CD4(+) type 1 T regulatory (Tr1) cells are induced in the periphery and have a pivotal role in promoting and maintaining tolerance. The absence of surface markers that uniquely identify Tr1 cells has limited their study and clinical applications. By gene expression profiling of human Tr1 cell clones, we identified the surface markers CD49b and lymphocyte activation gene 3 (LAG-3) as being stably and selectively coexpressed on mouse and human Tr1 cells. We showed the specificity of these markers in mouse models of intestinal inflammation and helminth infection and in the peripheral blood of healthy volunteers. The coexpression of CD49b and LAG-3 enables the isolation of highly suppressive human Tr1 cells from in vitro anergized cultures and allows the tracking of Tr1 cells in the peripheral blood of subjects who developed tolerance after allogeneic hematopoietic stem cell transplantation. The use of these markers makes it feasible to track Tr1 cells in vivo and purify Tr1 cells for cell therapy to induce or restore tolerance in subjects with immune-mediated diseases.

Key Points• Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation.• Haploidentical hematopoietic stem cell transplantation for hematologic malignancies.Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF-primed BM transplantation from an haploidentical family donor. Patients were transplanted in first or second complete remission (CR, standard-risk: n ‫؍‬ 45) or in > second CR or active disease (high-risk: n ‫؍‬ 35). The same regimen for GVHD prophylaxis was used in all cases. The cumulative incidence (CI) of neutrophil engraftment was 93% ؎ 0.1%. The 100-day CIs for II-IV and III-IV grade of acute GVHD were 24% ؎ 0.2% and 5% ؎ 0.6%, respectively. The 2-year CI of extensive chronic GVHD was 6% ؎ 0.1%. The 1-year CI of treatment-related mortality was 36% ؎ 0.3%. After a median follow-up of 18 months, 36 of 80 (45%) patients are alive in CR. The 3-year probability of overall and disease-free survival for standard-risk and high-risk patients was 54% ؎ 8% and 33% ؎ 9% and 44% ؎ 8% and 30% ؎ 9%, respectively. In multivariate analysis, disease-free survival was significantly better for patients who had standard-risk disease and received transplantations after 2007. We conclude that unmanipulated, G-CSF-primed BM transplantation from haploidentical family donor provides very encouraging results in terms of engraftment rate, incidence of GVHD and survival and represents a feasible, valid alternative for patients with high-risk malignant hematologic diseases, lacking an HLA identical sibling and in need to be urgently transplanted. (Blood. 2013;121(5): 849-857)

Summary:Twenty-six transplanted thalassemic patients out of 295 analyzed, showed the presence of persistent mixed chimerism, over a period of time varying between 2 and 11 years after BMT. Despite the presence of large numbers of residual host cells, these transplanted thalassemic patients no longer require red blood cell transfusions and have a functional graft, producing sufficient levels of hemoglobin A ranging from 8.3-14.7 g/dl. These ex-thalassemic patients with persistent mixed chimerism, although they did not achieve complete donor engraftment are no longer exposed to the risk of graft rejection. The mechanisms underlying this apparent state of tolerance or education in these patients are at the present time unknown. However, these observations may be useful for physicians involved in defining optimal strategies for clinical gene therapy, in utero hematopoietic stem cell transplantation and adoption of less toxic conditioning regimens in mini-transplantation. Bone Marrow Transplantation (2000) 25, 401-404. Keywords: persistent mixed chimerism after BMT In the early days of allogeneic bone marrow transplantation it was thought that ablation of all of the host stem cells was required to establish conditions for complete marrow engraftment of donor cells, ie complete chimerism (CC). Persistence of some host cells in the marrow along with donor cells, ie mixed chimerism (MC), was thought to presage rejection of the donor graft. 1 However, we have observed that MC is not unusual in our group of transplanted thalassemics that now numbers almost 900. While MC occasionally evolves into graft rejection, other patients seem to move into a state of persistent, stable MC where their Hb levels are generally sufficient to allow good quality of life without any red blood cell (RBC) transfusion support. 2 These observations led us to analyze MC in a group of 295 ex-thalassemic patients after bone marrow transplantation, all with a minimum follow-up of 2 years, in order to determine the incidence of MC at different periods after BMT and follow the evolution of MC over the time.Some of 26 patients with persistent MC had a proportion of donor engrafted cells no more than 20-30%, suggesting that low numbers of donor erythroid precursor cells are sufficient to produce high levels of beta-globin chain synthesis and hemoglobin. [3][4] These observations make possible the opportunity of investigating if the goal of 'cure' of thalassemic patients, aiming for establishing persistent MC rather than CC using less toxic myeloablative programs and consequently less regimen-related toxicity (RRT) may be achieved. [5][6][7]

The high probability of cure with little early or late morbidity and mortality suggests that patients with class 1 thalassemia who have HLA-identical donors available should be treated by bone marrow transplantation. However, this was not a controlled trial, so we cannot directly compare the outcome with that of conventional treatment.

When prepared for transplantation with busulfan (BU) 14 mg/kg and cyclophosphamide (CY) 120 to 160 mg/kg, patients with thalassemia in risk class 3, aged younger than 17 years, who receive transplants from HLA-identical donors, had a 30% incidence of transplant rejection with recurrence of thalassemia. This, relatively poor, outcome was ascribed to insufficient immune suppression or to inadequate eradication of the thalassemic marrow, or both. In an attempt to enhance both immune suppression and eradication of the thalassemic clones, hydroxyurea, azathioprine, and fludarabine were added to the BU and CY. This regimen, called protocol 26, was applied to 33 consecutive patients with class 3 thalassemia aged younger than 17 years and was well tolerated with 93% survival. The incidence of recurrent thalassemia after the transplantation decreased from 30%

HLA genotyping by next‐generation sequencing is now widely performed. We aimed at evaluating the performance of the One Lambda AllType kit using Thermo Fisher Scientific reagents on the Ion S5 XL platform. Reads were analyzed using the TypeStream Visual software. We performed 15 runs between April and September 2018 to type DNA at the HLA‐A/B/C/DRB1/3/4/5/DQA1/DQB1/DPA1/DPB1 loci from 340 samples and 15 positive controls. We observed only seven (0.1%) critical mistakes among the 6009 alleles typed, corresponding to two allele dropouts, one false heterozygous typing assignment, and four phasing abnormalities. Among the 1793 presumably new alleles detected by the analysis software, 11 displayed exon mismatches, of which nine were confirmed as new alleles and two had been described previously. Intron mismatches were observed among the remaining presumably new alleles, of which 371 were considered as probably new, and 1411 were rejected for at least one sequence feature such as homopolymers (n = 1206), nucleotide doublet repeats (n = 26), low read depth (<200 reads, n = 93), high background (>20%, n = 79), or phasing abnormalities (n = 7). A comparison of the AllType results with those obtained using other methods at the second‐field resolution level showed 99.5% (1497/1504) concordance for the HLA‐A/B/C/DRB1/DQB1/DPB1 loci. Similar agreement was observed between the HLA‐C or HLA‐DRB3/4/5 results and common linkage disequilibrium, with 96.6% (657/680) and 97.2% (530/545) concordance, respectively. Therefore, the AllType kit used with the Ion S5 XL platform displayed satisfactory performance for HLA typing in current clinical practice.

IL-10-producing CD4+ type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL-10 in the absence of IL-4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T-cell responses mainly via cytokine-dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- and perforin (PRF)-dependent mechanism that requires HLA class I recognition, CD54/lymphocyte function-associated antigen (LFA)-1 adhesion, and activation via killer cell Ig-like receptors (KIRs) and CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1-cell activation, is necessary for defining Tr1-cell target specificity. We also showed that high frequency of GZB-expressing CD4+ T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10-producing CD4+ T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.

With the aim to individuate alleles that may reflect a higher susceptibility to the disease, in the present study we analyzed the HLA allele frequency distribution in a group of 99 Italian patients affected by a severe or extremely severe form of COVID-19. After the application of Bonferroni's correction for multiple tests, a significant association was found for HLA-DRB1*15:01,-DQB1*06:02 and-B*27:07, after comparing the results to a reference group of 1017 Italian individuals, previously typed in our laboratory. The increased frequencies observed may contribute to identify potential markers of susceptibility to the disease, although controversial results on the role of single HLA alleles in COVID-19 patients have been recently reported.