the Met349Thr mutation to the maternal and the Arg719Trp de novo mutation to the paternal beta-myosin allele. Thus, the patient has no normal myosin. We interpret these findings in terms of compound heterozygosity of a dominant (Arg719Trp) and a recessive (Met349Thr) mutation. Whereas a single mutated Arg719Trp allele would be sufficient to cause HCM, the concurrent Met349Thr mutation alone does not apparently induce the disease. Nevertheless, it conceivably contributes to the particularly severe phenotype.