Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation.

BACKGROUND: After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation. DESIGN AND METHODS: Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4(+) and CD8(+) memory T cells. RESULTS: Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4(+) T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4(+) and CD8(+) T cells. CONCLUSIONS: Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4(+) and CD8(+) T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.

Functional reconstitution of the human chemokine receptor CXCR4 with G(i)/G (o)-proteins in Sf9 insect cells.

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) binds to the chemokine receptor CXCR4 that couples to pertussis toxin-sensitive G-proteins of the G(i)/G(o)-family. CXCR4 plays a role in the pathogenesis of autoimmune diseases, human immunodeficiency virus infection and various tumors, fetal development as well as endothelial progenitor and T-cell recruitment. To this end, most CXCR4 studies have focused on the cellular level. The aim of this study was to establish a reconstitution system for the human CXCR4 that allows for the analysis of receptor/G-protein coupling at the membrane level. We wished to study specifically constitutive CXCR4 activity and the G-protein-specificity of CXCR4. We co-expressed N- and C-terminally epitope-tagged human CXCR4 with various G(i)/G(o)-proteins and regulator of G-protein signaling (RGS)-proteins in Sf9 insect cells. Expression of CXCR4, G-proteins, and RGS-proteins was verified by immunoblotting. CXCR4 coupled more effectively to Galpha(i1) and Galpha(i2) than to Galpha(i3) and Galpha(o) and insect cell G-proteins as assessed by SDF-1alpha-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1alpha-stimulated GTP hydrolysis. SDF-1alpha stimulated [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) binding to Galpha(i2). RGS4 did not enhance GTPgammaS binding. Na(+) salts of halides did not reduce basal GTPase activity. The bicyclam, 1-[[1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100), acted as CXCR4 antagonist but was devoid of inverse agonistic activity. Halides reduced the maximum SDF-1alpha-stimulated GTP hydrolysis in the order of efficacy I(-) > Br(-) > Cl(-). In addition, salts reduced the potency of SDF-1alpha at activating GTP hydrolysis. From our data, we conclude the following: (1) Sf9 cells are a suitable system for expression of functionally intact human CXCR4; (2) Human CXCR4 couples effectively to Galpha(i1) and Galpha(i2); (3) There is no evidence for constitutive activity of CXCR4; (4) RGS-proteins enhance agonist-stimulated GTP hydrolysis, showing that GTP hydrolysis becomes rate-limiting in the presence of SDF-1alpha; (5) By analogy to previous observations made for the beta(2)-adrenoceptor coupled to G(s), the inhibitory effects of halides on agonist-stimulated GTP hydrolysis may be due to increased GDP-affinity of G(i)-proteins, reducing the efficacy of CXCR4 at stimulating nucleotide exchange.

No association between renin-angiotensin system gene polymorphisms and early and long-term allograft dysfunction in kidney transplant recipients.

BACKGROUND: Genes determining the activity of the renin-angiotensin system (RAS) may be alloantigen-independent factors influencing kidney allograft function. We determined if gene polymorphisms of the RAS are associated with early and long-term post-transplantation graft dysfunction in 405 Caucasian kidney recipients with graft survivals of >2 years. METHODS: We calculated the slopes of serum creatinine(-1)/year and urinary protein excretion/year to follow graft function over time. Subjects were genotyped for the deletion (D) polymorphism of the gene encoding angiotensin I-converting enzyme, the angiotensin II-receptor type1 gene 1166A-C polymorphism and the M235T polymorphism of the angiotensinogen gene. RESULTS: The frequencies of factors predicting graft function were similar in patients with different genotypes. None of the polymorphisms influenced need for dialysis in the first week after transplantation, occurrence of at least one rejection episode, the slope of serum creatinine(-1)/year or the slope of urinary protein excretion/year. Results were independent of blood pressure or the use of angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers or calcineurin inhibitors. The combination of genotypes did not influence the indicators of early and long-term graft dysfunction. CONCLUSIONS: Neither the investigated gene polymorphisms of the RAS in kidney allograft recipients nor their combinations have an impact on early and long-term graft dysfunction.