Quantitation of T-cell neogenesis in vivo after allogeneic bone marrow transplantation in adults

Following myeloablative therapy, it is unknown to what extent age-dependent thymic involution limits the generation of new T cells with a diverse repertoire. Normal T-cell receptor gene rearrangement in T-cell progenitors results in the generation of T-cell receptor rearrangement excision circles (TRECs). In this study, a quantitative assay for TRECs was used to measure T-cell neogenesis in adult patients with leukemia who received myeloablative therapy followed by transplantation of allogeneic hematopoietic stem cells. Although phenotypically mature T cells had recovered by 1 to 2 months after bone marrow transplantation (BMT), TREC levels remained low for 3 months after BMT. T-cell neogenesis became evident by 6 months, and normal levels of adult thymic function were restored at 6 to 12 months after BMT. Subsequent leukemia relapse in some patients was associated with reduced TREC levels, but infusion of mature donor CD4(+) T cells resulted in rapid restoration of thymic function. These studies demonstrate that T-cell neogenesis contributes to immune reconstitution in adult patients and suggest that thymic function can be manipulated in vivo. (Blood. 2001;98:1116-1121)     

T-Cell immune reconstitution in pediatric leukemia patients after allogeneic bone marrow transplantation with T-cell-depleted or unmanipulated grafts: evaluation of overall and antigen-specific T-cell repertoires

To evaluate the role of T-cell selection in the thymus and/or periphery in T-cell immune reconstitution after allogeneic bone marrow transplantation (allo-BMT), we have analyzed the overall and antigen-specific T-cell repertoires in pediatric allo-BMT recipients treated for leukemia. We observed a lack of overall T-cell receptor (TCR) diversity in the repopulating T cells at 3 months after allo-BMT, as was deduced from complementarity determining region 3 (CDR3) size distribution patterns displaying reduced complexity. This was noted particularly in recipients of a T-cell-depleted (TCD) graft and, to a lesser extent, also in recipients of unmanipulated grafts. At 1 year after allo-BMT, normalization was observed of TCR CDR3 size complexity in almost all recipients. Analysis of the antigen-specific T-cell repertoire at 1 year after BMT showed that the T cells responding to tetanus toxoid (TT) differed in TCR gene segment usage and in amino acid composition of the CDR3 region when comparing the recipient with the donor. Moreover, the TT-specific TCR repertoire was found to be stable within a given allo-BMT recipient, because TT-specific T cells with completely identical TCRs were found at 3 consecutive years after transplantation. These observations suggest an important role for T-cell selection processes in the complete restoration of the T-cell immune repertoire in children after allo-BMT.     

T-cell receptor excision circle and T-cell dynamics after allogeneic stem cell transplantation are related to clinical events

It is generally believed that homeostatic responses regulate T-cell recovery after peripheral stem cell transplantation (PSCT). We studied in detail immune recovery in relation to T-cell depletion and clinical events in a group of adult patients who underwent PSCT because of hematologic malignancies. Initially, significantly increased proportions of dividing naive, memory, and effector CD4(+) and CD8(+) T cells were found that readily declined, despite still very low numbers of CD4(+) and CD8(+) T cells. After PSCT, increased T-cell division rates reflected immune activation because they were associated with episodes of infectious disease and graft-versus-host disease (GVHD). T-cell receptor excision circles (TRECs) were measured to monitor thymic output of naive T cells. Mean TREC content normalized rapidly after PSCT, long before naive T-cell numbers had significantly recovered. This is compatible with the continuous thymic production of TREC(+) naive T cells and does not reflect homeostatic increases of thymic output. TREC content was decreased in patients with GVHD and infectious complications, which may be explained by the dilution of TRECs resulting from increased proliferation. Combining TREC and Ki67 analysis with repopulation kinetics led to the novel insight that recovery of TREC content and increased T-cell division during immune reconstitution after transplantation are related to clinical events rather than to homeostatic adaptation to T-cell depletion. (Blood. 2002;99:3449-3453)     

Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity

To evaluate the importance of the thymus for the reconstitution of immunity in recipients of a T-cell-depleted bone marrow, we measured the appearance of CD4(+)CD45RA(+)RO(-) naive T cells (thymic rebound), restoration of the diversity of the T-cell-receptor (TCR) repertoire and the response to vaccinations with tetanus toxoid (TT). Repopulation by CD4(+)CD45RA(+)RO(-) thymic emigrants varied among patients, starting at approximately 6 months after transplantation. Young patients reconstituted swiftly, whereas in older patients, the recovery of normal numbers of naive CD4(+) T cells could take several years. Restoration of TCR diversity was correlated with the number of naive CD4(+)CD45RA(+)RO(-) T cells. Moreover, the extent of the thymic rebound correlated with the patient's capacity to respond to vaccinations. Patients without a significant thymic rebound at the moment of vaccination (CD4(+)CD45RA(+)RO(-) T cells less than 30 microL) did not respond, or responded only marginally even after 3 boosts with TT. We conclude that during the first year after transplantation, the absence of an immune response is due mainly to the loss of an adequate T-cell repertoire. Restoration of the repertoire can come only from a thymic rebound that can be monitored by measuring the increase of CD4(+)CD45RA(+)RO(-) naive T cells. This will allow postponing revaccinations to a moment when the patient will be able to respond more effectively. This may be particularly useful in the elderly patient who, owing to low thymic activity, might not yet be able to respond 1 year after transplant when revaccinations are usually scheduled.     

Direct evidence for new T-cell generation by patients after either T-cell-depleted or unmodified allogeneic hematopoietic stem cell transplantations

Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P =.02). Recipients of T-cell-depleted allografts initially had lower TRECs than unmodified allograft recipients (P <.01), but the difference abated beyond 9 months. TREC level disparities did not achieve significance among adults with respect to type of allograft. Measurable, albeit low, TREC values correlated strongly with severe opportunistic infections (P <.01). This finding was most notable during the first 6 months after transplantation, when patients are at greatest risk but before cytofluorography can detect circulating CD45RA(+) T cells. Low TRECs also correlated strongly with extensive chronic graft-versus-host disease (P <.01). Recipients of all ages of either unmodified or T-cell-depleted allografts therefore actively generate new T cells. This generation is most notable among adult recipients of T-cell-depleted allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are significantly associated with morbidity and mortality after transplantation. T-cell neogenesis, appropriate to age but delayed in adult recipients of T-cell-depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.     

New tools in assessing immune reconstitution after hematopoietic stem cell transplantation

BACKGROUND AND OBJECTIVES: Immune functions are impaired after allogeneic stem cell transplantation for several months depending on the age of the recipient, initial pathology, degree of HLA and minor histocompatibility antigens mismatches, origin and manipulation of the graft (unmanipulated or T-cell depleted bone marrow transplantation, cord blood) and post-transplantation events (acute or chronic graft-versus-host disease, relapse and infectious complications). MATERIAL AND METHODS, RESULTS AND CONCLUSION: In addition to lymphocyte phenotyping and functional assays, new tools are now available to monitor specific aspects of the immune response in the follow-up of hematopoietic stem cell transplantation: reconstitution of T cell diversity (spectratyping or Immunoscope), thymic function (TREC or "T-cell receptor rearrangement excision DNA circles") and antigen-specific T cell responses (HLA tetramers).     

Evidence for adequate thymic function but impaired naive T-cell survival following allogeneic hematopoietic stem cell transplantation in the absence of chronic graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation (AHSCT) leads to a prolonged state of immunodeficiency characterized by low peripheral naive T-cell counts. To identify the mechanisms leading to this defect we quantitatively and qualitatively analyzed thymic function through quantification of T-cell receptor excision circle (TREC) frequencies (both the signal-joint TREC [sjTREC] and 6 different DbetaJbeta TRECs, by-products of T-cell receptor [TCR] alpha and beta gene rearrangement, respectively), in conjunction with immunophenotype and spectratype analyses in a cohort of patients sampled from 1 to 10 years following AHSCT. In this cohort, reduced thymic function was associated only with ongoing clinical chronic graft-versus-host disease (cGVHD). Nonetheless, the diversity of thymic production remained unchanged irrespective of the patient's cGVHD status. Interestingly, increased homeostatic proliferation was found in the naive T-cell compartment of cGVHD- patients who underwent transplantation. However, reduced expression of both the interleukin-7 receptor alpha (IL-7Ralpha) (CD127) chain and the antiapoptotic protein Bcl-2 was observed. Taken together, these data indicate that the inability to reconstitute the naive T-cell compartment for several years after AHSCT, in the absence of cGVHD, is a consequence of impaired naive T-cell survival rather than thymic dysfunction.     

Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation

Acute graft-versus-host disease (aGVHD) impairs thymus-dependent T-cell regeneration in recipients of allogeneic bone marrow transplants through yet to be defined mechanisms. Here, we demonstrate in mice that MHC-mismatched donor T cells home into the thymus of unconditioned recipients. There, activated donor T cells secrete IFN-gamma, which in turn stimulates the programmed cell death of thymic epithelial cells (TECs). Because TECs themselves are competent and sufficient to prime naive allospecific T cells and to elicit their effector function, the elimination of host-type professional antigen-presenting cells (APCs) does not prevent donor T-cell activation and TEC apoptosis, thus precluding normal thymopoiesis in transplant recipients. Hence, strategies that protect TECs may be necessary to improve immune reconstitution following allogeneic bone marrow transplantation.     

Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.     

Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation.

The duration of immunodeficiency following marrow transplantation is not known. Questionnaires were used to study the infection rates in 72 patients surviving 20 to 30 years after marrow grafting. Furthermore, in 33 of the 72 patients and in 16 donors (siblings who originally donated the marrow) leukocyte subsets were assessed by flow cytometry. T-cell receptor excision circles (TRECs), markers of T cells generated de novo, were quantitated by real-time polymerase chain reaction. Immunoglobulin G(2) (IgG(2)) and antigen-specific IgG levels were determined by enzyme-linked immunosorbent assay. Infections diagnosed more than [corrected] 15 years after transplantation occurred rarely. The average rate was 0.07 infections per patient-year (one infection every 14 years), excluding respiratory tract infections, gastroenteritis, lip sores, and hepatitis C. The counts of circulating monocytes, natural killer cells, B cells, CD4 T cells, and CD8 T cells in the patients were not lower than in the donors. The counts of TREC(+) CD4 T cells in transplant recipients younger than age 18 years (at the time of transplantation) were not different from the counts in their donors. In contrast, the counts of TREC(+) CD4 T cells were lower in transplant recipients age 18 years or older, even in those with no history of clinical extensive chronic graft-versus-host disease, compared with their donors. The levels of total IgG(2) and specific IgG against Haemophilus influenzae and Streptococcus pneumoniae were similar in patients and donors. Overall, the immunity of patients surviving 20 to 30 years after transplantation is normal or near normal. Patients who received transplants in adulthood have a clinically insignificant deficiency of de novo-generated CD4 T cells, suggesting that in these patients the posttransplantation thymic insufficiency may not be fully reversible.     

Incomplete T-cell immune reconstitution in two major histocompatibility complex class II-deficiency/bare lymphocyte syndrome patients after HLA-identical sibling bone marrow transplantation.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.     

Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) is followed by profound immunodeficiency. Thymic function is necessary for de novo generation of T cells after HSCT. Circulating CD45RA(+) naive T-cell levels are predictive of antigen-specific T-cell responses in the absence of graft-versus-host disease (GVHD). These T cells may not represent recent thymic emigrants, since naive T cells may maintain this phenotype if not antigen-activated. To accurately measure thymic output after HSCT and determine the factors that influence thymic function, T-cell receptor excision circles (TRECs) were examined in CD4(+) and CD8(+) cells from a cross-section of patients following HSCT. TREC levels rose weeks after HSCT and could be detected in patients 6 years after HSCT. TREC levels correlated with the frequency of phenotypically naive T cells, indicating that such cells were not expanded progeny of naive T cells present in the donor graft. Chronic GVHD was the most important factor that predicted low TREC levels even years after HSCT. Patients with a history of resolved GVHD had decreased numbers of TREC, compared with those with no GVHD. Because few adults had no history of GVHD, it was not possible to determine whether age alone inversely correlated with TREC levels. Recipients of cord blood grafts had no evidence of decreased TREC induced by immunosuppressive prophylaxis drugs. Compared with unrelated donor grafts, recipients of matched sibling grafts had higher TREC levels. Collectively, these data suggest that thymopoiesis is inhibited by GVHD. Larger studies will be needed to determine the independent contributions of age and preparative regimen to post-transplant thymopoietic capacity.     

Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients

The extent and rapidity with which T cells are regenerated from graft-derived precursor cells directly influences the incidence of infection and the T-cell-based graft-versus-tumor effect. Measurement of T-cell receptor excision circles (TRECs) in peripheral blood is a means of quantifying recent thymic T-cell production and has been used after transplantation in many studies to estimate thymus-dependent T-cell reconstitution. We hypothesized that the quality of thymic function before transplantation affects thymus-dependent T-cell reconstitution after transplantation. We used real-time polymerase chain reaction (PCR) to quantify signal-joint TRECs (sjTRECs) before and after transplantation. T-cell reconstitution was evaluated by T-cell receptor beta (TCRbeta) CDR3 size spectratyping. We tested 77 healthy sibling donors and 244 samples from 26 pediatric recipients of allogeneic hematopoietic stem cell transplantation (AHSCT). Blood from the healthy donors contained 1200 to 155,000 sjTREC copies/mL blood. Patients who had greater than 1200 copies/mL blood before transplantation showed early recovery of sjTREC numbers and TCRbeta repertoire diversity. In contrast, patients who had fewer than 1200 copies/mL blood before transplantation demonstrated significantly slower restoration of thymus-dependent T cells. We conclude that the rate of reconstitution of thymus-dependent T cells is dependent on the competence of thymic function in the recipients before transplantation. Therefore, pretransplantation measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution after transplantation.     

Evaluation of TCR Vbeta subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells

Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vbeta repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vbeta subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated ((60)Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34(+) cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vbeta repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vbeta usage was exhibited in the bone marrow of mice, which included TCR Vbeta 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vbeta subfamilies (Vbeta9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vbeta subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15-20 days. Oligoclonal expression or oligoclonal trend of some TCR Vbeta subfamilies (Vbeta10, 11 and Vbeta2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vbeta11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vbeta subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34(+) cells from CB. The restricted expression and clonal expansion of some CB T cell clones may be induced by tissue-antigens of NOD/SCID mice.     

Analysis of immune reconstitution in children undergoing cord blood transplantation

OBJECTIVE: The aim of this study was to investigate and compare immune reconstitution in allogeneic cord blood transplantation (CBT) and bone marrow transplantation (BMT) recipients. MATERIALS AND METHODS: Twenty-three children underwent CBT from either human leukocyte antigen-identical siblings (11 cases) or unrelated donors (12 cases) were enrolled in the study, together with 23 matched children receiving BMT. Patients were analyzed 2-3 and 12-15 months after transplant. Recovery of T-, B-, and NK-lymphocyte subsets, proliferative in vitro response to mitogens, as well as cytotoxic activities, were investigated. RESULTS: CBT recipients showed a marked increase in the number of B lymphocytes as compared with patients who underwent BMT (p < 0.001). The absolute number of CD3(+) and CD8(+) T cells, as well as the proliferative response to T-cell mitogens, recovered with time after transplantation, irrespective of the source of stem cells used. Recipients of unrelated CBT had a better recovery of CD4(+) T lymphocytes (p < 0.01). Among patients experiencing acute graft-versus-host disease (GVHD), children given CBT had a much greater production of CD4(+) CD45RA(+) T cells than BMT recipients (p < 0.005). Recovery of NK cell number and innate cytotoxic activities was fast, irrespective of the source of stem cells used. CONCLUSIONS: Despite the much lower number of lymphocytes transferred with the graft, recovery of lymphocyte number and function toward normal in CBT recipients was rapid and comparable to that observed after transplantation of bone marrow progenitors. This prompt immune recovery possibly was favored by the reduced incidence and severity of GVHD observed in children who underwent CBT.     

Human cord blood progenitors sustain thymic T-cell development and a novel form of angiogenesis

There is growing interest in using human umbilical cord blood (CB) for allogeneic bone marrow transplantation (BMT), particularly in children. Thus, CB has been identified as a rich source of hematopoietic progenitors of the erythroid, myeloid, and B-cell lineages. Whether CB blood cells engrafting in the BM space also comprise T-cell progenitors capable of trafficking to the thymus and reconstituting a functional thymopoiesis in young recipients is presently unknown. Here, we show that CB progenitors, engrafted in the BM of immunodeficient mice, sustain human thymopoiesis by generating circulating T-cell progenitors capable of homing to and developing within a human thymic graft. Surprisingly, development of CB stem cells in this in vivo model extended to elements of the endothelial cell lineage, which contributed to the revascularization of transplants and wound healing. These results demonstrate that human CB stem cell transplantation can reconstitute thymic-dependent T-cell lymphopoiesis and show a novel role of CB-derived hematopoietic stem cells in angiogenesis.     

Evaluation of TCR Vβ subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells

Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vβ repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vβ subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated (⁶⁰Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34⁺ cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vβ repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vβ usage was exhibited in the bone marrow of mice, which included TCR Vβ 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vβ subfamilies (Vβ9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vβ subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15–20 days. Oligoclonal expression or oligoclonal trend of some TCR Vβ subfamilies (Vβ10, 11 and Vβ2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vβ11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vβ subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34⁺ cells from CB. The restricted expression and clonal expansion of some CB T cell clones may be induced by tissue-antigens of NOD/SCID mice.     

Analysis of T-cell repertoire and mixed chimaerism in a patient with aplastic anaemia after allogeneic bone marrow transplantation

We analysed 26 T-cell receptor (TCR) beta chain subfamilies (VB) of a patient with aplastic anaemia (AA) who underwent allogeneic bone marrow transplantation (allo-BMT). The patient developed pancytopenia at d 80. The patient's T cells were skewed in 10 of 26 TCR-VB on d 83. These TCR-VB, especially VB15, which were almost entirely CD8-positive cells, were skewed throughout her clinical course. Chimaerism analysis of the CD8-positive cells indicated that they were of recipient origin. Therefore, some immune responses induced by the recipient CD8-positive T cells had an important role in pancytopenia in AA patients after allo-BMT.     

Flt3 ligand enhances thymic-dependent and thymic-independent immune reconstitution

Despite recent progress in our understanding of the biology of T-cell homeostasis, clinically available therapies to substantially improve immune reconstitution in patients sustaining T-cell depletion are lacking. T cells are regenerated via a dynamic interplay between thymopoiesis and thymic-independent homeostatic peripheral expansion (HPE). Using athymic mice subjected to T-cell depletion, we observed that HPE is critically dependent on dendritic cells (DCs) for presentation of antigen, raising the possibility that the availability of DCs might be limiting in vivo for HPE to occur efficiently. Indeed, flt3 ligand (flt3L) treatment of athymic mice subjected to T-cell depletion (without DC depletion) substantially enhanced HPE and improved immune competence. Following bone marrow transplantation (BMT) in athymic hosts, both dendritic cells and T cells were profoundly depleted and flt3L therapy restored DC numbers and enhanced HPE. In addition, thymus-bearing BMT recipients treated with flt3L regenerated increased numbers of thymic-dependent progeny with increased numbers of T-cell receptor excision circle (TREC)-positive T cells, indicating increased thymopoiesis. Therefore, flt3L is a potent immunorestorative agent that enhances both thymic-dependent and thymic-independent pathways of T-cell regeneration.