Spleen Compartments Research Articles

Graft-Versus-Host Disease Impairs Early B Lymphopoiesis in the Bone Marrow

Abstract 2976Graft-versus-host disease (GVHD) and infectious complications are main causes of non-relapse mortality after allogeneic stem cell transplantation (SCT). Impaired immune function after SCT is usually attributed to the immunosuppressive medication applied for GVHD prophylaxis or therapy. Using a major histocompatibility complex (MHC)–mismatched murine model of GVHD (C57BL/6→BALB/c), we now examined the influence of GVHD on B cell immunity after SCT in the absence of pharmacologic immunosuppression. Lethally irradiated BALB/c (H-2d) recipients were transplanted with T cell-depleted bone marrow (TCD BM; 2.5×106) from C57BL/6 (H-2b) donors and parallel groups received CD4+CD25− conventional donor T cells (Tconv; 0.25 × 106) 2d later. Mice that received TCD BM alone (n =10) did not develop GVHD and showed a rapid and complete reconstitution of B cells in peripheral blood (PB) (25 ± 7% CD19+ B cells at d21; 55 ± 5% at d100). Mice that received additional donor Tconv cells (n =12) developed severe GVHD and completely lacked donor and host B cells in PB until their early death or throughout the observation period of 100d (p<0.001). Animals that were protected from severe GVHD by the co-infusion of donor CD4+CD25+Foxp3+ regulatory T cells (Treg; 0.25×106 Treg transplanted together with TCD BM; 2d later transfusion of 0.25 × 106 Tconv) showed a delayed, but finally full reconstitution of their B cell compartment in PB (9 ± 12% CD19+ B cells at d21; 42 ± 17% at d100). Similarly, animals without GVHD after TCD BMT and animals protected from GVHD by co-transplanted Treg cells showed a complete reconstitution of their B cell compartment in spleen and BM at d100 (spleen: 26±4, 7×106 and 31 ± 9.3×106 CD19+ B cells, respectively; BM: 2, 2 ± 0, 3×106 and 2.9 ± 0.9×106 B cells, respectively). In contrast, B cells were not only undetectable in peripheral lymphoid organs in animals with severe GVHD but also in the BM, suggesting that B cell precursors were affected. To examine whether GVHD solely impedes B cell regeneration or actively contributes to B cell eradication, GVHD was induced after B cell reconstitution at d21 after TCD BMT by donor lymphocyte infusions (DLI). Within 1wk after the transfer of 8×106 or 12×106 donor CD4+ lymphocytes, a significant reduction of B cells in PB was detected (from 30.3 ± 5.2% to 10 ± 6.9% and 36.3 ± 9.2% to 5.9 ± 1.3%, respectively; n =4). Thus, GVHD not only affected B cell reconstitution, but even eradicated stem cell-derived B cells that were syngeneic to the GVHD-inducing T cells, suggesting that GVHD-induced inflammation contributed to B cell depletion. To examine the influence of GVHD on precursor cells, serial transplants were performed. Yet, TCD BM from both, animals with and without GVHD, reconstituted their B cell compartment upon secondary transplantation (n =18; 33.1 ± 14.8% vs. 32.4 ± 17% at d100), thereby proving that the stem cell compartment was not affected. Next, we examined the effect of GVHD on precursor cells. Multipotent BM precursors (lin−, Sca-1+, c-kit+ [LSK]) were not significantly different in GVHD animals (TCD BM plus Tconv; n =12) as compared to controls (TCD BM only; n =10; 3.5×103 ± 2.8×103 vs. 5.8×103 ± 2.5×103, respectively). However, common lymphoid precursors (CLP; Lin−, FLt3+, CD127+) in the BM were significantly reduced in animals with GVHD (0.3×103 ± 0.17×103) as compared to transplant recipients without GVHD (4.4×103 ± 2.2×103, p<0.001). These results suggest that the dysregulated production of pro-inflammatory cytokines during GVHD is toxic for early B cell precursors and/or that the alloresponse destroys the BM niche for developing B cells. As IFN- γ and TNF are known to be elevated in GVHD and to impair B lymphopoiesis even in a non-transplant setting, we generated mixed chimeras using BM from wt and cytokine receptor deficient animals. Yet, a selective B cell reconstitution from receptor deficient BM was not observed in GVHD, suggesting that neither of these cytokines is exclusively responsible for its toxic effects on B cell precursors. Taken together, our results show that GVHD not solely affects immune reconstitution by the well known destruction of secondary lymphoid organs, but it disturbs early lymphoid progenitors in the BM through inflammatory, but not necessarily allo-specific immune responses. Disclosures:No relevant conflicts of interest to declare.

Host-derived CD4+ T cells protect lethally irradiated B6.g7 mice from stem cell mediated transfer of autoimmunity. (115.26)

Abstract In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic TCR initiate disease by helping B cells to produce arthritogenic anti-GPI (glucose-6- phosphate isomerase) autoantibodies. We found that lethally-irradiated lymphocyte-deficient B6.g7 (I-Ag7+) Rag-/- mice reconstituted with K/BxN HSC (ckit+Lin-Sca-1hi) exhibit arthritis by week 4 and have anti-GPI Ab levels comparable to K/BxN mice. The normal B6.g7 mouse recipients of KBN HSC, however, showed only a mild arthritis with limited extent and duration. Titers of anti-GPI Abs in reconstituted B6.g7 mice were ~60 fold lower than in K/BxN mice. To investigate the basis of this attenuated autoimmunity, we examined the chimerism in the reconstituted B6.g7 mice. Although the B cell and myeloid compartments in blood and spleen had 98%±0.1 donor-derived cells, the CD4+ T cells were only 12%± 2.5 donor-derived. Further, host-derived CD4+ T cells in the reconstituted B6.g7 mice were 18%±3.5 CD25+FoxP3+ Treg cells compared to 8%±1.76 Tregs in donor-derived CD4+ T cells (P&amp;lt;0.024). Notably, attenuation of autoimmune diabetes and high percentage of host-derived Tregs (hTreg) were also observed when B6.g7 mice were reconstituted with HSCs from BDC2.5 transgenic mice. The hTreg were functional in vitro in standard assays. Our findings show that despite myeloablation, hTreg emerge in these mice and attenuate autoimmunity.

Physiologically Based Pharmacokinetic Model of Amphotericin B Disposition in Rats Following Administration of Deoxycholate Formulation (Fungizone®): Pooled Analysis of Published Data

The time course of tissue distribution of amphotericin B (AmB) has not been sufficiently characterized despite its therapeutic importance and an apparent disconnect between plasma pharmacokinetics and clinical outcomes. The goals of this work were to develop and evaluate a physiologically based pharmacokinetic (PBPK) model to characterize the disposition properties of AmB administered as deoxycholate formulation in healthy rats and to examine the utility of the PBPK model for interspecies scaling of AmB pharmacokinetics. AmB plasma and tissue concentration-time data, following single and multiple intravenous administration of Fungizone® to rats, from several publications were combined for construction of the model. Physiological parameters were fixed to literature values. Various structural models for single organs were evaluated, and the whole-body PBPK model included liver, spleen, kidney, lung, heart, gastrointestinal tract, plasma, and remainder compartments. The final model resulted in a good simultaneous description of both single and multiple dose data sets. Incorporation of three subcompartments for spleen and kidney tissues was required for capturing a prolonged half-life in these organs. The predictive performance of the final PBPK model was assessed by evaluating its utility in predicting pharmacokinetics of AmB in mice and humans. Clearance and permeability-surface area terms were scaled with body weight. The model demonstrated good predictions of plasma AmB concentration-time profiles for both species. This modeling framework represents an important basis that may be further utilized for characterization of formulation- and disease-related factors in AmB pharmacokinetics and pharmacodynamics.

A novel bacterial artificial chromosome-transgenic Podoplanin–Cre mouse targets lymphoid organ stromal cells in vivo

Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes (LNs). Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the Pdpn–Cre mouse revealed transgene expression in subsets of fibroblastic reticular cells and lymphatic endothelial cells in LNs. Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen expression in the genetically tagged stromal cells in Pdpn–Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn–Cre expression is well-suited to characterize the phenotype and to dissect the function of lymphoid organ stromal cells.

Soluble fibers and resistant starch ameliorate disease activity in an experimental model of inflammatory bowel disease

The goal of this study was to determine the efficacy of dietary fibers in preventing gut inflammation in interleukin‐10‐deficient (IL‐10−/−) mice. C57BL/6J wild type mice (n=90) and IL‐10−/− mice (n=185) were randomly assigned to either a control or isocaloric and isonitrogenous diets supplemented with PROMITOR™ soluble corn fiber (SCF), STA‐LITE III® polydextrose (PDX), biogum (BG), pullulan, PROMITOR™ resistant starch‐75 (RS‐75), SGF&amp;BG, RS‐75&amp;BG and inulin (4 g fiber/100 g diet). On day 47 spleen, mesenteric lymph nodes (MLN), duodenum, jejunum, ileum and colon were macroscopically and histologically evaluated. The spleen and Peyer's patches (PP) were collected in sterile conditions for isolating mononuclear cells, measuring the percentages of regulatory T cells (Treg) and cytokines produced by CD4+ T cells (i.e., IL‐12, IFN‐γ; and IL‐10). Dietary supplementation with RS‐75, SCF, RS‐75&amp;BG and inulin had a preventive effect in spontaneous colitis and ileitis and ameliorated disease activity, but only RS‐75 reduced inflammatory lesions in the MLN, ileum and colon. RS‐75, SCF and inulin significantly decreased IFN‐γ; production by effector CD4+ T cells from PP and RS‐75 increased the Treg compartment in spleen and PP. These data suggest certain soluble fibers and resistant starch may influence Treg and cytokine production to minimize gut inflammation. Supported by Tate &amp; Lyle Health &amp; Nutrition Sciences

Signals derived from intestinal microbial communities augment anti-viral immunity (46.11)

Abstract Alterations in intestinal microbial communities in humans are associated with enhanced susceptibility to inflammatory diseases suggesting that signals derived from commensal bacteria may influence the development and/or function of the immune system. Supporting this, germ-free (GF) mice exhibit reduced numbers of lymphocytes in the periphery and intestinal intraepithelial compartment. However, whether alterations in the composition of commensal bacteria influence immunity to infection remains poorly defined. To test this, mice housed under conventional (CNV) or GF conditions were infected with acute LCMV and the development of antigen-specific CD8+ T cell responses were analyzed. At day 7 p.i., GF mice exhibited a significant reduction in the frequency and number of LCMV-specific CD8+ T cells in multiple tissues including the spleen and intestinal intraepithelial compartment. The diminished CD8+ T cell response was not the result of inherent developmental defects in GF mice as depletion of intestinal bacteria in CNV mice via antibiotics also resulted in diminished LCMV-specific CD8+ T cell responses and impaired viral clearance. Further, adoptive transfer of CFSE-labeled LCMV-specific CD8+ T cells revealed a delay in the activation and expansion of LCMV-specific CD8+ T cells in antibiotic-treated mice. Collectively, these studies indicate that signals derived from intestinal bacteria aid in the induction of a rapid immune response required for immunity to viral infection.

Evidence for organ‐specific stem cell microenvironments

The X-linked Gata1(low) mutation in mice induces strain-restricted myeloproliferative disorders characterized by extramedullary hematopoiesis in spleen (CD1 and DBA/2) and liver (CD1 only). To assess the role of the microenvironment in establishing this myeloproliferative trait, progenitor cell compartments of spleen and marrow from wild-type and Gata1(low) mice were compared. Phenotype and clonal assay of non-fractionated cells indicated that Gata1(low) mice contain progenitor cell numbers 4-fold lower and 10-fold higher than normal in marrow and spleen, respectively. However, progenitor cells prospectively isolated from spleen, but not from marrow, of Gata1(low) mice expressed colony-forming function in vitro. Therefore, calculation of cloning activity of purified cells demonstrated that the total number of Gata1(low) progenitor cells was 10- to 100-fold lower than normal in marrow and >1,000 times higher than normal in spleen. This observation indicates that Gata1(low) hematopoiesis is favored by the spleen and is in agreement with our previous report that removal of this organ induces wild-type hematopoiesis in heterozygous Gata1(low/+) females (Migliaccio et al., 2009, Blood 114:2107). To clarify if rescue of wild-type hematopoiesis by splenectomy prevented extramedullary hematopoiesis in liver, marrow cytokine expression profile and liver histopathology of splenectomized Gata1(low/+) females were investigated. After splenectomy, the marrow expression levels of TGF-beta, VEGF, osteocalcin, PDGF-alpha, and SDF-1 remained abnormally high while Gata1(low) hematopoiesis was detectable in liver of both CD1 and DBA/2 mutants. Therefore, in the absence of the spleen, Gata1(low) hematopoiesis is supported by the liver suggesting that treatment of myelofibrosis in these animals requires the rescue of both stem cell and microenvironmental functions.

Pentostatin Facilitates Fully MHC-Disparate Murine Mini-Transplantation.

Abstract 3539Poster Board III-476Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy.Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures:No relevant conflicts of interest to declare.

Stereologic Analysis of Tissue Compartments of Gunshot-injured and Blunt-injured Spleen

The spleen is composed of several tissue compartments and the respective histoquantitative data are essential for complete understanding of immune or pathological processes in this organ. The aim of our study was to determine and compare the stereologic parameters of all tissue compartments of the gunshot-injured and blunt-injured human spleen. The model-based stereology with point-counting method was utilized to study the volume densities of red pulp, perifollicular zone, marginal zone, white pulp (follicles and periarteriolar lymphoid sheath), and connective tissue. The areal numerical density (the number of follicles per mm(2) of tissue section), the numerical density (the number of follicles per mm(3) of tissue) of lymphoid follicles and the mean follicle diameter were also determined. Our study provides stereological parameters for all tissue compartments of the human spleen. No morphometric differences were registered between tissue compartments of the blunt-injured and gunshot-injured spleen. As the gunshot-injured spleen was taken as presumably unstimulated in immunological regard, our results suggest that both gunshot-injured and blunt-injured organs may be used as models of the normal human spleen.

The Sle2c1 murine susceptibility locus controls T cell homeostasis through defective IL-2 production (49.15)

Abstract The function of the Sle2 mouse lupus susceptibility locus and its derived Sle2c1sublocus have been previously shown to be associated with a lower threshold of activation of the BCR leading to an accumulation of B1a cells in NZM2410 model. To explore whether these loci had any effects on other immune components, we introduced lpr as an amplifier into B6.Sle2 and its subcongenic strains, and analyzed systemically their cellular and functional phenotypes. Consequently it was found that the co-expression of Sle2 and lpr on a C57BL/6 background significantly increased lymphadenopathy primarily targeting T cells, as well as induced renal pathology which included heavy lymphoid infiltrates. Comparison of the 3 Sle2 subloci mapped the synergistic interaction with lpr to Sle2c. Further examination of both Fas-deficient and Fas-intact mice showed that Sle2c1 resulted in increased T cell proliferation, a reduced IL-2 production, and a reduced Foxp3+ regulatory T cell compartment in spleen and lymph nodes, phenotypes which are characteristic of lupus in human patients and mouse models. Our results demonstrate that the Sle2c1 locus contains a candidate gene which, in addition to regulate BRC activation threshold, also affects T cells homeostasis through altering IL-2 production and Treg cell balance.

The influence of commensal bacteria on anti-viral immunity (39.26)

Abstract Alterations in the composition of intestinal commensal bacteria in humans are associated with enhanced susceptibility to multiple inflammatory diseases suggesting that signals derived from commensal bacteria may influence the development and/or function of the immune system. Supporting this, germ-free or gnotobiotic mice exhibit reduced numbers of lymphocytes in the intestinal intraepithelial compartment. However, whether alterations in the acquisition or composition of commensal bacteria influence immunity to infection remains poorly defined. To test this, mice housed under conventional or gnotobiotic conditions were infected i.p. with Lymphocytic Choriomeningitis Virus (LCMV). At day 7 post-infection, gnotobiotic mice exhibited a significant reduction in the frequency and number of LCMV-specific CD8+ T cells in multiple tissues including the spleen and intraepithelial compartment. Furthermore, LCMV-specific CD8+ T cells from gnotobiotic mice were less capable of producing IFN-γ following LCMV peptide stimulation. In addition, at day 36 post-infection, when conventionally-housed mice have established a LCMV-specific CD8+ memory T cell population, gnotobiotic mice had a significantly impaired population of LCMV-specific CD8+ memory T cells. Diminished LCMV-specific CD8+ effector and memory T cell responses were not the result of inherent defects in gnotobiotic mice as oral administration of a cocktail of antibiotics to conventionally-housed mice also displayed a defective LCMV-specific CD8+ T cell response following infection. Collectively, these data suggest an integral role of commensal bacteria in influencing virus specific CD8+ T cell effector and memory responses.

Compartmentalization of allogeneic T-cell responses in the bone marrow and spleen of humanized NOD/SCID mice containing activated human resident myeloid dendritic cells

Human allogeneic (allo)-T-cell responses within recipient lymphoid tissues and the degree to which they are altered in the presence of activated tissue-resident dendritic cells (DC) remain unknown. This study examined allo-T-cell recruitment and the early allo-T-cell responses that occur in the bone marrow (BM) and spleen (SP) of humanized (hu) nonobese diabetic (NOD)/severe combined immunodeficient (SCID) recipients containing activated human tissue-resident myeloid DC (MDC). Human naïve allo-T cells were transferred into polyinosinic:polycytidylic acid [poly(I:C)]-treated or untreated huNOD/SCID recipients containing human tissue-resident DC derived from transplanted CD34(+) cells. Activation of human tissue-resident MDC mediated by poly(I:C) treatment, recruitment, proliferation, and effector differentiation of allo-T cells in the BM and SP of huNOD/SCID recipients were analyzed in vivo by flow cytometry. Poly(I:C) treatment induced transient activation of human MDC within a maximum of 8 hours, as evidenced in the BM by an increased proportion of MDC-expressing CD86 while in the SP by MDC expressing CD86 and producing interleukin-12. Poly(I:C)-pretreated huNOD/SCID recipients showed changes in the recruitment of allo-T cells in the BM and SP and developed different allo-T cell responses within the BM and SP compartments. In the BM, allo-T cells underwent multiple divisions and increased numbers of interferon-gamma(+) and tumor necrosis factor-alpha(+) effector cells, while the majority of splenic allo-T cells underwent a single division and had fewer effector allo-T cells. Our experimental transplantation model demonstrates that early allo-T-cell responses are regulated by compartmentalization in the BM and secondary lymphoid tissues; events potentially occurring after allotransplantation in human recipients.

The human spleen is a major reservoir for long-lived vaccinia virus–specific memory B cells

AbstractThe fact that you can vaccinate a child at 5 years of age and find lymphoid B cells and antibodies specific for this vaccination 70 years later remains an immunologic enigma. It has never been determined how these long-lived memory B cells are maintained and whether they are protected by storage in a special niche. We report that, whereas blood and spleen compartments present similar frequencies of IgG+ cells, antismallpox memory B cells are specifically enriched in the spleen where they account for 0.24% of all IgG+ cells (ie, 10-20 million cells) more than 30 years after vaccination. They represent, in contrast, only 0.07% of circulating IgG+ B cells in blood (ie, 50-100 000 cells). An analysis of patients either splenectomized or rituximab-treated confirmed that the spleen is a major reservoir for long-lived memory B cells. No significant correlation was observed between the abundance of these cells in blood and serum titers of antivaccinia virus antibodies in this study, including in the contrasted cases of B cell– depleting treatments. Altogether, these data provide evidence that in humans, the two arms of B-cell memory—long-lived memory B cells and plasma cells—have specific anatomic distributions—spleen and bone marrow—and homeostatic regulation.

Ablation of Gata1 in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis

AbstractThe transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre–mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre–mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.

Effects of intravenous and subcutaneous administration on the pharmacokinetics, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles

We have previously demonstrated that the immune response to an unmethylated cytidine–guanosine (CpG)-containing oligonucleotide (ODN) is greatly enhanced when encapsulated in a lipid nanoparticle (LN-CpG ODN). In this study, the pharmacokinetics, biodistribution and cellular uptake of LN-CpG ODN following intravenous (i.v.) and subcutaneous (s.c.) administration was characterized and correlated with immunostimulatory activity. It is shown that, despite dramatic differences in tissue distribution profiles and considerable differences in uptake by CD11c-positive, CD11b-positive, Mac-3-positive and CD45R/B220-positive cells following i.v. and s.c. administration, the resultant immune response is very similar with respect to levels of cellular activation (DX5, Mac-3, CD11b, CD45/B220, CD4, CD8 and CD11c) and cytolytic activity of immune cells [natural killer (NK) cells and monocytes/macrophages] in the spleen and blood compartments. Some differences in response kinetics and antibody-dependent cellular cytotoxicity (ADCC) activity were noted in the peripheral blood NK cell population. Analyses of particle biodistribution and cell types involved in uptake leads to the conclusion that the inherent ability of antigen-presenting cells (APCs) to sequester LN-CpG ODN results in efficient uptake of the particle, even when present at very low concentrations, leading to similar responses following i.v. and s.c. administration. These results contrast with the behavior of free CpG ODN, for which distinctly different immune responses are observed following i.v. or s.c. administration.

Distinct roles of lymphotoxin-β signaling and the homeodomain transcription factor Nkx2.3 in the ontogeny of endothelial compartments in spleen

The formation of peripheral lymphoid tissues is indispensable for the efficient recognition and elimination of external antigens by lymphoid and accessory cells of the adaptive immune system. The spleen is structurally arranged around various vascular beds with distinct endothelial phenotypes. Using immunohistochemistry, we investigated the postnatal developmental characteristics of the marginal sinus and its relationship with various red-pulp sinus subsets. We also determined the importance of the lymphotoxin beta receptor (LT beta R) and the role of the Nkx2.3 transcription factor for the formation of the splenic vasculature. Both the administration of soluble LT beta R-Ig fusion protein to neonates and the deletion of LT beta R or downstream signaling components (RelB and p52) of the NF-kappaB family inhibited the phenotypic maturation of marginal sinus but had no effect on the vascular compartmentalization of the red pulp. The integrity of the marginal sinus and the proper vascular segregation of the red pulp appeared to be controlled by Nkx2.3, as Nkx2.3-deficient mice exhibited an abnormal distribution of IBL-7/1(hi)/IBL-9/2(-) sinuses and a lack of IBL-7/1(lo)/IBL-9/2(+) vessels. Our data suggest that phenotypic heterogeneity among different vascular elements within distinct anatomical regions of the spleen differentially depends on developmental factors such as lymphotoxin signaling or Nkx2.3, whereas the marginal sinus is controlled by both pathways.

Shp-2 heterozygous hematopoietic stem cells have deficient repopulating ability due to diminished self-renewal

Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function. Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies. Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells. These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.

Anti-minor histocompatibility antigen specific CD8 memory cells respond differently to antigen and antigen-independent activation in the spleen and marrow compartments following HCT

Anti-minor histocompatibility antigen specific CD8 memory cells respond differently to antigen and antigen-independent activation in the spleen and marrow compartments following HCT

Antigen-Specific CD8+ Memory T Cells Survive, Function and Populate the Host Marrow Compartment Following Ablative TBI and Allogeneic BMT.

We are interested in investigating the survival and location of host CD8 memory (TM) cells following allogeneic hematopoietic cell transplants (HCT). The H60 antigen dominates the immune response in B6 mice primed with BALB.B antigens (B6BALB.B). In such primed recipients, transplant of BALB.B MiHA-disparate marrow BM induces CD8 TM responses that mediate resistance to bone marrow engraftment. Therefore an H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to detect host H60-specific CD8 TM in the spleen and marrow compartments. In the marrow compartment, the frequency of H60+ cells amongst the CD8+ T cell population was significantly (p = < 0.05) higher compared to spleen levels. In both BM and spleen, >90% of CD8+ H60+ cells expressed the central memory phenotype (CD44+, Ly6C+ CD25−, CD69−). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8+ TM must first survive the immediate post-HCT milieu in the hemopoietic compartments. We observed a dose-dependent increase in percent of CD8+ T cells expressing the H60 TCR in the spleen as well as bone marrow in B6BALB.B mice irradiated at 3.0, 6.0 (non-ablative) and 9.0 Gy (ablative) 24 hrs post-HCT. Five days post-HCT, CD8+ H60+ cells were also readily detectable. At this time, resistance to engraftment assessed by IL-3 progenitor assay was present in sensitized, ablatively conditioned recipient mice transplanted with 5 X 106 BALB.B or congenic H60 TCD-BM. We then utilized a “double transplant” model to determine the compartmental distribution and function of MiHA-specific TM at later intervals (14 days) post-HCT. B6 mice containing CD8 TM were ablatively conditioned and 24 hrs later received syngeneic B6 (Ly 5.2) BMT. Twelve days following this syngeneic HCT, the mice were irradiated at 4.5 Gy and administered a second HCT consisting of either syngeneic (Ly 5.1) or allogeneic HCT (5 X 106 TCD-BM). These recipients were assessed for donor progenitors 10 days later. At this time point, H60+ CD8 TM were again readily detectable in both compartments, indicating that these TM effectors survived ablative (and subsequent non-ablative) conditioning and were present at this time post-transplant. Resistance to allogeneic marrow engraftment as assessed by IL-3 progenitor assay was detected after this subsequent transplant with BALB.B BM. These findings indicate that host TM survive and function following ablative conditioning and HCT. To evaluate if the viability of host CD8 TM differed in the host compartments following HCT, Annexin V and 7-AAD staining was performed. A higher frequency of non-viable CD8+ H60+ T cells in the spleen (> 50%) compared to the BM (<30%) compartments was observed 2 weeks after allogeneic HCT. Such findings may reflect that activation-induced cell death in the spleen induced by antigen in the allogeneic HCT was greater than in the bone marrow. Thus, reduced apoptosis in the marrow microenvironment could reflect survival advantage for CD8+ TM in this compartment. Current studies are determining the effects of ablative conditioning on ex vivo effector function activity of MiHA-specific CD8+ TM.

Minor Histocompatibility Antigen-Specific CD8+ Memory T Cells in Hematopoietic Compartments Survive the Immediate Non-Ablative and Ablative TBI Milieu.

Minor histocompatibility (MiHA) antigens induce CD8+ T-cell responses that mediate resistance to bone marrow engraftment in an MHC-identical, MiHA-disparate marrow transplant model, in which recipients are sensitized to donor antigens (BALB.B à B6BALB.B) prior to BMT. The H60 H antigen has been shown to dominate the immune response in B6 mice primed with BALB.B antigens (B6BALB.B). We initially sought to determine the distribution and partially characterize the phenotype of H60-specific CD8+ cells in the blood, spleen and marrow compartments of B6 mice primed with 3 x 107 BALB.B lymphoid cells and ≥3 weeks later boosted with 2 x 107 cells. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to detect H60-specific CD8+ cells in these compartments. Eight days following the second immunization, the mean frequency of circulating H60-specific cells was 12.2% ± SE 0.88 of CD8+ cells (range: 5.6 – 20.5%). The frequency of splenic H60-specific CD8+ cells was equivalent to that of circulating antigen-specific cells, thus peripheral blood levels of H60-specific CD8+ cells appear to be representative of those resident in the spleen. Interestingly, in the marrow compartment, the frequency of H-60+ cells amongst the CD8 T cell population was higher compared to peripheral blood and spleen levels, suggesting that H60-specific CD8+ cells in this compartment may comprise both migrant cells from the periphery and resident cells in the marrow elicited during priming. In both BM and spleen, >90% of CD8+ H60+ cells expressed the memory phenotype(CD44+, Ly6C+) and as expected, did not express early activation markers (CD25, CD69). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8+ cell must first survive the immediate post-BMT milieu in the hemopoietic compartments. To examine this question, B6BALB.B mice irradiated at 3.0, 6.0 and 9.0 Gy were analyzed 24 hours later for the presence of H60-specific CD8+ cells in the spleen and marrow compartments. Although there was an expected irradiation dose-dependent decrease in absolute numbers of CD8+ H60+ cells in the two compartments, there was a dose-dependent increase in percent of CD8+ T cells expressing the H60 TCR in both compartments. This observation indicates enhanced survival of these antigen-specific CD8+ memory T cells post-conditioning. Preliminary results indicate that 24h post-BMT into 9.0 Gy TBI recipients, there was BrDU uptake in marrow and splenic CD8+H60+ T cells in B6BALB.B transplanted with 1 x 107 BALB.B BM-TCD. Approximately 80% of CD8+H60+ T cells in the marrow compartment of primed recipients of BALB.B cells exhibited proliferation by BrDU uptake. Thus, donor MiHA-disparate marrow grafts elicit antigen-driven proliferation early post-BMT by CD8+ memory T cells in both compartments consistent with the potential importance of these cells in mediating resistance against progenitor engraftment across these MiHA differences.