Characterization of the novel HLA-DRB3*02:192 allele by sequencing-based typing.

HLA-DRB3*02:192 differs from DRB3*02:02:01:02 by one nucleotide substitution in codon 204 in exon 4.

Characterization of the novel HLA-DQA1*05:05:14 allele by sequencing-based typing.

HLA-DQA1*05:05:14 differs from HLA-DQA1*05:05:01:04 by one nucleotide substitution in codon -8 in exon 1.

Identification of the novel HLA-DPA1*02:110:02 allele by next-generation sequencing.

The novel HLA-DPA1*02:110:02 allele differs from HLA-DPA1*02:01:01:06 by one nucleotide substitution in exon 4.

Characterization of the novel HLA-DPA1*01:03:40 allele by sequencing-based typing.

HLA-DPA1*01:03:40 differs from HLA-DPA1*01:03:01:02 by one nucleotide substitution in codon 60 in exon 2.

A new HLA-B allele, HLA-B*07:422.

The novel allele HLA-B*07:422 differs from HLA-B*07:02:01:01 by one nucleotide substitution in exon 4.

Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease.

BACKGROUND: Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However, since in most of the studies reported in literature the engraftment state was observed in the nucleated cells, in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit - erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow. DESIGN AND METHODS: The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit - erythroid colonies singly picked out after 14 days of incubation. RESULTS: The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%, 46%, 15% and 25% at day 1364, 1385, 1314 and 932, respectively. Similar results were obtained for the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit - erythroid colonies. In contrast, on the same days of observation, the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%, 100%, 73% and 90%. Conclusions Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant, although the biological mechanisms underlying these observations need further investigation.

A new HLA-B allele, HLA-B*35:481.

The novel allele HLA-B*35:481 differs from HLA-B*35:05:01 by two nucleotide substitution in exon 3.

Characterization of the novel HLA-DQA1*01:02:11 allele by sequencing-based typing.

HLA-DQA1*01:02:11 differs from HLA-DQA1*01:02:01:10 by one nucleotide substitution in codon 201 in exon 4.

Characterization of the novel HLA-DQA1*01:58 allele by sequencing-based typing.

HLA-DQA1*01:58 differs from HLA-DQA1*01:02:01:06 by one nucleotide substitution in codon 46 in exon 2.

Characterization of the novel HLA-DQA1*03:01:06 allele by sequencing-based typing.

HLA-DQA1*03:01:06 differs from HLA-DQA1*03:01:01:01 by one nucleotide substitution in codon 9 in exon 2.

Characterization of the novel HLA-DRB3*02:02:23 allele by sequencing-based typing.

HLA-DRB3*02:02:23 differs from HLA-DRB3*02:02:01:02 by one nucleotide substitution in codon 218 in exon 4.

A new HLA-DQA1 allele, HLA-DQA1*01:26.

The novel allele HLA-DQA1*01:26 differs from HLA-DQA1*01:01:01:01 by one nucleotide substitution in exon 2.

Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia.

BACKGROUND: Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood. DESIGN AND METHODS: The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor. RESULTS: Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro. CONCLUSIONS: Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.

Characterization of the novel HLA-DRB3*02:96 allele by sequencing-based typing.

HLA-DRB3*02:96 differs from HLA-DRB3*02:02:01:01 by one nucleotide substitution in codon 189 in exon 4.

Characterization of the novel HLA-C*07:708 allele by sequencing-based typing.

HLA-C*07:708 differs from HLA-C*07:01:01:01 by one nucleotide substitution at codon 258 in exon 4.

Characterization of the novel HLA-DRB3*02:02:17 allele by sequencing-based typing.

HLA-DRB3*02:02:17 differs from HLA-DRB3*02:02:01:01 by one nucleotide substitution in codon 179 in exon 3.

The novel HLA-DRB3*02:02:11 allele identified in an Italian individual.

HLA-DRB3*02:02:11 differs from HLA-DRB3*02:02:01:01 by a single synonymous substitution in exon 3'.

Mixed chimerism evolution is associated with T regulatory type 1 (Tr1) cells in a ß-thalassemic patient after haploidentical haematopoietic stem cell transplantation.

In a cohort of ß-Thalassemia (ß-Thal) transplanted with haploidentical-HSCT we identified one transplanted patient characterized by persistent mixed chimerism (PMC) for several months after HSCT. In this unique ß-Thal patient we assessed the donor engraftment overtime after transplantation, the potential loss of the non-shared HLA haplotype, and the presence of CD49b(+)LAG-3(+) T regulatory type 1 (Tr1) cells, previously demonstrated to be associated with PMC after HLA-related HSCT for ß-Thal. The majority of the patient's erythrocytes were of donor origin, whereas T cells were initially mostly derived from the recipient, no HLA loss, but an increased frequency of circulating Tr1 cells were observed. For the first time, we showed that when the proportion of residual donor cells decreases, the frequency of CD49b(+)LAG-3(+) Tr1 cells declines, reaching the levels present in healthy subjects. These findings confirm previous results obtained in transplant related settings for ß-Thal, and supported the central role of Tr1 cells in promoting and maintaining PMC after allo-HSCT.

Split chimerism between nucleated and red blood cells after bone marrow transplantation for haemoglobinopathies.

Previous studies have shown that a stable presence of both donor and recipient haematopoietic derived cells after allogeneic haematopoietic stem cell transplantation (HSCT) occurs in approximately ten percent of the patients affected by ß-Thalassemia. Once achieved this condition, defined as persistent mixed chimerism (PMC), the patients do not require additional red blood cells (RBCs) support and, regardless of the presence in some cases of an extremely low percentage of donor-derived nucleated cells, they are clinically cured by an incomplete, but functional graft. Most of the published papers have, however, investigated the impact of donor engraftment in the nucleated cells rather than in the mature erythrocytes. We have recently published a paper showing that in four long-term transplanted patients affected by hemoglobinopathies, characterized by the presence of few donor engrafted nucleated cells-both in the peripheral blood and in the bone marrow-the majority of the erythrocytes were of donor origin. Moreover we showed that the proportion of donor-derived erythroid precursors, determined by analyzing singularly picked-up burst-forming unit erythroid colonies, was equivalent to that observed in the mature nucleated cells rather than in the red blood cells. These results suggest that in patients characterized by the presence of PMC after HSCT a selective advantage of the donor erythroid precursors maturation might successfully contrast the problems bound to the recipient ineffective erythropoiesis. When genetically modified HSCT will be a possible option for treating Thalassemia Major, the co-existence of the repaired cells with those still expressing the genetic defect will be an expected scenario, not in an allogeneic, but in an autologous environment.

Relationship between mixed chimerism and rejection after bone marrow transplantation in thalassaemia.

BACKGROUND: Thalassaemia is a genetic disease that requires a hypertransfusion regimen to treat the anaemia caused by enhanced red blood cell destruction. The only radical cure for thalassaemia is to correct the genetic defect by bone marrow transplantation from an HLA-identical donor capable of producing and maintaining a normal haemoglobin level in the recipient. Complete donor haematopoiesis is not essential for sustained engraftment and the simultaneous presence of haematopoietic cells of both donor and recipient origin is not a rare event after a transplant. PATIENTS AND METHODS: The evolution of marrow engraftment of 93 transplanted thalassaemic patients, all from Middle East or Asian countries, was monitored by analysis of short tandem repeats. RESULTS: Forty-three of 93 (46%) patients experienced a status of mixed chimerism early after bone marrow transplantation. Results of further engraftment analysis in these patients showed in 27 complete donor engraftment; rejection occurred in seven, while eight maintained the presence of both host and donor-derived cells. Interestingly, five out of the seven patients who rejected their transplant showed more than 25% residual host cells early after transplantation. DISCUSSION AND CONCLUSION: Our study confirmed that the presence of large amounts of residual host cells within the first 2 months after a transplant is a risk factor for graft rejection also in a group of patients with wide ethnic heterogeneity, irregular transfusion regimens and/or poor chelation treatment. Ten percent of the transplanted thalassaemic patients maintained coexistence of donor and recipient cells, showing a stable functional graft, characterized by normal production of beta globin chains and high levels of haemoglobin. A mechanism responsible for peripheral tolerance induction, such as the production of specific regulatory T-cell clones, seems to play a key role in the induction of long-term tolerance after the transplant.