Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy

Background

CD4⁺ regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies.

Design and Methods

The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported.

Results

Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors.

Conclusions

Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.     

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.     

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.     

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Background

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells.

Design and Methods

We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease.

Results

In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells.

Conclusions

Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.Key words: CD127 positive selection, Treg, alloreactivity, donor lymphocyte infusion     

Hematopoietic stem and progenitor cells are differentially mobilized depending on the duration of Flt3-ligand administration

Background

Flt3-ligand is a cytokine that induces relatively slow mobilization of hematopoietic cells in animals and humans in vivo. This provides a time-frame to study hematopoietic stem and progenitor cell migration kinetics in detail.

Design and Methods

Mice were injected with Flt3-ligand (10 μg/day, intraperitoneally) for 3, 5, 7 and 10 days. Mobilization of hematopoietic stem and progenitor cells was studied using colony-forming-unit granulocyte/monocyte and cobblestone-area-forming-cell assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells was studied by transplantation of 1.5×10⁶ Flt3-ligand-mobilized peripheral blood mononuclear cells into lethally irradiated (9.5 Gy) recipients.

Results

Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of Flt3-ligand produced a steady increase in mobilized progenitor cells. Compared to Flt3-ligand administration for 5 days, the administration of Flt3-ligand for 10 days led to a 5.5-fold increase in cobblestone-area-forming cells at week 4 and a 5.0-fold increase at week 5. Furthermore, transplantation of peripheral blood mononuclear cells mobilized by 5 days of Flt3-ligand administration did not radioprotect lethally irradiated recipients, whereas peripheral blood mononuclear cells mobilized by 10 days of Flt3-Ligand administration did provide 100% radioprotection of the recipients with significant multilineage donor chimerism. Compared to the administration of Flt3-ligand or interleukin-8 alone, co-administration of interleukin-8 and Flt3-ligand led to synergistic enhancement of hematopoietic stem and progenitor cell mobilization on days 3 and 5.

Conclusions

These results indicate that hematopoietic stem and progenitor cells show different mobilization kinetics in response to Flt3-ligand, resulting in preferential mobilization of hematopoietic progenitor cells at day 5, followed by hematopoietic stem cell mobilization at day 10.     

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells

Background

Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants.

Design and Methods

We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8⁺ T and NK cells, and intracellular cytokine staining.

Results

Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8⁺ T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras.

Conclusions

In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8⁺ T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.     

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Background

Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease.

Design and Methods

We analyzed in detail CD8⁺ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8⁺ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens.

Results

Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones.

Conclusions

Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.     

Impact of hematopoietic chimerism at day +14 on engraftment after unrelated donor umbilical cord blood transplantation for hematologic malignancies

Background

Cord blood transplant is a feasible treatment alternative for adult patients with hematologic malignancies lacking a suitable HLA-matched donor. However, the kinetics of myeloid recovery is slow, and primary graft failure cannot be detected easily early after transplantation. We investigated the impact of hematopoietic chimerism status from unselected marrow cells 14 days after transplantation on predicting engraftment after a cord blood transplant.

Design and Methods

Seventy-one adult patients with hematologic malignancies undergoing single-unit unrelated donor cord blood transplantation after a myeloablative conditioning regimen were included in the study. All patients received conditioning regimens based on busulfan, thiotepa and antithymocyte globulin. Chimerism status was assessed analyzing short tandem repeat polymorphisms.

Results

The cumulative incidence of myeloid engraftment at 1 month was significantly lower in patients with mixed chimerism than in those with complete donor chimerism (55% vs. 94%; p<0.0001). For patients achieving myeloid recovery, the median time of engraftment was 16 days when donor chimerism at day + 14 was higher than 90%, compared with 24 days when donor chimerism was below this level (p<0.001). A donor chimerism level of 65% was found to be the best cut-off point for predicting primary graft failure, with a sensitivity of 97% and a specificity of 80%. The incidence of primary graft failure was 67% for patients with less than 65% donor chimerism at day +14 as compared to only 2% for those with more than 65% donor chimerism (p<0.001). Patients with mixed chimerism also had a lower cumulative incidence of platelet engraftment than those with complete chimerism (62% vs. 89%; p=0.01).

Conclusions

Donor-recipient chimerism status at day +14 predicts engraftment after a single-unit cord blood transplant in adults.     

Genetic variations in T-cell activation and effector pathways modulate alloimmune responses after allogeneic hematopoietic stem cell transplantation in patients with hematologic malignancies

Background

Recently, several important polymorphisms have been identified in T-cell activation and effector pathway genes and have been reported to be associated with inter-patient variability in alloimmune responses. The present study was designed to assess the impact of these genetic variations on the outcomes of allogeneic hematopoietic stem cell transplantation.

Design and Methods

We first investigated ten single nucleotide polymorphisms in six genes, CD28, inducible co-stimulator, cytotoxic T-lymphocyte antigen 4, granzyme B, Fas and Fas ligand, in 138 pairs of patients and their unrelated donors and a second cohort of 102 pairs of patients and their HLA-identical sibling donors.

Results

We observed that patients receiving stem cells from a donor with the cytotoxic T-lymphocyte antigen 4 gene CT60 variant allele (AA genotype) had a reduced incidence of grades II–IV acute graft-versus-host disease; however, they experienced early cytomegalovirus infection and relapsed more frequently, which suggested an interaction between the donor cytotoxic T-lymphocyte antigen 4 gene CT60 AA genotype and reduced T-cell alloreactivity. Furthermore, an unrelated donor with the granzyme B +55 variant genotype (AA) was an independent risk factor for development of grades II–IV acute graft-versus-host disease (P=0.024, RR=1.811). Among patients with acute myelogenous leukemia, those with the Fas -670 TT genotype were at higher risk of relapse (P=0.003, RR=3.823). The presence of these susceptible alleles in the donor and/or patient resulted in worse overall survival (54.9% versus 69.5%, P=0.029).

Conclusions

Our data suggest that genotype analysis of T-cell activation and effector pathway genes can be used for risk assessment for patients with hematologic malignancies before hematopoietic stem cell transplantation.Key words: CD28, inducible co-stimulator, cytotoxic T-lymphocyte antigen 4, granzyme B, Fas, Fas ligand, polymorphism, hematopoietic stem cell transplantation     

Successful generation of primary virus-specific and anti-tumor T-cell responses from the naive donor T-cell repertoire is determined by the balance between antigen-specific precursor T cells and regulatory T cells

Background

One of the major challenges in allogeneic stem cell transplantation is to find a balance between the harmful induction of graft-versus-host disease and the beneficial graft-versus-leukemia and pathogen-specific immune responses. Adoptive transfer of in-vitro generated donor T cells with specific anti-leukemic or pathogen-specific activity may be effective. However, in many cases this requires the in-vitro priming and expansion of antigen-specific precursor T cells from the naïve donor T-cell repertoire.

Design and Methods

Antigen-specific CD8 T cells were generated by co-culture of CD45RO-depleted, regulatory T cell-depleted donor peripheral blood mononuclear cells with autologous peptide-loaded dendritic cells, followed by two re-stimulations with peptide-loaded autologous monocytes. Responding T cells were isolated based on CD137 expression and further purified using peptide/major histocompatibility complex tetramers.

Results

Using this method we were able to reproducibly generate functionally high avidity T cells directed against multiple viral antigens and minor histocompatibility antigens from the naïve T-cell repertoire of seronegative, minor histocompatibility antigen-negative donors. Furthermore, we demonstrated that reduction of the regulatory T-cell frequency by depletion of CD45RO⁺ responder cells resulted in improved priming and expansion of antigen-specific precursor T cells.

Conclusions

In conclusion, we present a robust method for the in-vitro induction and isolation of antigen-specific T cells from the naïve repertoire. We demonstrate that the likelihood of successful generation of primary immune responses is determined by a delicate balance between the numbers of antigen-specific precursor T cells and the numbers and activation state of regulatory T cells locally at the site of priming of the immune response.     

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

Background

A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care.

Design and Methods

Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease.

Results

Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO⁺ CD27⁻) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25^high Foxp3⁺) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease.

Conclusions

Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.     

Persistence of recipient-type endothelium after allogeneic hematopoietic stem cell transplantation

Background

The possibility that allogeneic hematopoietic stem cell transplantation performed across the ABO blood group-barrier is associated with an increase of graft-versus-host disease, in particular endothelial damage, has not been elucidated so far. For this reason, we investigated the level of endothelial cell chimerism after allogeneic hematopoietic stem cell transplantation in order to delineate the role of hematopoietic stem cells in endothelial replacement.

Design and Methods

The frequency of donor-derived endothelial cells was analyzed in 52 hematopoietic stem cell transplant recipients, in 22 normal skin biopsies, in 12 skin samples affected by graft-versus-host disease, various tissues from five autopsies and four secondary solid tumors by ABH immunohistochemistry, XY fluorescence in situ hybridization and short tandem repeat analysis of laser captured endothelial cells.

Results

Skin biopsies from two patients transplanted with minor ABO-incompatible grafts (i.e. O in A) showed 3.3% and 0.9% H antigen-positive donor-derived endothelial cells by ABH immunohistochemistry. Tumor biopsies from two recipients showed 1.2% and 2.5% donor-derived endothelial cells by combined immunohistochemistry/ fluorescence in situ hybridization. All other skin samples, heart, liver, bone-marrow, and tumor tissues failed to reveal donor-type endothelial cells up to several years after ABO-incompatible hematopoietic stem cell transplantation.

Conclusions

Endothelial cell replacement by bone marrow-derived donor cells after allogeneic hematopoietic stem cell transplantation is a rare event. It does not seem to represent a major mechanism of physiological in vivo blood vessel formation, tumor neoangiogenesis, vascular repair after graft-versus-host disease episodes or acceptance of ABO-incompatible grafts.     

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1

Background

Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs.

Design and Methods

We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells.

Results

We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor.

Conclusions

Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.     

T-lymphoid differentiation potential measured in vitro is higher in CD34+CD38-/lo hematopoietic stem cells from umbilical cord blood than from bone marrow and is an intrinsic property of the cells

Background

Human bone marrow and umbilical cord blood are sources of allogeneic hematopoietic stem cells for transplantation, which is a life-saving treatment in a variety of diseases but is burdened by delayed T-cell reconstitution. Observational studies evaluating T-cell reconstitution in post-transplant recipients suggest that cord blood hematopoietic stem cells have a more effective capacity for T-cell reconstitution. This study focuses on the comparison of the capacity of cord blood and bone marrow hematopoietic stem cells to generate T cells in vitro.

Design and Methods

Hematopoietic stem cells were cultured in OP9-delta-like-1 and OP9-green fluorescent protein co-cultures to estimate T and myeloid generation capacity, respectively. Phenotypic markers of T-lineage or myeloid differentiation were measured by flow cytometry and used to analyze their kinetics as a function of culture time. Hematopoietic stem cells were labeled with carboxyfluorescein diacetate succinamidyl ester and analyzed after culture to track their phenotypic progression in consecutive generations. Mixed OP9-delta-like-1 co-cultures were done with either carboxyfluorescein diacetate succinamidyl ester-labeled bone marrow and unlabeled cord blood hematopoietic stem cells, or vice versa, to evaluate their mutual influence on T-lineage differentiation. The T-cell potential of hematopoietic stem cells was addressed quantitatively by limiting dilution analysis.

Results

Bulk cultures showed faster and more extensive T-cell differentiation by cord blood hematopoietic stem cells. Furthermore, the T-lymphoid differentiation capacity of cord blood and bone marrow hematopoietic stem cells can be discriminated very early based on the coordinated expression of CD34 and CD7. Mixing experiments with cord blood hematopoietic stem cells and bone marrow hematopoietic stem cells showed that these differences are cell intrinsic. Quantitative clonal analyses demonstrated that CD34⁺CD38^−/lo hematopoietic stem cells from cord blood contained a two-fold higher T-lineage generation capacity than CD34⁺CD38^−/lo bone marrow hematopoietic stem cells, whereas the myeloid differentiation was similar.

Conclusions

Our data shows that cord blood hematopoietic stem cells have higher T-lymphoid differentiation potential than bone marrow hematopoietic stem cells and that this property is cell autonomous.     

Severe events in donors after allogeneic hematopoietic stem cell donation

Background

The risk for donors of allogeneic hematopoietic stem cells transplants is generally considered negligible. Scattered reports of severe complications and a recent controversy on hematopoietic malignancies after granulocyte colony-stimulating factor administration have challenged this opinion.

Design and Methods

Three hundred and thirty-eight allogeneic transplant teams from 35 primarily European countries were asked to report numbers of fatalities, severe adverse events and hematologic malignancies occurring among their hematopoietic stem cell donors.

Results

Two hundred and sixty-two of the 338 teams (77.5%) responded to a first survey (1993–2002) and 169 of the 262 responder teams (65%) to a second survey (2003–2005). They had performed a total of 51,024 first allogeneic hematopoietic stem cell transplantations, of which 27,770 were bone marrow and 23,254 peripheral blood. They observed five donor fatalities, one after a bone marrow donation and four after peripheral blood donation (incidence 0.98 per 10,000 donations; 95% CI 0.32–2.29), 37 severe adverse events (7.25/10,000; 95% CI 5.11–9.99), of which 12 in bone marrow donors (4.32/10,000; 95% CI 2.24–7.75) and 25 in peripheral blood donors (10.76/10,000; 95% CI 6.97–15.85; p<0.05) and 20 hematologic malignancies (3.92/10,000; 95% CI 2.39–6.05), of which 8 after donating bone marrow and 12 after donating peripheral blood stem cells. The observed incidence rate of hematologic malignancies did not exceed the expected incidence in an age- and sex-adjusted general population.

Conclusions

Hematopoietic stem cell donation is associated with a small but definite risk of fatalities and serious adverse events. True incidences might be higher, due to potential underreporting by study design. A continuous, standardized donor follow-up is needed to define donor risk groups and to monitor intermediate and long-term sequelae.     

A randomized phase II study to evaluate tacrolimus in combination with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation

Background

There is evidence suggesting that sirolimus, in combination with tacrolimus, is active in the prevention of graft-versus-host disease. Sirolimus-based immune suppression may suppress alloreactive T cells, while sparing the survival and function of regulatory T cells.

Design and Methods

We conducted a randomized trial to compare the impact of sirolimus/tacrolimus against that of methotrexate/tacrolimus on the prevention of graft-versus-host disease and regulatory T-cell reconstitution.

Results

Seventy-four patients were randomized 1:1 to sirolimus/tacrolimus or methotrexate/tacrolimus, stratified for type of donor (sibling or unrelated) and the patients'' age. The rate of grade II-IV acute graft-versus-host disease at 100 days was 43% (95% CI: 27-59%) in the sirolimus/tacrolimus group and 89% (95% CI: 72-96%) in the methotrexate/tacrolimus group (P<0.001). The rate of moderate/severe chronic graft-versus-host disease was 24% (95% CI: 7-47%) in the sirolimus/tacrolimus group and 64% (95% CI: 41-79%) in the methotrexate/tacrolimus group (P=0.008). Overall survival and patient-reported quality of life did not differ between the two groups. On days 30 and 90 post-transplant, sirolimus-treated patients had a significantly greater proportion of regulatory T cells among the CD4⁺ cells in the peripheral blood, and isolated regulatory T cells were functional.

Conclusions

These data demonstrate that sirolimus/tacrolimus prevents grade II-IV acute graft-versus-host disease and moderate-severe chronic graft-versus-host disease more effectively than does methotrexate/tacrolimus, and supports regulatory T-cell reconstitution following allogeneic hematopoietic cell transplantation. Trial registration: ({"type":"clinical-trial","attrs":{"text":"NCT00803010","term_id":"NCT00803010"}}NCT00803010)Key words: tacrolimus, sirolimus, methotrexate, combination therapy, GVHD prophylaxis     

The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation

Background

T follicular helper (T_FH) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T_FH cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified.

Design and Methods

In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas.

Results

Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T_FH-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T_FH-associated molecules.

Conclusions

ICOS is a useful molecule for identifying T_FH cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T_FH-like profile) suggests its inclusion in the antibody panel for diagnosing T_FH-derived lymphomas. Our findings provide further evidence that the histological spectrum of T_FH-derived lymphomas is broader than previously assumed.     

Phenotypic Characteristics of PD-1 and CTLA-4 Expression in Symptomatic Acute Hepatitis A

Background/Aims

The immunoregulatory molecules programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with the dysfunction of antiviral effector T-cells, which leads to T-cell exhaustion and persistent viral infection in patients with chronic hepatitis C and chronic hepatitis B. Little is known about the role of PD-1 and CTLA-4 in patients with symptomatic acute hepatitis A (AHA).

Methods

Peripheral blood mononuclear cells were isolated from seven patients with AHA and from six patients with nonviral acute toxic hepatitis (ATH) during the symptomatic and convalescent phases of the respective diseases; five healthy subjects acted as controls. The expression of PD-1 and CTLA-4 on T-cells was measured by flow cytometry.

Results

PD-1 and CTLA-4 expression during the symptomatic phase was significantly higher in the T-cells of AHA patients than in those of ATH patients or healthy controls (PD-1: 18.3% vs 3.7% vs 1.6%, respectively, p<0.05; CTLA-4: 23.5% vs 6.1% vs 5.9%, respectively, p<0.05). The levels of both molecules decreased dramatically during the convalescent phase of AHA, whereas a similar pattern was not seen in ATH.

Conclusions

Our findings are consistent with a viral-protective effect of PD-1 and CTLA-4 as inhibitory molecules that suppress cytotoxic T-cells and thereby prevent the destruction of virus-infected hepatocytes in AHA.