Bernard Chapuis

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells.

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene‐targeting therapies since they differentiate into various cell‐lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non‐hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45+ leukocytes. After culture, leukocytes were replaced by a homogenous layer of adherent CD45− CD14− CD34− CD11b− CD90+ HLA‐ABC+ cells. Culture doubling time (mean = 4 days, range 1.6–6.7 days) was not correlated with patient age (27–81 years, n = 16). Amplified cultures supported long‐term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 106‐fold) were not age‐related. All 14 clones analyzed (from five patients, ages 27–78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM‐derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC‐based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC. J. Cell. Physiol. 198: 110–118, 2004.

Invasive aspergillosis. Clinical features of 35 proven cases at a single institution.

Thirty-five patients with clinical features and histologically or microbiologically proven infection met predetermined stringent criteria for invasive aspergillosis over a 5-year period at our institution. Underlying conditions included hematologic malignancy, solid tumor, bone marrow and solid organ transplantation, and immunosuppressive therapy. The majority of patients (94%) presented with respiratory symptoms and abnormal pulmonary chest radiography; only 40% had neutropenia at time of infection. Invasive aspergillosis was suspected in only 21 cases (60%). Concomitant infections were present in 83% of patients. Half of patients had pathogenic or potentially pathogenic microorganisms other than Aspergillus spp. isolated from pulmonary specimens at time of aspergillosis. Aspergillus spp. were recovered from sputum in 75% of patients and from bronchoalveolar lavage in only 52%. Invasive aspergillosis is an unexpectedly unrecognized disease with poor outcome; overall mortality was 94% in our series. The lack of sensitivity of diagnostic procedures, together with the high frequency of concomitant infections, delays the time of diagnosis. Early diagnostic tests are needed, and presumptive antifungal therapy among high-risk patients is mandatory.

CD45 isoform phenotypes of human T cells: CD4(+)CD45RA(-)RO(+) memory T cells re-acquire CD45RA without losing CD45RO.

We have studied the alterations in CD45R phenotypes of CD4+CD45RA–RO+ T cells in recipients of T cell‐depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re‐expression of CD45RA by former CD4+CD45RA– T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA+ T cells through the thymic pathway. We found that CD4+CD45RA–RO+ recipient T cells could re‐express CD45RA but never reverted to a genuine CD4+CD45RA+RO– naive phenotype. Even 5 years after transplantation, they still co‐expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower (∼ 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4+CD45RA–RO+ T cells may re‐express CD45high isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co‐expression of CD45RO. Therefore, it is likely that the long‐lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.

Total body irradiation before allogeneic bone marrow transplantation: is more dose better?

PURPOSE This study was performed to retrospectively assess the potential influence of total-body irradiation (TBI) dose on overall survival in patients undergoing allogeneic bone-marrow transplants (BMT) for hematologic malignancies. METHODS AND MATERIALS Between October 1984 and December 1996, 116 patients were conditioned with high-dose chemotherapy and fractionated TBI before allogeneic BMT. The median age was 34 years (range 3-60). The TBI dose was given in 6 fractions, twice-a-day, over 3 days before BMT. The total dose was 10 Gy in 24 patients, 12 Gy in 66 patients, and 13.5 Gy in 26 patients. RESULTS TBI dose was inversely correlated with overall survival. Five-year survival was 62% for patients conditioned with 10 Gy, 55% for patients conditioned with 12 Gy, and 46% for patients conditioned with 13.5 Gy. Age at BMT was also independently correlated with survival, with the best outcome for patients < 40 years old. CONCLUSION A TBI dose (fractionated) > 10 Gy may not necessarily be associated with a better outcome in patients undergoing allogeneic bone-marrow transplant for hematologic malignancies.

HA-1 and the SMCY-derived peptide fidsyicqv (H-Y) are immunodominant minor Histocompatibility antigens after bone marrow transplantation.

BACKGROUND Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.

Donor Lymphocyte Infusion for the Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

The results of donor lymphocyte infusion (DLI) for treatment of relapse after bone marrow transplantation (BMT) are reviewed. Durable complete remission can be achieved at the molecular level for a majority (more than 70%) of patients with CML, when treated at early relapse. Results are less favourable for acute leukemias, although useful responses have been reported. Data are scarce though promising for myelodysplastic syndromes and multiple myeloma. Major treatment-associated toxicities are GVHD and bone marrow aplasia. The latter complication can be predicted by evaluating the level of residual donor-derived hematopoiesis. Modification of infused cells (CD8 negative selection or transduction with a suicide gene), addition of peripheral blood stem cells, and early implementation of escalating doses may counteract the complications and increase the response rate. Response rate is variably influenced by the presence of chronic GVHD after initial BMT, T-cell depleted BMT, underlying disease and stage at relapse, and the level of mixed chimerism. DLI is a direct demonstration of the graft-versus-leukemia effect (GVL). Because GVL after BMT is sometimes the predominant cause of cure, it may be advisable in such situations to redirect the conditioning regimens for BMT towards engraftment and less immediate cytotoxicity.

T-cell repertoire complexity after allogeneic bone marrow transplantation

We analyzed the T-cell repertoire in patients transplanted with bone marrow from an HLA identical sibling by determining the TCR diversity through Vbeta-CDR3-size spectratyping with Vbeta/Cbeta- and Vbeta/Jbeta-specific primers. Using the Vbeta/Cbeta primers, we observed limited TCR diversity only in recipients of a T-cell-depleted graft, whereas the TCR diversity of patients transplanted with an unmanipulated graft seemed to be indistinguishable from the one of a normal individual. However, with Vbeta/Jbeta-specific primers, increase of the resolution by approximately 10-fold also allowed the detection of imbalances in the TCR repertoire of recipients of an unmanipulated graft. This demonstrates that when high numbers of T cells are cotransfused with marrow, the TCR repertoire is more complete but still not as complete as in normal individuals, thereby emphasizing the important role of coinfused mature T cells in the restoration of the T-cell compartment after bone marrow transplantation.

Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR αβ lymphocytes

A subset of CD8+ T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8+ T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7+ memory cells and CCR7– effector cells. Since NKG2A can only be induced on naive CD8+ T cells while CD94– memory cells are refractory, it is likely that commitment to the CD94:NKG2A+ subset occurs during the first encounter with antigen. CCR7+CD94:NKG2A+ T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN‐γ. These cells occur as a separate CD94:NKG2A+ T cell lineage with a distinct TCR repertoire that differs from that of the other CD8+CD94– T cells activated in situ.

Impact of high-resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland

It is currently unknown what degree of human leukocyte antigen (HLA)-mismatching is acceptable in unrelated donor hematopoietic stem cell transplantation (UD-HSCT). Mismatches at some loci may be more permissive than others. We have analyzed the effect of high-resolution HLA-matching on outcome of all 214 consecutive recipients of UD-HSCT carried out in Switzerland. All typing was by the Swiss reference laboratory. Donor–recipient pairs were HLA-10/10 matched (n=130) or mismatched for either HLA-A/-B/-DRB1/multiple loci (n=33; (HLA-A/-B=10); (-DRB1=8); (multiple=15)); HLA-C (n=29) or HLA-DQ/-DRB3 (n=22; (DQ=16); (-DRB1=6)). The median follow-up was 32 months. Survival probabilities (±95% confidence interval) at 3 years were 57 (±10)% for recipients of HLA 10/10-matched transplants, 53 (±22)% for recipients of HLA-DQ/-DRB3-mismatched transplants, 44 (±20)% for recipients of HLA-C-mismatched transplants and 0% for recipients of transplants mismatched at HLA-A/-B/-DRB1/multiple loci (P<0.0001). In multivariate analyses, HLA compatibility was the variable most significantly associated with survival and treatment-related mortality. We found important differences in survival in recipients of UD-HSCT with best results for transplants from 10/10 matched donors. Single mismatches at HLA-DQ/-DRB3 were well tolerated, mismatches at HLA-C had intermediate results and mismatches at HLA-A/-B/-DRB1/multiple loci resulted in poor survival.

Increased frequency of DR2 in patients with aplastic anaemia and increased DR sharing in their parents

68 patients with aplastic anaemia, their parents and healthy siblings were typed for HLA‐A, B and DR antigens. Among the patients there is an overrepresentation of DR2. The parents of affected children show an increased compatibility at the DR locus. This situation could favour the expression of recessive gene(s) involved in haematopoiesis and located in the HLA locus.