F1 Donors Research Articles

The Effect of Donors Used in In vivo Maternal Haploid Technique on Haploid Induction Rate

Maize is an important plant grown to obtain grain and silage, and is used in human and animal nutrition. In conventional maize breeding studies, inbred line development studies are carried out for at least 7 years if a single generation is obtained in a year, while it is possible to develop 100% homozygous lines in a short period of 2 years with the in vivo maternal haploid technique. The in vivo maternal haploid technique is widely used in advanced maize breeding programs. The choice of donor or source material to be used for haploid induction depends on the purpose of the breeding program. Generally, breeders use F1 or F2 populations as source material for haploid induction. In this study; 30 F1 genotypes and their F2s were crossed with the inducer line. The putative haploid seed was identified by considering the R1-nj color marker, and the haploid induction rate was determined. The effect of the generations of the donor genotypes on the haploid induction rate was compared by performing an independent sample test, and the haploid induction rate obtained from the F1 donors was found to be higher than the haploid induction rate of the F2 donors. It was determined that there was a change in the haploid induction rate as the genotypes changed within the F1 and F2 donor groups.

Anti-HLA-A2-CAR Tregs prolong vascularized mouse heterotopic heart allograft survival

Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99days, whereas median survival with polyclonal Treg infusion was 35days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors, A2-CAR Tregs slightly increased median graft survival from 11 to 14days, which was further extended to >100days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients.

In utero exposure to alloantigens primes alloimmunization to platelet transfusion in mice.

Platelet transfusions remain a mainstay of treatment for many patients with thrombocytopenia, but can lead to alloantibodies to Human Leukocyte Antigens (anti-HLA) resulting in inadequate responses to subsequent platelet transfusions (refractoriness), as well as complicate transplantation. Despite substantial decreases in alloimmunization with the implementation of leukoreduction, a significant percentage of patients still become alloimmunized following platelet transfusions. It remains unclear why some patients make anti-HLA antibodies, but others do not make anti-HLA antibodies even with chronic transfusion. Antecedent pregnancy correlates with risk of alloimmunization due to platelet transfusion in humans - however, isolation of pregnancy as a single variable is not possible in human populations. A tractable murine model of pregnancy and transfusion was engineered by breeding C57BL/6 (H-2b ) dames with BALB/c (H-2d ) sires. After pregnancy, female mice were transfused with leukoreduced platelets from F1 (H-2b/d ) donors that expressed the same paternal major histocompatibility complex (MHC) H-2d alloantigens as the sires. Control groups allowed isolation of pregnancy or transfusion alone as independent variables. Alloimmunization was determined by testing serum for antibodies to H-2d MHC alloantigens. No alloantibodies were detected after pregnancy alone, or in response to transfusion of platelets alone; however, significant levels of alloantibodies were detected when pregnancy was followed by transfusion. These findings isolate antecedent pregnancy as a causal contribution to increased frequencies of alloimmunization by subsequent platelet transfusion in mice and provide a platform for ongoing mechanistic investigation.

Haploidentical mixed chimerism cures autoimmunity in established type 1 diabetic mice.

Clinical trials are currently testing whether induction of haploidentical mixed chimerism (Haplo-MC) induces organ transplantation tolerance. Whether Haplo-MC can be used to treat established autoimmune diseases remains unknown. Here, we show that established autoimmunity in euthymic and adult-thymectomized NOD (H-2g7) mice was cured by induction of Haplo-MC under a non-myeloablative anti-thymocyte globulin-based conditioning regimen and infusion of CD4+ T cell-depleted hematopoietic graft from H-2b/g7 F1 donors that expressed autoimmune-resistant H-2b or from H-2s/g7 F1 donors that expressed autoimmune-susceptible H-2s. The cure was associated with enhanced thymic negative selection, increased thymic Treg (tTreg) production, and anergy or exhaustion of residual host-type autoreactive T cells in the periphery. The peripheral tolerance was accompanied by expansion of donor- and host-type CD62L-Helios+ tTregs as well as host-type Helios-Nrp1+ peripheral Tregs (pTregs) and PD-L1hi plasmacytoid DCs (pDCs). Depletion of donor- or host-type Tregs led to reduction of host-type PD-L1hi pDCs and recurrence of autoimmunity, whereas PD-L1 deficiency in host-type DCs led to reduction of host-type pDCs and Helios-Nrp1+ pTregs. Thus, induction of Haplo-MC reestablished both central and peripheral tolerance through mechanisms that depend on allo-MHC+ donor-type DCs, PD-L1hi host-type DCs, and the generation and persistence of donor- and host-type tTregs and pTregs.

Distinct roles for major and minor antigen barriers in chimerism-based tolerance under irradiation-free conditions.

Eliminating cytoreductive conditioning from chimerism‐based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade [CB] and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell‐mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC‐mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell‐mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.

Induction of Haploidentical Mixed Chimerism Cures Autoimmune Type 1 Diabetes

Abstract Type 1 diabetes is an autoimmune disease in association with particular MHC, and induction of MHC-matched mixed chimerism cannot reverse autoimmunity. Haploidentical HCT has gradually become a common practice for treating non-malignant diseases, but it remains unknown whether induction of haploidentical mixed chimerism can reverse autoimmunity. In the current studies, we induced mixed chimerism in NOD mice (H2-Ag7) by transplanting bone marrow and CD4+ T-depleted spleen cells from haploidentical (B6 × B6g7) F1 donors (H2-Ab/g7). We found that haploidentical mixed chimerism eliminated insulitis as well as prevented in pre-diabetic and cured diabetes in new-onset WT NOD mice. Interestingly, although I-As was thought to be autoimmune susceptible, induction of haploidentical mixed chimerism with I-Ag7/s transplants also effectively eliminate insulitis. Furthermore, the mixed chimerism markedly reduced the percentage of host-type but not donor-type CD4+CD8+ thymocytes and increased CD11c+CD8−SIRPα+CD11b+ migratory DC that can mediate thymic negative selection in the chimeric thymus. In the peripheral, percentage of tetramer+ autoreactive host-type Tcon cells was markedly reduced and part of the residual T cells became PD-1hiIL-7Rαlo anergic/exhausted, while percentage of host-type CD4+Foxp3+ Treg cells significantly increased. Additionally, in the pancreas of mixed chimeric BDC2.5 NOD mice, Treg cells among tetramer+ autoreactive T cells increase 5–10 fold. Taken collectively, haploidentical mixed chimerism reverses autoimmunity and eliminating insulitis in NOD mice by enhancing DC mediated negative selection in thymus and augmenting expansion of Treg, especially antigen specific Treg cells, in the periphery.

134 Effects of Gas Tension During Culture upon Development of Bovine Embryos from Beef (Nellore) and Dairy (Girolando) Breeds

Culture of bovine embryos is a critical step during in vitro embryo production (IVEP) and, as such, has been the focus of numerous studies on cattle IVEP. Improvements of culture conditions to mimic the in vivo maternal microenvironment involves studying the optimal gas tension for pre-implantation embryonic development. In the commercial conditions, there is great variability in results, in part because of the difference between breeds and donors. The objective of this study was to evaluate the effects of culture in high or low oxygen tension upon the development of embryos from a crossbred dairy breed (Girolando F1; Gir × Holstein) and a beef Bos indicus breed, Nellore. We collected data from an IVEP commercial operation located in a tropical area of southeastern Brazil (Minas Gerais State) from February to May 2017. The study was designed in a 2 × 2 factorial arrangement of treatments: 2 O2 tensions during culture (5%, low O2 v. 20%, high O2) and 2 breeds (Nellore, beef v. Girolando F1, dairy). Thus, the following 4 groups were studied: Nellore-high O2 (n = 86 donors), Nellore-low O2 (n = 107 donors), Girolando F1-high O2 (n = 114 donors), and Girolando F1-low O2 (n = 110 donors). Outcome variables were the number of cleaved embryos 72 h post-insemination (hpi), cleavage rate relative to the total number cumulus–oocyte complexes (COC) put in culture, number and percentage of blastocysts 192 hpi relative to the structures kept in culture. Variables that were not normally distributed were transformed using the formula log(y + 0.05). Data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) for the main effects of gas tension (low v. high O2) and breed (Girolando F1 v. Nelore). Results are shown as mean ± SEM. Gas tension affected the number of cleaved embryos (10.52 ± 0.92 v. 8.33 ± 0.72 for high and low O2, respectively; P < 0.01) and cleavage rates (40.58 ± 2.49 v. 44.41 ± 2.88 for high and low O2; P < 0.01 in Nellore), but did not affect these variables in Girolando F1 donors (13.23 ± 1.33 v. 10.76 ± 0.76 cleaved embryos, for high and low O2; P = 0.63; 58.01 ± 2.00 v. 60.19 ± 1.97 cleavage rate, for high and low O2; P = 0.80). Nonetheless, the number and percentage of blastocysts were not affected by gas tension in either breed. Results for Nellore were 4.99 ± 0.56 v. 3.51 ± 0.38 blastocysts in high and low O2, respectively (P = 0.051) and 41.92 ± 3.91% v. 39.81 ± 3.77% blastocysts, in high and low O2 (P = 0.11). For Girolando F1, numbers of blastocysts were 5.84 ± 0.66 v. 4.24 ± 0.39 in high and low O2 (P = 0.19) and percentage of blastocysts 49.14 ± 2.97% v. 49.11 ± 3.40% in high and low O2 (P = 0.46). These results suggest that oxygen tension during culture affects IVEP differently depending on breed. The initial period of culture, recognised as critical in IVEP, seemed more sensitive to high O2 tension, particularly in Nellore.

Desempenho de doadoras leiteiras mestiças F1 (Gir x Holandês) no sistema de produção in vitro de embriões

RESUMO O Brasil atualmente é detentor do terceiro maior rebanho de vacas leiteiras do mundo, composto, em sua maioria, de animais mestiços F1 (Gir X Holandês), os quais são bem adaptados às áreas tropicais. O objetivo do presente estudo foi investigar a eficiência da ovum pick-up (OPU) e da produção in vitro de embriões oriundos de doadoras Girolando com sêmen sexado de touros provenientes de duas raças, Gir e Holandesa. Foram utilizados dados referentes a 232 animais de diversos rebanhos, totalizando 4334 oócitos recuperados. Os oócitos foram classificados, e as estruturas viáveis (GI, GII e GIII) foram utilizadas para produção in vitro de embriões (PIVE). Foi observada uma média de oócitos recuperados para as doadoras F1 de 18,14±1,33. Além disso, notou-se que não houve diferença entre os dois cruzamentos utilizados, considerando média de total de embriões (4,96±0,40 e 6,56±0,76) para o cruzamento F1 X HPB (Holandês preto e branco) e F1 X Gir, respectivamente. Portanto, as doadoras F1 apresentaram potencial como doadoras de oócitos no sistema de produção in vitro de embriões, independentemente da raça do touro cujo sêmen foi utilizado.

Multivariate analysis of backcross progeny of Passiflora L. (Passifloraceae) for pre-breeding genotype selection.

The Ward-MLM procedure was used to evaluate genetic variation in four backcross progenies and in their parents, hybrid F1 HD13 and donor parent Passiflora sublanceolata. Sixteen quantitative descriptors and five qualitative characteristics of relevance to ornamental flower production were assessed. Using the pseudo-F and pseudo-T² criteria, we identified four groups among these plants in two evaluation periods. In both evaluations, the BC1 plants showed greater dissimilarity to their recurrent parent, but showed high genetic similarity with the P. sublanceolata parent. The first two canonical variables produced by the Ward-MLM procedure accounted for over 90% of the variation in both evaluation periods, enabling the representation of diversity through two-dimensional graphics. Groups II and IV were formed in the first assessment period. Groups I and IV formed in the second period and showed plants with selection potential. We found that it was essential to use both qualitative and quantitative variables for this analysis. Assessments of quantitative descriptors indicate that the selection of BC1 plants can be performed in any of the four progenies. Because of the similarities observed for some floral descriptors between BC1 and the P. sublanceolata parent, a second generation backcross was not recommended. However, the selection of BC1 plants for evaluation and direct use as an ornamental cultivar, or as a resource in other breeding programs, can be recommended.

Donor CD4 T Cells Trigger Costimulation Blockade-Resistant Donor Bone Marrow Rejection Through Bystander Activation Requiring IL-6

Bone marrow (BM) transplantation under costimulation blockade induces chimerism and tolerance. Cotransplantation of donor T cells (contained in substantial numbers in mobilized peripheral blood stem cells and donor lymphocyte infusions) together with donor BM paradoxically triggers rejection of donor BM through undefined mechanisms. Here, nonmyeloablatively irradiated C57BL/6 recipients simultaneously received donor BM (BALB/c) and donor T cells under costimulation blockade (anti-CD154 and CTLA4Ig). Donor CD4, but not CD8 cells, triggered natural killer-independent donor BM rejection which was associated with increased production of IL-6, interferon gamma (IFN-γ) and IL-17A. BM rejection was prevented through neutralization of IL-6, but not of IFN-γ or IL-17A. IL-6 counteracted the antiproliferative effect of anti-CD154 in vitro. Rapamycin and anti-lymphocyte function-associated antigen 1 negated this effect of IL-6 in vitro and prevented BM rejection in vivo. Simultaneous cotransplantation of (BALB/cxB6)F1, recipient or irradiated donor CD4 cells, or late transfer of donor CD4 cells did not lead to BM rejection, whereas cotransplantation of third party CD4 cells did. Transferred donor CD4 cells became activated, rapidly underwent apoptosis and triggered activation and proliferation of recipient T cells. Collectively, these results provide evidence that donor T cells recognizing the recipient as allogeneic lead to the release of IL-6, which abolishes the effect of anti-CD154, triggering donor BM rejection through bystander activation.

A New Murine Model Of Chronic Graft Versus Host Disease

Chronic graft-versus-host-disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with highly variable clinical presentations. The pathophysiology of cGVHD remains to be further understood. The utilization of murine models to study cGVHD has contributed to the understanding of cGVHD, and highlights its mechanistic complexity. Here, we report a new murine cGVHD modle with obvious lung tissue damage in these mice resulted in the development of bronchiolitis obliterans (BO), which is pathopneumonic for cGVHD.Recipients 8 weeks BALB/c (H-2d) female mice, which irradiated with 700cGy dose of linear accelerator, then were injected with bone-marrow cells (1×107) and spleen cells (5×106) from (C57BL/6 x BALB/c) F1 donors (H-2bd haplotype) male mice. The observed items post-transplantation included hematopoietic reconstruction, implant, and general condition. Clinical scores were assessed every 3 days after +14 d. At + 135 d, mice were killed to evaluate the pathological changes of major target organs.Mice in transplantation groups were in hematopoietic reconstruction at +7 d, and all survived to the end point (+135 d). Chromosomes of recipient mice were in full donor chimera. Clinical scores of cGVHD group have been more than 0.6 since +90 d. These mice develop pathologic manifestations in several organs including lung, skin, and liver. Biopsy-proven BO incidence was 100%, and pathological scores of cGVHD group were 5.33±1.55, which significantly higher than those of transplantation control groups (P < 0.05).To date, most models involve parent-into-F1 combinations the transfer of MHC-mismatched cells resulted in a phenotype that resembles clinical systemic lupus erythematosus (SLE), termed SLE-cGVHD. However, some of the models do not use any pretransplantation conditioning and no obvious lung tissue damage. We exchanged parent for recipients, and provide a total boby irradiation as pretransplantation conditioning. New MHC-mismatched murine cGVHD model was easily obtained, and more relevancy to the clinical features of cGVHD. This model provides an ideal study model of clinical cGVHD pathogenesis and treatment strategies. NOTESThe project was sponsored by grants from National Natural Science Foundation (No. 81270648 and 30972790), National Public Health Grand Research Foundation (No.201202017), and Guangdong Natural Science Foundation (No. S2012010009560). Disclosures:No relevant conflicts of interest to declare.

Group A Pak Proteins Are Essential for HSC/P Engraftment.

AbstractAbstract 2624Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in critical hematopoietic stem and progenitor (HSC/P) functions such as cell migration, adhesion, proliferation, and survival. Conditional deletion of Rac1 in the hematopoietic cells results in defects in proliferation and engraftment while Rac2 germline deletion leads to enhanced cell death and increased HSC/P mobilization, most likely resulting from decreased adhesion via altered integrin signalling (Gu et al. Science, 2003). Although, Rac1 and Rac2 proteins have been shown to regulate distinct biological processes, simultaneous deletion of both Rac1 and Rac2 reveal overlapping functions in HSC/P survival, mobilization, engraftment, and retention (Cancelas et al. Nat. Med., 2006). Our studies now focus on indentifying potential downstream effector proteins of Rac that are important for normal HSC/P function(s). The best characterized Rac effector proteins are the Pak protein kinases, including Group A (Pak1, Pak2, and Pak3) Paks. Pak proteins not only have an intrinsic kinase activity but also regulate actin-dependent functions. Previous studies have demonstrated inhibition of Rac in HSC/P was associated with reduced phosphorylation of Pak1 (Cancelas et al. Nat. Med., 2006). However, the exact role Pak proteins play in HSC/P function(s) has yet to be elucidated. To further identify the possible role(s) of Pak proteins in hematopoiesis, freshly isolated lineage-, Sca-1+; c-Kit+ (LSK) cells from BoyJ(CD45.1)/C57Bl6J(CD45.2) F1 donor mice were transduced with retroviral vectors containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function, or empty vector control (Mieg3); both constructs co-express GFP. 6.0×104 GFP+ CD45.1/CD45.2 LSK cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1) bone marrow (BM) into lethally irradiated C57Bl/6J (CD45.2) recipients. Percent chimerism was measured from 3-weeks up to 14-weeks post BM transplant. We found that PID transduced LSK cells were incapable of contributing to recipient hematopoietic reconstitution compared to Mieg3 transduced controls at 14 weeks post-transplant (0/9 PID transduced demonstrated >1% engraftment vs 9/9 Mieg3 transduced which had >10% engraftment, p<0.05). To explore the underlying mechanism of this engraftment failure, we cultured transduced LSK in vitro for up to 12 days and compared growth of GFP+ vs GFP- cells. The growth of PID transduced LSK was essentially identical to non-transduced cells for the initial 7 days of in vitro culture. However between day 7 and day 10, the PID LSK cells displayed decreased growth kinetics (Fig. 1). The reduced growth in vitro was correlated with decreased proliferation as measured by BrdU incorporation. There was no change in cell survival, as assessed by AnnexinV staining. Furthermore, LSK cells transduced with a dominant negative Pak construct (DN-Pak), a more general inhibitor of Pak activity, showed defects in cytoskeletal rearrangements after stimulation with the HSC/P chemoattract, SDF-1α. These data suggest that Pak proteins may be critical downstream effectors of Rac GTPases and defective Pak activation may mechanistically link the role of Rac GTPases in regulation of both cytoskeletal and proliferative functions of HSC/P during engraftment. [Display omitted] Disclosure:Sanchez-Aguilera:Children's Hospital Boston: Employment.

Gametoclonal and Somaclonal Variation among Head Cabbage Androgenic Lines of R1 and R2 Generations Obtained from Jaguar F1 Hybrid

Head cabbage androgenic genotypes of R1 and R2 generations derived from Jaguar F1 donor hybrid wereevaluated according to their agroeconomical characters, ability for generative propagation, self-incompatibility,intraline uniformity and stability of morphological characters. Most of R1 and R2 genotypes had desiredmorphological characters. However, in R1 generation 21 lines showed lack of internal uniformity probably due tothe somaclonal variation. Genotypes of R1 and R2 generation from different embryos were more diversified thangenotypes derived from the same embryo according to self-incompatibility, ability for seed setting, vegetativeperiod, head shape, internal sump length and mass of head. Rigid selection of androgenic material at R0, R1 andR2 pedigree according to absence of somaclonal variation, agroeconomical traits, self-compatibility and seedsetting ability seemed to be crucial for the deriving stable and suitable for the breeding genotypes of headcabbage.

Transplantation tolerance induced by regulatory T cells: In vivo mechanisms and sites of action

The mechanisms by which CD4 +CD25 +Foxp3 + T (Treg) cells regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer model, we analyzed the in vivo expansion, trafficking, and effector function of alloreactive T cells and donor-specific Treg cells, in response to a full-thickness skin allograft. Fluorescent-labeled CD4 +CD25 − and antigen-specific Treg cells were transferred alone or co-injected into syngeneic BALB/c-Nude recipients transplanted with skins from (C57BL/6×BALB/c) F1 donors. Treg cells divided in vivo, migrated and accumulated in the allograft draining lymph nodes as well as within the graft. The co-transfer of Treg cells did not modify the early activation and homing of CD4 +CD25 − T cells in secondary lymphoid organs. However, in the presence of Treg cells, alloreactive CD4 +CD25 − T cells produced significantly less IFN-γ and were present in reduced numbers in the secondary lymphoid organs. Furthermore, time-course studies showed that Treg cells were recruited into the allograft at a very early stage after transplantation and effectively prevented the infiltration of effector T cells. In conclusion, suppression of rejection requires the early recruitment to the site of antigenic challenge of donor-specific Treg cells, which then mainly regulate the effector arm of T cell alloresponses.

Mesenchymal Stem Cells (MSC) Are Not Intrinsically Immune Privileged: Direct Demonstration in TCR Transgenic Mice and by Imaging of Allogeneic Luciferase+ MSC in Immune Competent Vs Immune Deficient Mice

Mesenchymal stem cells (MSC) can induce a broad array of immunomodulating mechanisms. Furthermore, several studies have advocated that MSC can be transplanted across allogeneic barriers without eliciting an immune response. This notion was based on clinical case reports or animal studies using highly sensitive techniques such as polymerase chain reaction, fluorescent in-situ hybridization or enhanced green fluorescent protein, enabling detection of rare cells in different tissues. However, a recent study comparing syngeneic and allogeneic MSC demonstrated that while the former cells induced tolerance to allogeneic bone marrow (BM) the use of donor type allogeneic MSC was counteractive leading to enhanced rejection of the BM cells. Thus it was indicated for the first time that allogeneic MSC might induce immune memory rather than tolerance to donor type cells. In the present study we directly addressed this possibility by infusing intravenously MSC isolated from H2db (C57BLxBalb) F1 donors, into TCR transgenic mice (the 2C model, C57BL/6 background), in which CD8+ T cells express a TCR transgene against H2d. Mice in the control group were infused with phosphate buffered saline (PBS). Thirty days after the first immunization, the mice were re-challenged with MSC and 5 days later peripheral blood CD8+ T cells were examined by FACS for the acquisition of a memory phenotype (CD122+, CD44+and CD62Llow). This assay revealed a significantly elevated level of memory CD8 T cells (6.7±0.45 %) in the re-challenged mice compared to that found in the control group of naïve mice (0.4 ± 0.5 %, P<0.01). Further evidence for induction of immune memory by MSC was directly demonstrated by non-invasive imaging of bone marrow derived MSC isolated from Luciferase+ (Luc+) transgenic FVB-L2G85 mice (MSC-Luc+). Thus, while MSC (0.9–1.8 *108 MSC/Kg) infused intravenously or intraperitonealy into immune competent Balb/c mice survived longer (27% survival at 35 days) compared to adult fibroblast (Fib-Luc+ ) (9% survival at 15 days, p<0.01), this prolonged survival of MSC is significantly shorter compared to that exhibited in immune deficient Balb-Nude and NOD-SCID recipients (100% survival at 120 days, p<0.01), indicating that the MSC cannot evade immune rejection although capable of delaying it. The enhanced survival of MSC in Balb-Nude mice strongly indicates that rejection of these cells in normal Balb/c mice is mediated by T cells. Remarkably, rejection was found upon infusion of about 100- fold more MSC, compared to the cell number, which can currently be generated ex-vivo for transplantation in humans (around 1*106/Kg). Infusion of a lower number of MSC (4 *107 MSC/Kg) was found to be even less effective (9% survival at 15 days, p<0.01). To define whether the allogeneic rejection of MSC-Luc+ or Fib-Luc+ is associated with induction of immune memory, we re-challenged mice previously rejecting 2*106 Fib-Luc+ or MSC-Luc+ cells, with Fib-Luc+ cells. Thus, 30 days after rejection of the first inoculums the recipients were implanted with a second transplant of 2*106 Fib-Luc+ cells. Our data reveals that graft rejection was significantly more rapid in re-transplanted Balb/c mice. While a significant density of Fib-Luc+ cells can be detected in all transplanted recipients at day two, survival at day 5 was reduced to 27% or 18% in mice primed with Fib or MSC, respectively, compared to 81% in naive recipients (p<0.01). Survival of Fib-Luc+ cells in re-challenged mice was further reduced at day 9 (9% or 0% in mice previously receiving Fib or MSC, respectively) compared to 72% survival in naive recipients, p<0.01). Collectively, these results demonstrate that MSC are not intrinsically immune privileged and under allogeneic settings these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (‘off the shelf‘) MSC is commonly advocated for a variety of clinical applications, our results strongly suggest that long term survival of allogeneic MSC likely represents a major challenge. Further studies attempting to overcome rejection of donor MSC in the context of hematopoietic stem cell transplantation or in conjunction with co-stimulatory blockade are warranted.

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: analysis of the role of donor T cells in the induction of autoimmunity

The injection of (C57BL/6 x BALB/c) F1 spleen cells into BALB/c newborn mice leads to activation of persisting F1 donor B cells and development of a lupus-like syndrome in tolerized BALB/c mice. This syndrome is characterized by hypergammaglobulinaemia, high levels of anti-DNA and anti-Sm antibodies, circulating immune complexes and deposits of immunoglobulin in renal glomeruli. The role of donor T cells in this model was investigated by injecting the newborn mice with F1 cells depleted in different T cell subsets by using specific monoclonal antibodies (MoAbs). Tolerance, as shown by an absence of H-2b-specific CTL alloreactivity and persistence of immunoglobulin bearing the donor allotype were observed in mice injected with F1 cells previously depleted in the CD4+ and/or CD8+ T cell subsets as well as in those which received Thy-1+-depleted F1 spleen cells. In these mice, a typical autoimmune syndrome was found, including splenomegaly and lymphadenopathy, anti-ssDNA and anti-aortic myosin IgG antibodies and renal deposition of immunoglobulin. However, some quantitative changes were seen: the levels of anti-aortic myosin antibodies were lower in mice tolerized with CD4+-depleted F1 cells than in those receiving untreated F1 cells. Conversely, higher levels of these autoantibodies were observed in mice tolerized with CD8+-depleted F1 cells. These results suggest that mature donor T cells are not necessary neither for the establishment of neonatal tolerance to alloantigens nor for the activation of F1 donor B cells in the production of the autoimmune syndrome in tolerant mice, but they may contribute in the regulation of the expression of autoreactive B cell clones.

Immunodeficiency in RFM/(T6xRFM)F1 mouse chimaeras with lethal host-versus-graft syndrome.

Rather than central tolerance, the perinatal inoculation of related F1 hybrid spleen cells into inbred mice may result in host-versus-graft (HVG) reactions manifested as transient autoimmunity, or as a lethal immunodeficiency syndrome. RFM/(T6xRFM)F1 chimaeras with lethal disease die in 30 days with lymphosplenomegaly, immune complexes and impaired immune responses. The present studies used in vitro proliferation assays to show that the HVG reaction caused hyperplasia sufficient to account for the lymphosplenomegaly, while also causing severe impairment of splenic and nodal cell responses to concanavalin A (Con A) and to bacterial lipopolysaccharide (LPS). By 25 days, HVG mice could not distinguish between self and non-self as judged by mixed lymphocyte reactions (MLR) to RFM, (T6xRFM)F1 and third party A/J cells. There were no indications that host cells reactive to F1 donor cells had undergone clonal deletion, anergy or expansion. Flow cytometry revealed that donor T lymphocytes achieved stable engraftment, mostly in the nodes, despite the HVG reaction. Taken together with previous observations, these studies showed that HVG reactions in young parent F1/chimaeras can result in an immunodeficiency state which is characterized by an early appearing, profound and persistent impairment of both host and donor T and B cell functions. The results suggest that HVG reactions can contribute directly to immune deficits seen after clinical allogeneic bone marrow transplantation.

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/c×C57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4+ and CD8+ T cells 7 and 21 days after donor cell transfer. The populations of CD8+CD45RBlow and CD8+CD44high cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8+ T cells leading to enhanced graft survival.

Effects of donor oocytes and culture conditions on development of cloned mice embryos.

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 x DBA/2) and B6CBAF1 (C57BL/6 x CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos.

Rat laryngeal transplant model: technical advancements and a redefined rejection grading system.

The rat laryngeal transplant model, introduced in 1992, laid the basic science foundation that contributed to the first successful human larynx transplant in 1998. Over 1,500 rat transplants later, numerous modifications have improved the model, increasing the initially reported evaluability rate of 50% to almost 100%. The observed histologic rejection process has been altered, as well. We report the technique modifications, as well as the results of a new study using nonimmunosuppressed, allogenic transplantations, in order to define a new rejection grading system. Using the updated model, we performed 50 transplantations between LBN(f1) donor rats and Lewis recipients. Larynges from 8 groups of 5 to 10 animals were harvested at intervals between 1 and 20 days after transplantation. The larynges were examined grossly and microscopically in a blinded manner for evidence of rejection. A multivariable linear regression model was used to define a new rejection grading scale. All 50 animals survived their assigned posttransplantation period. No animals exhibited vascular thromboses, for an evaluability rate of 100%. Histologic criteria in 7 categories and gross criteria in 5 categories demonstrated increased rejection proportional to the amount of time after transplantation. The equation [Group = -2.209 + 0.465 x (Size) + 0.901 x (VA Flow) + 0.613 x (Muscle) + 1.040 x (Thyroid)] reproducibly grades rejection on the basis of gross and histologic findings. New modifications to the rat laryngeal transplant model have conferred greatly improved animal survival rates and anastomotic patency rates. Additionally, the observed rates of rejection have been reduced in comparison to those of initial studies. This updated rejection staging system will be used to compare immunosuppressive regimens in future rat laryngeal transplant studies.