Relevance of KIR gene matching in unrelated hematopoietic stem cell transplantations.

The aim of this collaborative study was to evaluate the impact of killer cell immunoglobulin-like receptor (KIR) gene disparities on unrelated hematopoietic stem cell transplantations (HSCT) outcome. To address this question, we have determined the presence or absence of 14 functional KIR genes in HLA-matched (n= 164) or HLA-mismatched (n= 100) donor/recipient pairs and investigated whether KIR gene disparities had an impact on both the occurrence of acute graft-vs-host-disease incidence and overall survival. In a univariate analysis, our preliminary results suggest a detrimental effect of a few KIR gene disparities on patient survival that should be avoided in unrelated HSCT.

Induction of transplantation tolerance in humans using fetal cell transplants.

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.

Outcome and long-term follow-up of alloreactive donor lymphocyte infusions given for relapse after myeloablative allogeneic hematopoietic stem cell transplantations (HSCT).

In order to study efficacy, toxicity and the long-term results of donor lymphocyte infusions (DLI), we retrospectively analyzed DLI given for relapse after conventional allogeneic hematopoietic stem cell transplantation (HSCT) in 30 patients with a median delay of 107.5 months after transplant and 58 months after DLI. After DLI, 15 patients established full donor chimerism, three patients developed grade III and one grade IV acute GVHD. A total of 15 patients achieved a disease response. Among the 14 patients with chronic myeloid leukemia (CML), 11 are alive at the last follow-up: five are in complete molecular response (CMR) and two in complete cytogenetic response (CCR) with no other intervention after DLI, three in CMR after imatinib mesylate given after DLI and one in complete hematological response after imatinib mesylate and reduced-intensity conditioning allogeneic SCT performed after DLI. At the time of the last follow-up, 19 (63%) patients died and 11 (37%) remain alive. The 3-year probability of survival for the entire population, CML patients and non-CML patients, was 60, 93, 62% after transplantation, and 48, 80 and 48% after DLI, respectively. A multivariate analysis demonstrated a significantly worse survival rate after transplantation for female recipients, advanced disease and acute leukemia before transplantation.

A new HLA-B44 allele (B*44020102S) with a splicing mutation leading to a complete deletion of exon 5.

Using a combination of serology and polymerase chain reaction with sequence-specific primer (PCR-SSP), we have identified in a volunteer bone marrow donor a new HLA class I antigen within the B44 serotype. This human leukocyte antigen (HLA)-B44 variant was typed as 'blank' by microlymphocytotoxicity, whereas the B*44020101 allele was identified by PCR-SSP. A family study confirmed the Mendelian segregation of this blank antigen identified on one of the maternal haplotype transmitted to her child. The DNA sequence of B*44new, now referred to as B*44020102S, performed from the promoter region to the 3' untranslated region revealed a single nucleotide difference (A/G) compared to B*44020101 at the end of intron 4 in the acceptor-splicing site. This mutation leads to an incorrect splicing characterized by the deletion of exon 5 that encodes the transmembrane domain of the HLA antigen. Indeed, full-length complementary DNA sequencing revealed a complete absence of exon 5. Fluorescence-activated cell sorter analysis confirmed the absence of expression of HLA-B44 on the cell surface in the donor, compared to the HLA-B44 positive control. The isoelectric focusing analysis failed to reveal the presence of an HLA-B44 antigen in the donor, showing that no normal HLA-B*44020101 allele was synthesized. The new B*440201010102S allele is a soluble form of B44 without any detectable cell-surface expression. It can thus be considered as a soluble antigen, a form apparently inactive and unfit for antigen presentation.

Outcome of 27 patients with Hurler's syndrome transplanted from either related or unrelated haematopoietic stem cell sources.

Over the last 15 years, we have performed a total of 30 haematopoietic stem cell transplants on 27 children suffering from Hurler's syndrome. These children were of median age 11 months at the time of diagnosis and 25 months at the time of transplantation. The phenotype was severe in 21 cases (78%). The donor was familial in 13 cases: nine genotypically identical, one phenotypically identical father and three HLA-mismatched donors. Unrelated donors were selected in 17 cases: four phenotypically identical and 13 with 1-4 HLA mismatches. The conditioning regimen generally consisted of busulphan 600 mg/m(2) plus cyclophosphamide (Endoxan) 260 mg/kg and cyclosporin with methotrexate for GvHD prophylaxis. Rabbit anti-thymocyte globulin (Thymoglobuline) was given for all unrelated or familial mismatched transplantations. The median nucleated cell dose infused was 6.00 x 10(8) TNC/kg. No bone marrow (apart from one) was T cell depleted. For first transplants, engraftment was observed in 23/27 patients (pts) (85%). Primary graft failure was observed in 4/27 patients (16%), two were retransplanted from an unrelated donor, one with success. Four patients have died. The primary cause of death was infection in three cases (TRM : 11%) and disease progression in one case, after primary graft failure. Of the 23 living patients, two have disease progression after graft failure and 21 (78%) have functional grafts with a favourable long-term outcome after a median follow-up of 4.7 years, having either full or mixed chimaerism. Among surviving patients with functional grafts, 13 (62%) were transplanted from unrelated donors of whom 10 (77 %) had HLA disparities. There was a remarkably low incidence of GvHD. In our experience, haematopoietic stem cell transplantation using an HLA-matched familial donor or an HLA-matched or -mismatched unrelated donor without T cell depletion or irradiation can achieve a favourable outcome in Hurler's syndrome, with improved cognitive function, but with a limited effect on the corneas and skeleton.

Multiple sclerosis-associated retrovirus particles cause T lymphocyte-dependent death with brain hemorrhage in humanized SCID mice model.

A retroviral element (multiple sclerosis-associated retrovirus, MSRV) defining a family of genetically inherited endogenous retroviruses (human endogenous retrovirus type W, HERV-W) has been characterized in cell cultures from patients with multiple sclerosis. Recently, MSRV retroviral particles or the envelope recombinant protein were shown to display superantigen activity in vitro, but no animal model has yet been set up for studying the pathogenicity of this retrovirus. In the present study, the pathogenicity of different sources of MSRV retroviral particles has been evaluated in a hybrid animal model: severe combined immunodeficiency (SCID) mice grafted with human lymphocytes and injected intraperitoneally with MSRV virion or mock controls. MSRV-injected mice presented with acute neurological symptoms and died within 5 to 10 days post injection. Necropsy revealed disseminated and major brain hemorrhages, whereas control animals did not show abnormalities (P <.001). In ill animals, reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed circulating MSRV RNA in serum, whereas overexpression of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma was evidenced in spleen RNA. Neuropathological examination confirmed that hemorrhages occurred prior to death in multifocal areas of brain parenchyma and meninges. Further series addressed the question of immune-mediated pathogenicity, by inoculating virion to SCID mice grafted with total and T lymphocyte-depleted cells in parallel: dramatic and statistically significant reduction in the number of affected mice was observed in T-depleted series (P <.001). This in vivo study suggests that MSRV retroviral particles from MS cultures have potent immunopathogenic properties mediated by T cells compatible with the previously reported superantigen activity in vitro, which appear to be mediated by an overexpression of proinflammatory cytokines.

Cyclosporin A inhibits dendritic cell maturation promoted by TNF-alpha or LPS but not by double-stranded RNA or CD40L.

Here, we investigated the influence of cyclosporin A (CsA) on dendritic cell (DC) generation. With this aim, human DC were propagated from monocytes in serum-free medium with granulocyte macrophage-colony stimulating factor and interleukin-4. DC were then exposed to tumor necrosis factor alpha (TNF-alpha) for maturation. Our results show that CsA does not impair commitment of monocytes into DC, as assessed by loss of CD14 and increase of CD40 and CD1a. However, TNF-alpha-induced DC maturation was affected, as CsA-treated DC expressed lower levels of human leukocyte antigen and costimulatory molecules but sustained levels of CD1a, and less DC expressed DC-lysosomal-associated-membrane-protein (LAMP) and CD83. Accordingly, CsA inhibited the allostimulatory and accessory cell functions of DC. Surprisingly, when other maturation stimuli were used, we observed that CsA significantly inhibited maturation induced by lipopolysaccharides but not by polyribocytidylic acid or CD40 ligand, as assessed by DC phenotype and functions. Therefore, our results indicate that CsA may differentially affect DC maturation.

Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.

A retroviral element (MSRV) defining a family of genetically inherited endogenous retroviruses (HERV-W) has recently been characterized in cell cultures from patients with multiple sclerosis (MS). To address the possible relationship with MS, direct detection of circulating virion RNA was proposed but revealed technically difficult to perform in standardized conditions, in the face of multiple endogenous HERV-W copies. A parallel approach has evaluated MSRV potential pathogenicity in relation to characteristic features of multiple sclerosis, in particular, T-lymphocyte-mediated immunopathology. We report here that MSRV particles induce T-lymphocyte response with a bias in the Vbeta16 chain usage in surface receptor, whatever the HLA DR of the donor. A recombinant MSRV envelope-but not core-protein reproduced similar nonconventional activation. Molecular analysis of Vbeta CDR3 showed that Vbeta16 expansions are polyclonal. Our results thus provide evidence that MSRV envelope protein can trigger an abnormal immune response with similar characteristics to that of superantigens.

Generation of helper and cytotoxic CD4+T cell clones specific for the minor histocompatibility antigen H-Y, after in vitro priming of human T cells by HLA-identical monocyte-derived dendritic cells.

BACKGROUND: There is now convincing evidence that minor histocompatibility antigens (mHag) may play a significant role in the pathogenesis of graft-versus-host disease after HLA-identical bone marrow transplantation. Indeed, in this clinical situation, T cells specific for mHag have been isolated. Here, we addressed whether one can generate mHag-specific T cells in vitro, without any in vivo immunization, among healthy blood donors. METHODS: We used monocyte-derived dendritic cells (Mo-DCs) as antigen presenting cells to induce primary responses between healthy HLA-identical siblings, in mixed lymphocyte dendritic cell reactions (MLDCRs). RESULTS: We show that CD4+ T-cell clones, specific for the mHag H-Y, can be generated in vitro. These clones were derived from a gender-mismatched positive MLDCR pair of HLA-identical siblings and were restricted by the HLA DQB1*0502 molecule. In addition, these CD4+ T clones were also able to lyse allogeneic targets with the same pattern of restriction and specificity than helper function. Finally, acute myeloid leukemia (AML) blast cells were susceptible to lysis by these clones. CONCLUSIONS: Altogether, these results predict that Mo-DCs could help to generate class II-associated, mHag-specific, T-cell lines or clones in vitro, between healthy blood donors, without any need of transplantation-mediated immunization.

Outcome of unrelated bone marrow donor searches in 174 children resulting in 45 patients transplanted in the HLA-matched and -mismatched situation.

The aim of the study was to evaluate the outcome of unrelated bone marrow donor (UBMD) searches initiated for 174 children between 1986 and 1997. Seven patients were registered twice so that a total of 181 UBMD searches took place. At the time of registration, patients suffered from hematological malignancies (n = 121), non-malignant hemopathies (n = 26) and inborn errors (n = 34). Forty-five of the patients (26%) were given transplants from unrelated donors of whom 26 (58%) were HLA-mismatched transplants. Our strategy accepted HLA mismatches at the time of donor selection, using Thymoglobuline as part of the conditioning regimen. Of the 45 patients given unrelated donor transplants, overall survival was 60% at 3 years and concerned 27 patients of whom 14 were from HLA-mismatched donors. Disease-free survival for hematological malignancies was 65% in HLA-matched transplants and 50% in HLA-mismatched transplants. For some patients (16%) urgency led us to use alternative options: non-identical related donor (n = 14), autograft (n = 10), related cord blood transplant (n = 4). For others, UBMD searches were stopped because of favorable evolution (n = 29), death (n = 24), disease progression (n = 22) or other reasons (n = 21). By the end of the follow-up period, 88 patients had died (50%), 75 (43%) are currently alive with or without being transplanted of whom eight are still having active searches and 11 are no longer contactable. In conclusion, in severe disease in children, an immediate transplant from a partially matched donor might be preferable to a prolonged search for a full match. Consequently, this strategy increases the number of patients for whom a suitable donor can be found. We have chosen this option in order not to delay BMT; in so doing we have obtained encouraging results which include high overall survival, low incidence of acute GVHD grade III-IV and low percentage of relapse even in mismatched pairs.

Distinct subsets of dendritic cells resembling dermal DCs can be generated in vitro from monocytes, in the presence of different serum supplements.

We recently demonstrated that dendritic cells (DCs) can be generated from monocytes in the presence of high concentrations of human serum (HS), provided the extra-cellular pH is maintained at plasma values. Because monocyte-derived DCs (Mo-DCs) can also be generated in the presence of fetal calf serum (FCS) or serum-free medium, we have investigated whether these different culture supplements influence DC generation. With this aim, purified monocytes were cultured with GM-CSF plus IL-4 for 6 days and were further exposed to TNF-alpha for 2 additional days, in the presence of HS, autologous plasma (AP), FCS, or X-VIVO 20, a serum-free medium. Our results show that good yields of functionally mature DCs can reproducibly be obtained in the presence of HS or AP, as assessed by CD83 and CD86 up-regulation, dextran-FITC uptake, allogeneic MLR assays and the induction of an autologous response. Interestingly, the effect of serum on DC generation was probably not only quantitative, but also qualitative, since (i) the majority of HS- or AP-cultured DCs expressed CD83 with very weak levels of CD1a, whereas CD83+ DCs cultured in FCS or X-VIVO were mostly CD1a++; (ii) HS- and AP-cultured DCs were much more granular and heterogeneous than FCS- or X-VIVO-cultured DCs, and (iii) the presence of Birbeck-like granules was preferentially observed in HS- or AP-cultured DCs, as assessed by electron microscopy. That these different cells resemble dermal DCs (DDCs) was further supported by the observations that most of the cells displayed intracytoplasmic FXIIIa in the absence of Lag antigen, and expressed E-cadherin at very low levels. Altogether, our results indicate that starting from the same monocytic population, different subsets of DCs can be generated, depending on the culture conditions. Thus, HS or AP favors the generation of fully mature DCs that resemble activated dermal DCs, whereas FCS, or X-VIVO preferentially leads to the generation of less mature CD1a++ dermal-like DCs.

Sequence of four new HLA-Cw alleles: a possible role of interallelic recombination.

In order to extend our current understanding of HLA-C polymorphism, four new alleles have been cloned and sequenced: Cw*1801 in a donor of mixed origin, Cw*02024 in a Senegalese individual, Cw*1205 and Cw*1604 in European Caucasoid blood donors. HLA-Cw*1801, which most likely results from an interallelic recombination between Cw*0704 and 0401 alleles, was not associated with B*8101, but with either B*4403 or B18. The Cw*02024 allele differs from Cw*02022 by a silent mutation in exon 3. Both Cw*1801 and Cw*02024 appear to be rather frequent in populations of African origin but have not yet been detected in Caucasoids. HLA-Cw*1604 differs from Cw*1601 by two nucleotides at codon 156 leading to a Gln to Trp substitution. This new Cw16 subtype was subsequently identified in three additional unrelated families, all of South-European origin, and presented an unusual association with B*4402 in all cases. HLA-Cw*1205 is a composite allele with the alpha1 domain of Cw*1602 and the alpha2 domain of Cw*1203. It appears to be rare, at least in European Caucasoids. Three of these four alleles may have resulted from gene conversion-like or interallelic recombination events.

Correlation between clinical response to interleukin 2 and HLA phenotypes in patients with metastatic renal cell carcinoma.

HLA phenotypes were characterized for 79 patients with metastatic renal cell carcinoma treated with interleukin 2 (IL-2). HLA-A32 was associated with a clinical response (P = 0.025). The frequency of HLA-A3 and/or A32 was higher among responders than non-responders (P = 0.008). Thus, these results suggest that, in vivo, IL-2 may enhance cellular-mediated immunity against a tumour antigen and that some MHC molecules are more efficient than others for endogenous tumour antigen presentation.

Implication of HLA-DR residues at positions 67, 71, and 86 in interaction between HLA-DR11 and peptide HA306-320.

To get further insight into the role of three polymorphic DR residues located in one alpha-helix of the HLA-DR binding groove, we studied how natural substitutions at positions 67, 71, and 86 on DR11 molecules influence MHC binding and/or T cell recognition of peptide HA306-320 and of monosubstituted peptide analogues. Our results show that: 1) Reactivities of all HA306-320-specific T cell clones tested are decreased by DR substitution at position 86 and can even be lowered by additional substitutions at position 71, and at positions 71 plus 67, indicating that these three residues are functionally important. 2) The functional effects of substitutions at positions 67, 71, and/or 86 cannot be explained by a decreased affinity of HA306-320 for the substituted DR11 molecules, as determined in binding assays. 3) More likely, they are explained by modifications of the conformation, orientation, or location of the peptide once bound in the HLA groove, because each individual DR substitution at positions 86, 71, and 67 differentially affects the binding ability of the same panel of 50 monosubstituted analogues. 4) This interpretation is reinforced by the identification of a small set of monosubstituted analogues that can compensate the functional effects of DR substitutions at positions 86, 86 plus 71, or 86 plus 71 plus 67, and thus restore T cell reactivities. All together these results strongly suggest that residues 67, 71, and 86 play a key role in interactions with HA306-320, probably by modifying the way the peptide is bound within the binding groove of HLA-DR11. Using the same DR11.1-restricted clones, we identified putative T cell and DR contact residues of HA306-320 by comparing DR binding and T cell-activating capacity of the peptide analogues. This analysis suggests that: 1) Residues 310, 311, 312, 313, and 316 are putative TCR contacts. 2) Peptide HA306-320 anchors to DR11.1 molecules mainly via residue Y-309, possibly at the vicinity of DR residue 86, whereas peptide residues 315 and 317 constitute minor aggregotopes that would be at the vicinity of DR residues 71 and/or 67. 3) Finally, residues 308, 310, and 314 might also be on the MHC side of the DR-peptide-TCR complex.

Chimerism and tolerance to host and donor in severe combined immunodeficiencies transplanted with fetal liver stem cells.

We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.

The bare lymphocyte syndrome.

The bare lymphocyte syndrome is a combined immunodeficiency resulting from the lack of expression of either class I or class II HLA antigens at the cell surface. The main clinical manifestations are infections of the respiratory or the digestive tract. The immunodeficiency involves the absence of antibody formation and the absence of cell-mediated response, to specific antigen, contrasting with virtually normal transplant immunity to allogeneic determinants. The responsible gene(s) is not born by chromosome 6. The best treatment appears, at the present time, to be in utero stem cell transplantation into the sick fetus, and it may, in the future, be gene therapy.