不同预处理移植后树突状细胞早期重建及与移植物中CD34+细胞相关性分析

目的：比较不同预处理异基因造血干细胞移植（allo-HSCT）后早期树突状细胞（DCs）亚群重建情况，以及移植物中CD34^＋细胞是否影响移植后早期DCs亚群重建。方法：采用三色流式细胞仪动态检测不同预处理移植后早期外周血树突状细胞亚群DC1、DC2水平。结果：移植后早期清髓性移植患者体内DCs亚群数量非常低，常规移植组移植后14天与半相合移植组相比，DC1、DC2均无统计学意义（P〉0．05）。非清髓性移植组（NST）DC1、DC2高于清髓性移植组，两者相比具有统计学意义（P〈0．05）。在30天和60天，所有组DC1、DC2略有波动，但是幅度不大。以输入的CD34^＋细胞数平均分为三组，三组患者DC1、DC2移植后14、30和60天均无统计学意义（P〉0．05）。结论：NST后患者早期DCs重建较清髓性干细胞移植患者早，而常规移植和半相合移植早期DCs重建较慢，二者无差别。移植物中的CD34^＋细胞不影响移植后早期DCs亚群重建。     

Cord blood CD34+ cells cultured with FLT3L, stem cell factor, interleukin-6, and IL-3 produce CD11c+CD1a-/c- myeloid dendritic cells

Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34(+) cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c(+)CD16() CD1a()/c() MDCs from CB CD34(+) cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33(+)CD14()CD15() precursors with a mean of 4 x 10(6) cells generated from 1-4 x 10(4) CB CD34(+) cells or myeloid precursors after 2 weeks. After 8-12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34(+) cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.     

Mitochondrial DNA sequence heterogeneity of single CD34+ cells after nonmyeloablative allogeneic stem cell transplantation

We applied a single-cell method to detect mitochondrial DNA (mtDNA) mutations to evaluate the reconstitution of hematopoietic stem cells (HSCs) and committed progenitor cells after nonmyeloablative allogeneic stem cell transplantation in humans. In a total of 1,958 single CD34(+) cells from six human leukocyte antigen-matched sibling donor and recipient pairs, individual CD34(+) clones were recognized based on the observed donor- or recipient-specific mtDNA sequence somatic alteration. There was no overall reduction of mtDNA heterogeneity among CD34(+) cells from the recipient after transplantation. Samples collected from two donors over time showed the persistence of certain CD34(+) clones marked by specific mutations. Our results demonstrate the feasibility of distinguishing donor and recipient individual CD34(+) clones based on mtDNA mutations during engraftment. HSCs were not limited in number, and similar mtDNA heterogeneity levels suggested representation of the total stem cell compartment during rapid hematopoietic reconstitution in the recipient. Disclosure of potential conflicts of interest is found at the end of this article.     

Reduced blood BDCA-2+ (lymphoid) and CD11c+ (myeloid) dendritic cells in systemic lupus erythematosus

Type 1 IFN is thought to be implicated in the autoimmune process of SLE. Plasmacytoid dendric cells (DC), which are natural IFN-alpha producing cells, play a pivotal epipathogenic role in SLE. The present study was undertaken to investigate the phenotypic characteristics of peripheral blood DC in SLE patients in comparison with those of healthy controls. Samples from 20 SLE patients and 18 healthy controls were studied. Three-colour flow cytometry was performed to identify myeloid DC, as CD11c(+) lineage marker(-), and HLA-DR(+) cells and plasmacytoid DC, as BDCA-2(+) linage marker(-), and HLA-DR(+) cells. We used the whole blood 'lyse/no-wash' procedure, which allows precise counting of peripheral blood DC. BDCA-2(+) plasmacytoid DC and CD11c(+) myeloid DC were reduced in SLE patients compared with controls. Similarly, BDCA-3(+) DC were reduced in SLE patients. These results indicated that SLE patients had a reduced number of both BDCA-2(+) plasmacytoid DC and CD11c(+) myeloid DC. These alternations of the DC subset may drive the autoimmune response in SLE.     

Host-Derived CD8+ Dendritic Cells Protect Against Acute Graft-versus-Host Disease after Experimental Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a frequent life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT) and induced by donor-derived T cells that become activated by host antigen-presenting cells. To address the relevance of host dendritic cell (DC) populations in this disease, we used mouse strains deficient in CD11c⁺ or CD8α⁺ DC populations in a model of acute GVHD where bone marrow and T cells from BALB/c donors were transplanted into C57BL/6 hosts. Surprisingly, a strong increase in GVHD-related mortality was observed in the absence of CD11c⁺ cells. Likewise, Batf3-deficient (Batf3^-/-) mice that lack CD8α⁺ DCs also displayed a strongly increased GVHD-related mortality. In the absence of CD8α⁺ DCs, we detected an increased activation of the remaining DC populations after HSCT, leading to an enhanced priming of allogeneic T cells. Importantly, this was associated with reduced numbers of regulatory T cells and transforming growth factor-β levels, indicating an aggravated failure of peripheral tolerance mechanisms after HSCT in the absence of CD8α⁺ DCs. In summary, our results indicate a critical role of CD8α⁺ DCs as important inducers of regulatory T cell–mediated tolerance to control DC activation and T cell priming in the initiation phase of GVHD.     

Ex vivo characterization of human thymic dendritic cell subsets

Interactions between thymic dendritic cells (DC) and thymocytes are critical for proper development of T-cells. We identified human thymic DC populations on the basis of CD123, CD11c and CD14 expression. High levels of CD123 (IL-3R) and CD45RA defined the plasmacytoid DC (pDC) subset. Human thymic CD11c(+) DC expressed CD45RO and myeloid-related markers (CD13, CD33 and CD11b). CD11c(+) DC could be separated into two main subsets based on differential expression of CD14: CD11c(+) CD14(-) and CD11c(+) CD14(+) cells. Spontaneous production of IL-10 and IFNgamma without exogenous stimulation, was observed in the three DC subsets. Important phenotype modifications were observed in pDC cultures supplemented with IL-3. A down-regulation of CD123 and appearance of myeloid markers such as CD11b and CD11c on CD45RA(+) cells was noticed within the first 48h; at a later time there was a shift from CD45RA to CD45RO expression, as well as appearance of CD14 expression. CD11c(+) cells emerging in pDC culture did not express high levels of HLA-DR, CD83 and co-stimulatory molecules. This suggests an in vitro evolution of human thymic pDC toward a myeloid phenotype found in the CD11c(+) subset of thymic DC.     

Cytomegalovirus-specific CD4+ and CD8+ T-cells follow a similar reconstitution pattern after allogeneic stem cell transplantation.

Cytomegalovirus (CMV) is a common herpes virus that can cause significant morbidity and mortality in immunocompromised individuals, particularly those undergoing allogeneic stem cell transplantation (SCT) for hematological malignancies. Recent studies have examined the kinetics of CMV-specific CD8+ T-cell reconstitution after SCT transplantation and have found virus-specific cytotoxic T-lymphocyte regeneration to be dependent on CMV serologic status and CMV reactivation events. However, the reconstitution kinetics of CMV-specific CD4+ T-cells under these same circumstances were not addressed. In this study, we used HLA class I peptide tetramer for CMV pp65 and cytokine flow cytometry to follow the reconstitution of both CD4+ and CD8+ CMV-specific T-cells after allogeneic SCT. We found that following SCT in which both donors and recipients are CMV seropositive, virus-specific CD4+ T-helper cells show the same reconstitution kinetics as CD8+ cytotoxic T-cells. Following CMV reactivation, a synchronous but temporary increase in both CD4+ and CD8+ CMV-specific lymphocytes occurs. The pattern repeats itself after subsequent episodes of CMV reactivation. These data imply that both CD4+ and CD8+ lymphocytes are necessary for an efficient immune response to CMV and suggest that CD4+ and CD8+ CMV-specific T-cells are required for the complete restoration of CMV immunity. These findings may have important implications in the development of CMV-specific adoptive immunotherapy strategies.     

Reduced graft-versus-host disease in C3-deficient mice is associated with decreased donor Th1/Th17 differentiation

Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is mediated by the activation of recipient dendritic cells and subsequent proliferation of donor T cells. The complement system was recently shown to modulate adaptive immunity through an interaction of the complement system and lymphocytes. Complement proteins participate in the activation of dendritic cells, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3(-/-)) had significantly lower GVHD-related mortality and morbidity compared with wild-type recipient mice. The numbers of donor-derived T cells, including IFN-γ(+), IL-17(+), and IL-17(+)IFN-γ(+) subsets, were decreased in secondary lymphoid organs of C3(-/-) recipients. Furthermore, the number of recipient CD8α(+)CD11c(+) cells in lymphoid organs was reduced. We conclude that C3 regulates Th1/17 differentiation in bone marrow transplantation, and define a novel function of the complement system in GVHD.     

CD3+/Tregs ratio in donor grafts is linked to acute graft-versus-host disease and immunologic recovery after allogeneic peripheral blood stem cell transplantation

Graft-versus-host disease (GVHD), mediated by mature T cells present in the donor graft, remains a major complication after allogeneic peripheral blood stem cell transplantation (PBSCT). Regulatory T cells (Tregs) (CD4(+)CD25(high)Foxp3(+)) are believed to maintain tolerance and to inhibit GVHD after allogeneic PBSCT (allo-PBSCT). In this study, we analyzed the graft CD3(+)/Tregs ratio (gCD3/Tregs R) and evaluated its impact on acute GVHD (aGVHD) and immunologic recovery after myeloablative allo-PBSCT. We analyzed 65 consecutive patients who underwent transplantation with unmanipulated peripheral blood stem cells from an HLA-identical related donor (n = 45) or an HLA-identical unrelated donor (n = 20). The median CD3(+) and Tregs doses administered were 256 × 10(6)/kg of body weight (range, 67-550 × 10(6)/kg) and 12 × 10(6)/kg (range, 2-21 × 10(6)/kg), respectively; the median gCD3/Tregs R value was 18 (range, 8-250). Patients were subdivided into a high gCD3/Tregs R (≥36) group (HR; n = 26) and a low gCD3/Tregs R (<36) group (LR; n = 39). The incidence of aGVHD (grade II-IV) was lower in the LR group compared with the HR group (8/39 [20%] versus 22/26 [84%]; P < .001). Median cytomegalovirus-specific CD8(+) T lymphocytes were significantly higher in the LR group than in the HR group at 1 month (2 cells/μL versus 0 cells/μL; P < .001), 2 months (6?cells/μL versus 1 cell/μL; P < .001), and 3 months (15 cells/μL versus 3 cells/μL; P < .001) months. Moreover, cytomegalovirus infection/disease was observed in 15% of patients in the LR group versus 69% of patients in the HR group (P < .001). At multivariate logistic regression, gCD3/Tregs R was correlated both with aGVHD (odds ratio, 2.50; 95% confidence interval, 1.30-4.50; P = .05) and with cytomegalovirus infection/disease (odds ratio, 2.35; 95% confidence interval, 0.9-5.00; P = .05). Taken together, our data may suggest that the balance in favor of graft Tregs content is able to mediate protective effects against aGVHD and to maintain an optimal microenviroment for the reconstitution of functional immunity.     

Evolution of responding CD4+ and CD8+ T-cell repertoires during the development of graft-versus-host disease directed to minor histocompatibility antigens.

Graft-versus-host disease (GVHD) can be induced in lethally irradiated mice after allogeneic bone marrow transplantation between major histocompatibility complex-matched strains expressing multiple minor histocompatibility antigen differences. In the B6 --> BALB.B irradiation model, both CD4(+) and CD8(+) donor T cells have the capacity to mediate lethal GVHD. Previously, CDR3-size spectratyping was used to analyze these T-cell responses at a single early time point (day 5) after transplantation and revealed clonal or oligoclonal expansions of the V beta 2, 4, and 6 to 14 families for the CD4(+) response and of the V beta 4, 6, 8 to 11, and 14 families for the B6 CD8(+) response. Appropriate positive selection of these T-cell receptor V beta-skewed CD4(+) and CD8(+) T-cell subsets and their subsequent transfer into lethally irradiated BALB.B recipients resulted in fatal GVHD induction. In contrast, BALB.B mice transplanted with nonskewed V beta CD4(+) T cells survived, with minimal symptoms of GVHD. This study was undertaken to investigate the evolution of the donor/antihost minor histocompatibility antigen T-cell repertoire responses throughout the course of GVHD development. The results indicated that a number of V beta families were consistently involved throughout the course of GVHD, whereas some V beta families exhibited skewed expansions only in either the early or late stages of disease. In addition, sequence analysis of relevant representative skewed CDR3 bands from the CD4(+) V beta 11(+) and the CD8(+) V beta 14(+) families, both of which exhibited strong consistent responses, demonstrated increased use of the J beta 2.5 and J beta 2.4 segments, respectively, thus identifying the T-cell receptor specificities involved.     

Antiviral immunity and T-regulatory cell function are retained after selective alloreactive T-cell depletion in both the HLA-identical and HLA-mismatched settings.

Nonselective T-cell depletion reduces the incidence of severe graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, but the cost is delayed and disordered antigen-specific immune reconstitution and increased infection. We use a method of selective depletion of alloreactive T cells expressing the activation marker CD69 after coculture with stimulator cells in a modified or standard mixed lymphocyte reaction. The technique has been shown to reduce alloreactivity while retaining third-party responses in vitro and, in a mismatched murine model, led to donor T-cell engraftment with a virtual absence of graft-versus-host disease and increased survival. We show in a human HLA-mismatched and unrelated HLA-identical setting that this technique retains >80% of specific cellular antiviral activity by cytomegalovirus-tetramer analysis and cytomegalovirus/Epstein-Barr virus peptide-stimulated interferon-gamma ELISpot assay. Furthermore, CD4(+) CD25(+) T-regulatory cells are not removed by this method of selective allodepletion and retain their function in suppressing allogeneic proliferative responses. Preservation of antiviral cytotoxic T lymphocytes in selectively allodepleted stem cell grafts would lead to improved antiviral immunity after transplantation. The retention of immunosuppressive CD4(+) CD25(+) T-regulatory cells could lead to more ordered immune reconstitution and further suppress alloreactive responses after transplantation.     

Phenotypic Characterization of Five Dendritic Cell Subsets in Human Tonsils

Heterogeneous expression of several antigens on the three currently defined tonsil dendritic cell (DC) subsets encouraged us to re-examine tonsil DCs using a new method that minimized DC differentiation and activation during their preparation. Three-color flow cytometry and dual-color immunohistology was used in conjunction with an extensive panel of antibodies to relevant DC-related antigens to analyze lin(-) HLA-DR(+) tonsil DCs. Here we identify, quantify, and locate five tonsil DC subsets based on their relative expression of the HLA-DR, CD11c, CD13, and CD123 antigens. In situ localization identified four of these DC subsets as distinct interdigitating DC populations. These included three new interdigitating DC subsets defined as HLA-DR(hi) CD11c(+) DCs, HLA-DR(mod) CD11c(+) CD13(+) DCs, and HLA-DR(mod) CD11c(-) CD123(-) DCs, as well as the plasmacytoid DCs (HLA-DR(mod) CD11c(-) CD123(+)). These subsets differed in their expression of DC-associated differentiation/activation antigens and co-stimulator molecules including CD83, CMRF-44, CMRF-56, 2-7, CD86, and 4-1BB ligand. The fifth HLA-DR(mod) CD11c(+) DC subset was identified as germinal center DCs, but contrary to previous reports they are redefined as lacking the CD13 antigen. The definition and extensive phenotypic analysis of these five DC subsets in human tonsil extends our understanding of the complexity of DC biology.     

Comparative analysis of the morphological, cytochemical, immunophenotypical, and functional characteristics of normal human peripheral blood lineage(-)/CD16(+)/HLA-DR(+)/CD14(-/lo) cells, CD14(+) monocytes, and CD16(-) dendritic cells

Human peripheral blood (PB) CD14(lo)/HLA-DR(+) cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16(+)/HLA-DR(+) cells compared to both PB CD14(+) monocytes and CD16(-) DC. In contrast to CD14(+) monocytes, purified CD16(+)/HLA-DR(+) cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and alpha-naphthyl acetate esterase. Normal human PB CD16(+)/HLA-DR(+) cells also displayed phenotypic characteristics different from those of CD14(+) monocytes: they lacked the CD64 Fcgamma receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14(+) monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16(-) DC, CD16(+)/HLA-DR(+) cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complement-receptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16(+)/HLA-DR(+) cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16(-) DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-gamma-stimulated PB CD16(+)/HLA-DR(+) cells produced significant amounts of IL1beta, IL6, IL12, TNFalpha, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16(+)/HLA-DR(+) cells than in CD14(+) monocytes. In addition, upon comparing CD16(+)/HLA-DR(+) cells with CD33(+++)/CD16(-) DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/cell were significantly different in both cell subsets. In summary, our results show that CD16(+)/HLA-DR(+) cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16(-) DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow.     

Screening of the HLDA9 panel on peripheral blood dendritic cell populations

Dendritic cells (DC) are a heterogeneous population of bone marrow derived leucocytes that are essential in the initiation of primary T lymphocyte responses. DC are identified as Lineage negative, HLA-DR(+) blood cells that can be further subdivided by CD11c to distinguish CD11c(+) DC and the CD11c(-) plasmacytoid DC. Plasmacytoid DC are the primary IFNα producing cells and express CD303, CD304 and CD123. The CD11c(+) myeloid DC can be divided into populations by CD1c, CD16 and CD141 expression. Despite DC being a functionally unique population, they share many cell surface antigens with myeloid lineage cells and B lymphocytes. We used flow cytometry to screen fresh human blood DC populations with the HLDA9 panel of 63 directly labelled mAb which included mAb specific for a number of B lymphocyte antigens. Of this panel, 23 mAb did not bind Lin(-)HLA-DR(+) DC and 10 bound all four populations. Eight mAb bound to the three CD11c(+) DC populations whilst no mAb tested bound to only pDC. Some of the mAb expected to bind to DC populations failed in this analysis. Overall, this screening highlighted similarities between the CD11c(+) DC subsets and the relatively immature state of peripheral blood DC.     

T-cell receptor Vbeta repertoire after myeloablative and reduced intensity conditioning allogeneic haematopoietic stem cell transplantation

T cells play an important role in the adaptive immune system. After haematopoietic stem cell transplantation (HSCT), T-cell function is impaired. This is reflected by the emergence of opportunistic infections, infections that are often difficult to treat because of the patient's insufficient immune function. T-cell receptor reconstitution was studied using CDR3 spectratyping to analyze the diversity of the T-cell repertoire at 3, 6 and 12 months after myeloablative and reduced intensity conditioning (RIC) HSCT in 23 patients. Immune function in vitro was tested by lymphocyte stimulation at 3, 6 and 12 months after HSCT. Lower diversity in the CDR3 repertoire was demonstrated in CD4+ cells after RIC HSCT at 3 and 6 months and in CD8+ cells at 3 months compared with healthy donors. After myeloablative HSCT, lower diversity was seen at 3, 6 and 12 months in CD4+ cells and at 6 and 12 months in CD8+ cells after HSCT. Acute and chronic graft-versus-host-disease (GVHD) did not affect diversity. Responses to phytohaemagglutinin (PHA), Concanavalin A (Con A) and Staphylococcus aureus protein A were significantly lower compared with healthy donors during the first 6 months after RIC HSCT. After myeloablative HSCT, lymphocyte response to Con A was significantly lower at 3 months compared with healthy donors. Decreased responses to cytomegalovirus and varicella zoster virus antigens were seen in patients suffering from acute GVHD grade II or chronic GVHD. The T-cell repertoire is skewed under the first year after HSCT, and immune reconstitution after HSCT with myeloablative and RIC conditioning seems to be comparable. GVHD, infections and age are more important for immune reconstitution than type of conditioning.     

Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts

CD4(+)CD25(+) regulatory T cells (Tregs) are well known to suppress immunopathology induced in lymphopenic animals following T cell reconstitution, including acute graft-versus-host disease (GVHD) post-bone marrow transplantation. The regulatory potential of this subset in nonlymphopenic hosts and in chronic, Th2-mediated GVHD is less clear. We have generated alloantigen-specific cells from CD4(+)CD25(+) populations stimulated with MHC-disparate dendritic cells and found them to express a stable Treg forkhead box p3(+) phenotype with enhanced suppressive activity mediated by cell contact. When transferred into nonlymphopenic F1 hosts, nonspecific Tregs proliferated as rapidly as CD4(+)CD25(-) cells but displayed distinct growth kinetics in vitro. Tregs, expanded in response to alloantigen in vitro, displayed greatly enhanced suppressive activity, which was partially antigen-specific. They were effective inhibitors of chronic GVHD, blocking donor cell engraftment, splenomegaly, autoantibody production, and glomerulonephritis. CD25(+) and CD25(-) cells were equally susceptible to inhibition by immunosuppressive drugs targeting TCR signaling and rapamycin, but Tregs were resistant to inhibition by dexamethasone. The data indicate that alloantigen-driven expansion, rather than homeostatic proliferation, is key to the effectiveness of CD4(+)CD25(+) Tregs in GVHD and suggest that cellular therapy with alloantigen-induced Tregs in combination with glucocorticoid treatment would be effective in prevention of chronic GVHD after immune reconstitution.     

IL-7 Is the Limiting Homeostatic Factor that Constrains Homeostatic Proliferation of CD8+ T Cells after Allogeneic Stem Cell Transplantation and Graft-versus-Host Disease

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4⁺ T cell HP during GVHD. However, it is not clear whether these alterations to the peripheral CD4⁺ T cell niche also contribute to impair CD8⁺ T cell regeneration during GVHD. We found that IL-7 therapy was sufficient for restoring CD8⁺ T cell HP in GVHD hosts while forcing DC regeneration with Flt3-L had only a modest effect on CD8⁺ T cell HP in IL-7 treated mice. Using bone marrow chimeras, we showed that HP of naïve CD8⁺ T cells is primarily regulated by MHC class I on radio-resistant stromal cells, yet optimal recovery of CD8⁺ T cell counts still requires expression of MHC class I on both radio-resistant and radio-sensitive hematopoietic cells. Thus, IL-7 level is the primary limiting factor that constrains naïve CD8⁺ T cell HP during GVHD, and accessibility of MHC class I on stromal cells explains how IL-7 therapy, as a single agent, can induce robust CD8 ⁺ T cell HP in the absence of DCs.     

Absolute values of dendritic cell subsets in bone marrow,cord blood,and peripheral blood enumerated by a novel method

Dendritic cells (DCs) are pivotal in inducing immunity or alternatively downregulating immune reactivity. In humans, the opposing phenotypic subsets of CD11c(+)/CD123(-) "myeloid" DCs and CD123(+)/CD11c(-) "lymphoid" DCs have been proposed to orchestrate these immune responses. In this study we determined the absolute numbers of both subsets in three resting hematopoietic tissues by employing a novel flow cytometry method, eliminating processing steps and calculations based on mononuclear cell percentages. Internal bead standards along with the cells of interest were simultaneously acquired directly from unmanipulated whole blood specimens. We found significant differences (p < 0.001) between the mean absolute numbers of CD123(+)/CD11c(-) lymphoid DCs among the three sources, with the fewest present in peripheral blood (8.2/ micro l), about 50% more in cord blood (12.2/ micro l), and fivefold more in bone marrow (40.2/ micro l). Cord blood and bone marrow CD11c(+)/CD123(-) myeloid DC counts did not differ from each other (23.5/ micro l and 28.9/ micro l, respectively) but peripheral blood contained significantly fewer (15.5/ micro l, p = 0.006). CD11c(+)/CD123(-) DCs had significantly higher surface expression of HLA-DR (p < 0.001) in all three sources. To test for association with the DC subsets, T, B, and natural killer (NK) lymphocytes were also enumerated. In bone marrow only, significant correlations were found between the size of the CD123(+)/CD11c(-) lymphoid DC pool and NK cells (p = 0.0029) and B cells (p = 0.0033). These data support the hypothesis that a common CD123(+)/CD11c(-) DC, NK cell, and B cell progenitor is resident in marrow, and this cell may be identical to the common lymphoid progenitor previously described in mice and/or the human CD34(+)/Lin(-)/CD10(+) progenitor.     

CD57+ T cells augment IFN-gamma production in a one-way mixed lymphocyte reaction and their expansion after stem cell transplantation in paediatric patients

To clarify the immune response of CD57+ T cells (most of them are CD8+) in peripheral blood (PB) against alloantigens in order to elucidate the T helper 1 (Th 1) immune response, we assessed the role of CD57+ T cells in IFN-gamma (one of the representative Th 1 cytokines) production in a one-way mixed lymphocyte reaction (MLR). In this study, we showed that CD57+ T cells in responder cells were essential for effective IFN-gamma production in allogeneic MLR due partly to the augmentation of the alloresponse of regular T cells. Furthermore, IFN-gamma production in MLR correlated with the proportions of CD57+ T cells in PB regardless of the responders' age. We also showed that the extent of the expansion of CD57+ T cells in paediatric patients after haematopoietic stem cell transplantation (HSCT) was markedly lower than that in adult patients. In addition, CD57+ T cells purified and activated with a combination of cytokines showed a greater cytotoxicity than regular T cells against human umbilical vein endothelial cells. Because IFN-gamma production in one-way MLR is a useful predictor of graft-versus-host disease (GVHD), especially in the acute phase that occurs after allogeneic HSCT, our findings suggested that CD57+ T cells play a role in the development of GVHD and thus may explain the reason as to why a higher donor age is associated with an increased risk of developing GVHD while, in addition, the incidence of severe GVHD in paediatric patients is lower than that in adult patients.