Natural killer cell deficiency in patients with non-Hodgkin lymphoma after lung transplantation.

BACKGROUND: Post-transplant non-Hodgkin lymphoma (NHL) is a well-recognized complication of solid-organ transplantation, and pharmacologic suppression of adaptive immunity plays a major role in its development. However, the role of natural killer (NK) cells in post-lung transplant de novo NHL is unknown. METHODS: Extensive phenotypic analyses of NK cells from patients diagnosed with NHL after liver or lung transplantation were conducted with multicolor flow cytometry. Polyfunctionality assays simultaneously assessed NK cell degranulation (CD107a) and intracellular cytokine production (interferon-γ and tumor necrosis factor-α) in the presence of NHL target cells. RESULTS: The development of de novo NHL is linked to NK-cell maturation defects, including overexpression of NKG2A and CD62L and down-modulation of inhibitory killer immunoglobulin-like receptors and CD57 receptors. More importantly, in patients who developed NHL after lung transplantation, we observed a specific down-modulation of the activating receptors (NKp30, NKp46, and NKG2D) and a sharp decrease in perforin expression and degranulation against NHL target cells. CONCLUSIONS: Our results suggest that accumulation of abnormal NK cells could play a role in the outgrowth of NHL after lung transplantation, independently of the immunosuppressive regimen.

Transplantation-induced cancers: Emerging evidence that clonal CMV-specific NK cells are causal immunogenic factors.

Solid cancers are a major adverse outcome of liver transplantation. Recent reassessments have revealed insights into causal factors, primarily centering on modulations of the natural killer (NK) cell compartment in liver transplant recipients. In the presence of cytomegalovirus, the clonal expansion of differentiated NK cells could restrict the diversity of the NK repertoire favoring the development of certain tumors.

Expansion of CMV-mediated NKG2C+ NK cells associates with the development of specific de novo malignancies in liver-transplanted patients.

Solid cancers are a major adverse outcome of orthotopic liver transplantation (OLT). Although the use of chronic immunosuppression is known to play a role in T cell impairment, recent insights into the specificities of NK cells led us to reassess the potential modulation of this innate immune cell compartment after transplantation. Our extensive phenotypic and functional study reveals that the development of specific de novo noncutaneous tumors post-OLT is linked to unusual NK cell subsets with maturation defects and to uncommon cytokine production associated with the development of specific cancers. Remarkably, in CMV(+) patients, the development de novo head/neck or colorectal tumors is linked to an aberrant expansion of NK cells expressing NKG2C and a high level of intracellular TNF-α, which impact on their polyfunctional capacities. In contrast, NK cells from patients diagnosed with genitourinary tumors possessed a standard immature signature, including high expression of NKG2A and a robust production of IFN-γ. Taken together, our results suggest that under an immunosuppressive environment, the interplay between the modulation of NK repertoire and CMV status may greatly hamper the spectrum of immune surveillance and thus favor outgrowth and the development of specific de novo tumors after OLT.

A single amino-acid change in a highly conserved motif of gp41 elicits HIV-1 neutralization and protects against CD4 depletion.

BACKGROUND: The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. METHODS: Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. RESULTS: Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. CONCLUSIONS: These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

Shaping of iNKT cell repertoire after unrelated cord blood transplantation.

Invariant natural killer T (iNKT) cells have a pivotal role in immune regulation, tumor surveillance, and the induction of allograft tolerance. In this report, we analyze the recovery of iNKT cells after unrelated cord blood transplantation (UCBT) of adult patients with high-risk acute myeloid leukemia. We found that iNKT cells were reconstituted within 1 month after UCBT, at the same time as NK cells and before conventional T cells. These iNKT cells displayed a unique primed/central memory CD4(+)CD45RO(+)CCR7(+)CD62L(+) phenotype soon after the transplant. Interestingly, the functional competence of these cells was poor, except for their high GM-CSF production capacity. However, this post-graft functionally immature state was transient and all of the patients tested had fully functional iNKT cells 3 to 6 months post-UCBT and high cytolytic capacity for destroying primary CD1d(+) myeloid blast cells. Our results raise the possibility that iNKT cells might play a key role in graft-versus-leukemia activity after UCBT.

Length variability of telomeric repeat sequences of human herpesvirus 6 DNA.

The telomeric repeat sequences (TRS) located near both ends of human herpesvirus 6 (HHV-6) genome are unique structures of unknown function among human herpesviruses. The goal of the present study was to investigate the variability of TRS copy number among different laboratory strains and HHV-6-infected clinical specimens regarding the two variants A and B of HHV-6. DNA obtained from infected cells was submitted to a PCR assay designed to amplify the part of genome containing TRS specifically either for HHV-6A or HHV-6B. Amplicons were analyzed by electrophoresis on agarose gel with ethidium bromide staining and nucleotide sequencing. The number of TRS copies was highly variable among the distinct laboratory strains and clinical specimens studied, ranging from 15 up to more than 180. However, this number was constant for a given strain after serial propagation in cell cultures as well as in different samples from the same subject. This permitted to detect a mixed infection with two distinct strains of HHV-6A within the same patient. The PCR-based analysis of HHV-6 TRS has a limited sensitivity but is highly specific, which provides the opportunity to include it in the set of molecular tools dedicated to the study of HHV-6 epidemiology.

Variability of gB and gH genes of human herpesvirus-6 among clinical specimens.

The isolates of human herpesvirus-6 (HHV-6), a betaherpesvirus closely related to human cytomegalovirus (HCMV), are classified as either variants A (HHV-6A) or B (HHV-6B) but their intravariant variability has not been studied extensively so far. The full-length genes of envelope glycoproteins gB and gH from 40 distinct HHV-6-DNA-positive specimens and 11 laboratory strains were amplified using PCR, and their nucleotide sequence determined. Nucleotide divergences were observed at 156 (6.2%) and 98 (4.7%) positions in the case of gB and gH genes respectively. Phylogenetic analysis, including reference strain sequences, confirmed the unambiguous distinction between HHV-6A and HHV-6B for both genes. In the case of HHV-6B isolates, two subgroups of gB gene (designated as gB-B1 and gB-B2) and two subgroups of gH gene (gH-B1 and gH-B2) were identified but the phylogenetic trees of both genes were not fully congruent with each other. The analysis of gB and gH protein sequences showed that 26 and 39 critical amino acid changes respectively permitted the unambiguous distinction between HHV-6A and HHV-6B. Among HHV-6B isolates, gB and gH gene subgroups were characterized by specific amino acid signatures made of six, and two residues respectively. The linkage unbalance between amino acid signatures as well as the distribution of crucial nucleotide changes strongly suggested the occurrence of intravariant recombination within gB gene among HHV-6B isolates. These results indicate that, as in the case of HCMV, homologous recombination may contribute to the genetic variability of HHV-6.

Human herpesvirus-6 (HHV-6) DNA in plasma reflects the presence of infected blood cells rather than circulating viral particles.

BACKGROUND: The presence of HHV-6 DNA in plasma or serum is considered a good marker of active infection. However, it is ignored whether this DNA corresponds to virus particles produced by lymphoid tissue infection or virus-free DNA released from infected circulating blood cells. OBJECTIVES: To investigate whether HHV-6 DNA in whole plasma is nonencapsidated and its amount is correlated to cellular and human herpesvirus-7 (HHV-7) DNA loads in plasma subfractions as well as in corresponding peripheral blood mononuclear cells (PBMCs). STUDY DESIGN: Whole plasma samples from immunocompromised patients were submitted to a DNase-resistance test. Plasma samples from a second group of patients were split up into three subfractions: P1 (pellet of clarification), P2 (pellet of ultracentrifugation), and S (supernatant of ultracentrifugation). HHV-6, HHV-7, and cellular DNA loads were determined in each fraction and PBMCs using specific real-time PCR. RESULTS: Among 14 whole plasma samples, the majority of HHV-6 DNA detected was unprotected against DNase, i.e. nonencapsidated. The study of 35 other plasma samples revealed that cellular DNA was present in all subfractions from all samples whereas HHV-6 DNA was detected in 13 P1, 12 P2, 10 S fractions, and HHV-7 DNA in only one P1 fraction. Accordingly, median HHV-6 DNA load was significantly higher in P1 than in P2 and S fractions. The detection of HHV-6 DNA in plasma subfractions was statistically associated with a higher HHV-6 viral load in PBMCs (p