Adenovirus infection and disease in paediatric haematopoietic stem cell transplant patients: clues for antiviral pre-emptive treatment.

Human adenovirus (HAdV) infections constitute a major cause of morbidity in paediatric haematopoietic stem cell transplant (HSCT) patients. New antiviral treatments offer promising perspectives. However, it remains challenging to identify patients at risk for disseminated infection, and who should receive early antiviral intervention. We conducted a longitudinal study of allogeneic HSCT recipients, including weekly HAdV monitoring, to determine the risks factors associated with HAdV infection and dissemination, and to assess whether HAdV loads in stools may be used as surrogate markers for HAdV dissemination. Between September 2010 and December 2011, out of 72 patients, the cumulative incidence rates at day 100 of HAdV digestive infection, systemic infection and related disease were 35.9%, 24.0%, and 18.3%, respectively. In multivariate analysis, the risk factors for HAdV digestive and systemic infection were cord blood and in vitro T-cell depletion. Graft-versus-host disease (GVHD) grade >2 was also associated with systemic infection. In patients with HAdV digestive shedding, GVHD grade >2 and HAdV load in stools were the only risk factors for systemic infection. The median peak levels of HAdV in stool were 7.9 and 4.0 log10 copies/mL, respectively, in patients with HAdV systemic infection and in those without. HAdV monitoring in stools of paediatric HSCT recipients receiving cord blood or in vitro T-cell depleted transplants helps to predict patients at risk for HAdV systemic infection. Our results provide a rationale for randomized controlled trials to evaluate the benefit of anti-HAdV pre-emptive treatments based on HAdV DNA levels in stools.

[Vaccination post hematopoietic stem cell transplantation: which vaccines and when and, how to vaccinate? An SFGM-TC report]. / Vaccinations post-allogreffe de cellules souches hématopoïétiques: lesquels? Quand? Comment?

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. Here we report our results and recommendations regarding vaccination post Hematopoietic Stem Cell Transplantation with practical focus on which vaccines to use and when and how to vaccinate?

Cellular immune parameters associated with spontaneous control of CMV in children who underwent transplantation.

CD4(+) T-cell functions that best correlate with CMV control were evaluated by studying the relationship between CMV infection and CMV-specific immune recovery as determined by proliferation assay and intracytoplasmic-IFNgamma assay. A total of 30 children (mean age: 8.30 years) who received an allogeneic hematopoietic SCT (HSCT) were included. In total, 13 recipients were seronegative before HSCT. None developed CMV infection or CMV-specific immunity. A total of 17 recipients were seropositive: (i) four patients spontaneously controlled CMV. The median of CMV-specific IFNgamma-secreting CD4 T cells was 9.13/microl at month 3 in these four patients and three of the four patients evidenced optimal proliferative responses since month 1; (ii) in 10 patients who received anti-CMV chemotherapy because of prolonged viremia, lower (P=0.016) IFNgamma responses (0.39/microl), together with delayed and/or depressed proliferative responses, were observed; (iii) finally, one patient with early CMV-associated disease had undetectable proliferative and IFNgamma responses until month 3. In conclusion, both intense IFNgamma responses and early proliferative responses seem to be associated with optimal CMV control.

HLA-associated genetic resistance and susceptibility to type I diabetes in French North Africans and French natives.

The distribution of human leukocyte antigen (HLA)-DRB1-DQA1-DQB1 haplotypes was analyzed separately in two distinct French ethnic groups with type I diabetes (T1D), i.e. French North African migrants (n= 64, mean age at diagnosis = 8.25 years) and ancient French natives (n= 60, mean age at diagnosis = 7.42 years). HLA associations were determined by calculating odds ratios (ORs) between patients and two ethnic-matched control populations. Results show highly similar ORs for the conservative DRB1*0301-DQA1*0501-DQB1*0201 haplotype of susceptibility (OR: 3.22 and 3.93 in migrants and natives, respectively) and the DRB1*1501-DQA1*0102-DQB1*0602 haplotype of resistance (OR: 0.05 and 0.03, respectively). In contrast, among the more variable DRB1*04-DQB1*0302 haplotypes of susceptibility, the DRB1*0402 (OR: 3.10 and 32.84) and 0405 (OR: 5.90 and 16.25, respectively) were associated with T1D in migrants and natives, whereas an increase of DRB1*0401, a rare allele in migrants, was significant in natives only. Also, among the DRB1*11-DQA1*0505-DQB1*0301 haplotypes of resistance, the OR observed for DRB1*1104-DQA1*0505-DQB1*0301, common in migrants, was lower (OR: 0.08) than for DRB1*1101-DQA1*0505-DQB1*0301 (OR: 0.32), common in natives. How DRB1*11 subtypes might affect differently the risk conferred by DQA1*0505-DQB1*0301 will be discussed.

[Tonsillectomy in 2005]. / L'amygdalectomie en 2005.

During the past years, the number of tonsillectomies (only palatine tonsils are taken off) has decreased, indications for surgery have changed. A multi-disciplinal group of paediatricians tried to elaborate the state of the art in the field. Tonsils are the first line defense of high respiratory tract. The immune functions of their lymphoid tissue are multiple: mucosal antigens capture, presentation to lymphocytes, antigens specific proliferation of lymphocytes T and B, differentiation of lymphocytes in effectors lymphocytes and immune lymphocytes. Epithelial cells on the tonsils' surface express non-specific defense. These facts explain partly tonsils' hypertrophy. Tonsillectomy has no general immune consequences. In 2002, in France, 75,000 tonsillectomies were realized, of which 90% were in children. Tonsil's hypertrophy is the major indication, mandatory when sleep apnoeas exist. The main historical tonsillectomy indication for recurrent tonsillitis should decrease due to a more precise diagnostic (rapid test at bed site), an efficient antibiotics therapy and better care for pain. Other indications are scarce. Surgery, feasible from 9 months of age, requires a brief general anaesthesia and has very few contra-indications. The technique, operator dependent, relies on his experience. The only potentially severe complication is an haemorrhage due to scab fall between the eighth and twelfth days. It requires explanation and a written note given to parents. The possibility of lack of feeding and voice modification, usually transitory, should be known. Multiple consequences of tonsillectomy especially allergy have been alleged. Since the years 1980, it is well established that pre-existing allergy or asthma are not a contraindication. More, its deleterious impact on allergic children has not been demonstrated. Last, a gain of weight post-tonsillectomy is possible and could become a risk if excessive.

Listing of common HLA alleles and haplotypes based on the study of 356 families residing in the Paris, France, area: implications for unrelated hematopoietic stem cell donor selection.

In this study we have identified frequent human leukocyte antigen (HLA)-A, -B, -C,-DRB1, and -DQB1 alleles, frequent HLA-B/C, HLA-DRB1/DQB1 two-allele associations, and the most common HLA-A/B/C/DRB1/DQB1 five-locus haplotypes in a population residing in the Paris, France, area. The study was carried out in 356 families of children awaiting hematopoietic stem-cell transplantation (HSCT), with the selection criterion that haplotypes could be assigned with certainty to both the patient and at least one parent. Parental haplotypes were HLA-A, -B serologically typed, and HLA-C, -DRB1, -DQB1 broadly typed by polymerase chain reaction-sequence-specific oligonucleotide probe. The alleles of the most frequent haplotypes were subsequently defined at a high-resolution level by polymerase chain reaction-sequence-specific primer. The results on the distribution of common alleles and common allele associations demonstrated similarities with the previously published data in Caucasian populations, as expected from the geographic origin of the studied population. More importantly, this study provides the largest listing of common B/C and DRB1/DQB1 associations and of common five-allele haplotypes defined with certainty in a Caucasian population to date. These results can be used to help estimate the likelihood of finding a suitable donor in unrelated HSCT and to delineate search strategies for potential donors.

Common genomic HLA haplotypes contributing to successful donor search in unrelated hematopoietic transplantation.

The aim of the study was to identify the most frequent HLA haplotypes in order to optimize donor searches in unrelated hematopoietic stem cell (HSC) transplantation. Pediatric patients from the north of France who underwent initial HLA typing for donor search in our center were included. Patients and family members were broadly typed for HLA class I and II. Patients were further DNA typed at the sequence level for HLA-A, -B, -Cw, -DRB1, and -DQB1 alleles. In 200 of 207 patients HLA haplotypes were assigned by the mode of inheritance. The most common haplotypes were defined based on frequencies over 0.75%. Searches for unrelated donors were completed for 86 patients lacking a family donor. Matching criteria were either the optimal level of 10 alleles or a one-HLA class I mismatch as a second choice. Rates of successful search reach 85% for patients (n=20) who express at least one common five-allele (HLA-A/B/Cw/DRB1/DQB1) haplotype, but also 77% for more patients (n=53) who express at least one of the 20 most frequent three-allele (HLA-A/B/Cw) haplotypes. Success rates are clearly less (39%) in patients lacking these haplotypes. The use of these data to delineate search strategies is discussed.

Immune reconstitution after haematopoietic transplantation with two different doses of pre-graft antithymocyte globulin.

Antithymocyte globulin is widely used before haematopoietic transplantation with HLA-matched unrelated donors or mismatched relatives to prevent rejection and graft-versus-host disease (GVHD). However, optimal dosage is still under debate. Thirty-one consecutive children, mainly with haematological malignancies, were transplanted in a single institution with such donors, selected by HLA-A -B compatibility by serology and DRB1* by DNA typing. Antithymocyte globulin (Thymoglobuline; Sangstat) was infused at days -3, -2, -1. Total dosage varied: 16 patients received a median of 7.5 mg/kg (2.5 to 10.5: low-dose group), and 15 a median of 15.5 mg/kg (14.4 to 19.4: high-dose group). Post-transplant GVHD prophylaxis consisted of cyclosporine, short-course methotrexate and steroids. CD3(+), CD4(+) and CD19(+) cell reconstitution was slower in the high-dose group. Median time to reach 100 CD4(+) cells was 8 months vs 4 months (P = 0.03). Median time to normal CD19(+) cells was 16 months vs 8 months (P = 0.01). CD16(+)CD56(+) and CD8(+) cell reconstitution was similar. Nine patients in the high-dose group and two in the low-dose group experienced life-threatening opportunistic infections (P = 0.009). Although obtained from a limited number of patients, our data suggest that a higher pre-graft dose of antithymocyte globulin may negatively influence immune reconstitution.

Characterization of the B*5002-Cw*0602-DRB1*0406-DQB1*0402 Haplotype: impact on the unrelated-bone marrow donor search strategy.

Improvements in HLA-typing by DNA-based methods now allow accurate genotyping of unrelated bone marrow (UBM) donors and recipients and also definition of haplotypes. In this regard, B*5002 has been predicted in linkage disequilibrium with Cw*0602, DRB1*0406 and DQB1*0402 based on the frequency of allele coexistence. Here, we confirm this assumption by HLA genotyping of four informative families and three unrelated individuals. In the four families, the extended haplotype HLA-B*5002, -Cw*0602, -DRB1*0406, DQB1*0402 can be definitely assigned by the mode of heritance. Furthermore, this association of alleles was also found in the three B*5002 unrelated individuals. Knowledge of the frequent linkage disequilibrium of these rare alleles can improve UBM donor search strategies.

Naive CD4+ T cell subset is the predominant population involved in cytolysis through Fas-ligand in human at birth.

Here we show that Fas ligand, a death factor that plays a key role in peripheral T cell tolerance and homeostasis, can be induced in peripheral blood mononuclear cells from both newborns (cord) and adults. Indeed, stimulation of both populations by ionomycin and phorbol 12-myristate 13-acetate or through the T cell receptor (anti-CD3) leads to a selective lysis of targets transfected with Fas but not of the parental cell line. This lysis clearly involves a Fas-based mechanism inasmuch as it was correlated with the appearance of Fas ligand transcripts and competitively inhibited by a Fas-Fc fusion protein. Yet, the use of separated T cell populations revealed that both CD4+ and CD8+ T cells express this function in adults whereas the CD4 subset is primarily involved in lysis by a Fas-based mechanism in cord. Altogether these results suggest that distinct T cell subsets might be recruited to exert Fas-based lysis during T cell ontogeny. Furthermore, it is tempting to speculate that CD4+ T cells are primarily involved in peripheral T cell homeostasis and tolerance in early life because they are committed to exert Fas-based lysis before any antigen-priming. Finally, the model of Fas ligand exploration described herein might be of great help for the identification of Fas ligand dysregulation in various diseases in infants, including those patients with autoimmune lymphoproliferative syndrome in which Fas is functional.

Defective IL2 gene expression in newborn is accompanied with impaired tyrosine-phosphorylation in T cells.

Here we confirmed that IL2 mRNA expression in CD3-stimulated T cells is defective at birth. Because protein-tyrosine phosphorylation is an important part of signaling through CD3 and plays a key role in IL2 transcription, we further investigated whether impaired IL2 response to CD3 in newborns would be accompanied with an alteration of tyrosine phosphorylation. In this purpose, CD3-induced tyrosine phosphorylation was evaluated comparatively in newborn and adult cells by immunoblotting of total cellular extract with an antiphosphotyrosine antibody. Results show that, in both peripheral lymphocytes or purified CD4 T cells from both cord and adult, CD3 stimulation could induce small even significant tyrosine-phosphorylation. Tyrosine phosphorylation occurs as soon as 2' following CD3 ligation and was still evident up to 15-20'. Yet, by using a highly sensitive method to analyze CD3-induced accumulation of phosphorylated substrates, which consisted in adding pervanadate, an inhibitor of phosphatases, during the last 2 min of CD3 stimulation, we showed that the intensity of tyrosine phosphorylation was clearly decreased in cord cells. From these results, it is tempting to speculate that suboptimal capacities of cord T cells to up-regulate tyrosine phosphorylation might contribute to defective IL2 production in neonates.

Intermittent hemophagocytic lymphohistiocytosis is a regular feature of lysinuric protein intolerance.

We describe 4 cases of lysinuric protein intolerance, which all fulfilled the diagnostic criteria for hemophagocytic lymphohistiocytosis. Mature histiocytes and neutrophil precursors participated in hemophagocytosis in the bone marrow. Moreover, serum levels of ferritin and lactate dehydrogenase were elevated, hypercytokinemia was present, and soluble interleukin-2 receptor levels were increased up to 18.6-fold. The diagnosis of lysinuric protein intolerance should therefore be considered in any patient presenting with hemophagocytic lymphohistiocytosis.

Cytokine mRNA and protein expression in a mixed leukocyte reaction before and after allogeneic transfusions. Groupe Coopératif de Transplantation d'Ile de France.

BACKGROUND: The precise mechanism by which pretransplant blood transfusions may favorably influence the graft outcome in human transplantation remains unknown. Here, we explored whether the mechanism might be related to an alteration of cytokine response to transplantation antigens. METHODS: Eight patients awaiting kidney transplantation were selected to receive a single planned pretransplant blood transfusion. Before transfusion and 7 days after transfusion, peripheral blood mononuclear cells from these patients were isolated and in vitro stimulated in a one-way mixed leukocyte reaction (MLR) by using allogeneic fixed Epstein Barr virus-transformed cells as stimulators. RESULTS: The use of a semiquantitative reverse-transcriptase polymerase chain reaction cycle technique to analyze cytokine mRNAs revealed that allostimulation by donor cells clearly induced accumulation of interleukin (IL)-2, IL-4, interferon (IFN)-gamma, and IL-10 mRNA in peripheral blood mononuclear cells collected both before and after transfusion (eight of eight patients). However, both T helper 1 (IFN-gamma) and T helper 2 (IL-4) cytokine responses were more elevated after transfusion in eight of eight patients, as were IL-2 responses in five of eight patients. Such up-regulation of cytokine responses by transfusion was mostly directed against blood donor cells. Indeed, after stimulation by third-party cells, this up-regulation was both inconstant (two of three patients) and of less intensity, and no change was detected after stimulation by autologous cells (three of three patients). CONCLUSIONS: That IL-2, IL-4, and IFN-gamma responses to donor cells were increased by transfusion was further supported by results on cytokine secretion showing increased levels of IL-2 (P < 0.05), IFN-gamma (P = 0.054), and IL-4 (P < 0.05) proteins in supernatants of posttransfusion MLR as compared with pretransfusion MLR. In contrast, transfusion-induced changes in the amount of IL-10 mRNAs were not obvious and were quite variable from one patient to another.

Expansion of polyclonal B-cell precursors in bone marrow from children treated for acute lymphoblastic leukemia.

In a series of 12 patients (mean age: 3 years at diagnosis) receiving chemotherapy for acute lymphoblastic leukemia, bone marrow examinations performed during hematopoietic recovery following treatment-induced agranulocytosis or completion of maintenance treatment showed at least 15% of non malignant immature cells which were sometimes hardly distinguishable from leukemic cells. No comparable data was observed in patients treated with G-CSF. The cytological features of these cells as well as their immunophenotyping were defined. Results showed that the majority of cells expressed HLA-DR, CD19, CD10 and cytoplasmic IgM but not the CD34 markers. This predominant and homogeneous pre-B cell population which likely represents the expansion of a minor population detectable in normal bone marrow is phenotypically indistinguishable from leukemic cells. The pattern of IgH gene rearrangements studied by PCR amplification of the CDRIII region showed that these cells were polyclonal. Except in one patient, minimal residual disease was not detected using probes specific for IgH or TCR gene rearrangement of the malignant clone. In children during the hematopoietic recovery after chemotherapy, immature marrow cells in great numbers, even with an highly homogeneous immunophenotype identical to the malignant clone's, are not sufficient for the diagnosis of relapse.

Cytotoxic T lymphocyte changes after HLA-DR match and HLA-DR mismatch blood transfusions.

In the present study, we analyzed transfusion-induced cytolytic T lymphocyte (CTL) changes in patients who received either a one-HLA-DR-match or a zero-HLA-DR-match pretransplant blood transfusion. Twenty-four nonimmunized naive patients awaiting kidney transplantation received one planned, HLA-typed blood transfusion. The frequencies of CTL precursors (CTLp) directed against blood donor cells and controls were evaluated before and sequentially at days 7, 28, and 60 after transfusion. Results showed that sharing one HLA-DR between donor and recipient did not prevent CTL sensitization. Indeed, (1) An increase of donor-specific CTLp frequencies was observed in 8 of 11 patients who received a zero HLA-DR match (71%), as well as in 9 of 13 patients who received a one-HLA-DR-match (69%) transfusion. (2) This increase occurred with similar kinetics in the two groups, as it was highly significant 7 days after transfusion (P=0.002 and P=0.0035 in the first and second groups, respectively) but transient; CTLp frequencies returned to pretransfusion levels by day 60 after transfusion in both groups. (3) Finally, the magnitude of this increase was similar in the DR-match and DR-mismatch groups. Thus, if it is confirmed, in clinical practice, that one DR match between the blood donor and the patient would improve the tolerance effect of pretransplant blood transfusion, this phenomenon is unlikely to be related to the prevention of CTL sensitization.

Binding analysis of 95 HIV gp120 peptides to HLA-DR1101 and -DR0401 evidenced many HLA-class II binding regions on gp120 and suggested several promiscuous regions.

To identify HLA-DR-binding peptides within the human immunodeficiency virus (HIV)-1 proteins. 95 overlapping synthetic peptides representing the entire sequence of gp120-LAI were screened for their capacity to bind to two HLA-DR molecules with distant sequences (DR0401 and DR1101). By using a cell surface competitive binding assay, 56 DR-binding peptides were identified, of which 35 bound to both DR1101 and DR0401. A highly significant concordance was evidenced by statistical analysis between binding of peptides to one and to the other DR molecule, suggesting a high proportion of promiscuity among gp120 peptides, even though no clear sequence pattern accounting for such promiscuity was found. DR-binding peptides were located along the entire gp120 sequence. Yet, the majority of them (42 among 56) were concentrated in seven multiagretopic regions that were arbitrarily defined as regions containing four or more overlapping continuous peptides binding to DR1011 and/or DR0401. A good correlation was found between DR-binding regions or DR-binding peptides defined in this study and promiscuous T helper gp120 epitopes previously described in seropositive individuals. All these results suggest that the identification of multiagretopic DR-binding regions may be a great help for the predicition of protein determinants that have the likelihood of being promiscuous T helper epitopes in humans.

Binding of ALA-substituted analogs of HA306-320 to DR1101, DR1301, and DR0402 molecules: correlation of DR-peptide interactions with recognition by a single TCR.

We tested the hypothesis that a cross-reactive T-cell clone could recognize HA306-320 peptide complexed to autologous HLA-DR1101, and also to allogenic HLA-DR0402 and HLA-DR1301 molecules, because of similar orientations of HA306-320 side chains in the groove of the three DR molecules. To approach peptide orientations in each HLA groove we compared the capacity of Ala-monosubstituted analogs to bind and be presented by DR1101, DR0402, and DR1301. Results indicated that the orientation of HA306-320 in DR1101 was grossly similar to the known orientation of HA307-319 in DR0101. Data suggested many similarities in peptide orientations in DR0402 and DR1301 as well. However, differences in binding were also observed. Ala substitution of Y309 had much less effect on peptide binding to DR1301 and DR0402 than to DR1101 and Ala-substitution of T314 increased affinity for DR1301 but not for DR1101 and DR0402. These alterations of peptide-DR interactions were probably communicated to the upper peptide surface. Indeed, the levels of T-cell clone reactivities against analogs mutated at positions predicted to face the TCR were lower when complexed to allogeneic DR molecules than when complexed to DR1101. Yet these epitopic alterations are likely subtle, since the decreased reactivity of the clone to allogeneic molecules could be compensated by peptide substitution at Y309, predicted to face the MHC.

Sharing of four DR-beta sequence motifs between HLA-DRB1*1601 and DRB1*1101 correlates with frequent degenerate T-cell recognition of HA306-320 peptide complexed to these two molecules.

This paper shows that the seven HA306-320 specific T-cell clones isolated from one individual recognize the peptide complexed to both autologous HLA-DRB1*1101 and allogeneic HLA-DRB1*1601 (or DRB5*0201) molecules. For each T-cell clone, a single T-cell receptor (TCR) is involved in the recognition of these two different peptide-DR complexes as evidenced by cold target competition experiments. Yet, the seven T-cell clones express several different TCRs as judged by V beta-J beta usage and fine specificities. Furthermore, one representative clone has the same fine specificity for HA306-320 analogues mutated at epitopic residues irrespective of the use of DR1101 or DR1601 APC. These results suggest that structural differences between DRB1*1101 and DRB1*1601 (or DRB5*0201) do not dramatically influence the orientation of HA306-320 in the grooves such that most residues interacting with TCRs are conserved. In another individual, the same pattern of restriction, i.e. DR1101 + DR1601, was found for several HA306-320 specific clones. Two additional patterns, DR1101 + DR0801 and DR1101 + DR0801 + DR1601, were identified. By comparing DR sequences the authors found that DRB1*1101 and DRB1*1601 share four important motifs, i.e. beta 85-86, beta 67-71, beta 57 and beta 28-31 supposed to line three distinct HLA-DR pockets. Three of these motifs are also shared with DRB1*0801. All the results further support that the motif similarities allow the peptide to adopt very similar orientations in the cross-reacting DR molecules.

Preferential usage of the T-cell receptor by influenza virus hemagglutinin-specific human CD4+ T lymphocytes: in vitro life span of clonotypic T cells.

Human helper T-cell (Th) responses to influenza A virus were studied by analyzing T-cell receptor V beta gene diversity in hemagglutinin-specific Th lymphocytes. The T-lymphocyte population from peripheral blood became quickly oligoclonal when stimulated in vitro with the HA306-329 peptide, and T-cell receptor V beta 3 genes were mainly expanded. Moreover, specific junctional region oligonucleotide probes corresponding to hemagglutinin-specific clones were used to estimate temporal diversity during antigenic stimulations.