Nicholas Zavazava

HLA-G has a concentration-dependent effect on the generation of an allo-CTL response

Human leucocyte antigen (HLA) ‐G is expressed on trophoblast cells during pregnancy, suggesting a role in protection of the semiallogeneic fetus. Published data suggest that HLA‐G protects a cell against natural killer cell lysis. It has been hypothesized that HLA‐G may also protect the fetus by preventing allo‐cytotoxic T lymphocyte (CTL) responses. To test this hypothesis, we assayed the effects of various concentrations of purified HLA‐G on CTL response in a mixed lymphocyte culture (MLC) system. We found that concentrations ≥ 0·1 µg/ml of HLA‐G suppressed the allo‐CTL response by 30–100% over the control, but, paradoxically, concentrations of 0·01–0·05 µg/ml of HLA‐G augmented the allo‐CTL response by 25–50% over the control. Concentrations ≤ 0·001 µg/ml HLA‐G had no effect. Addition of HLA‐G to preprimed allo‐CTL effector cells did not affect their killing ability. Allo‐CTL suppressive doses of HLA‐G induced a T helper type 2 (Th2) cytokine response, whereas allo‐CTL‐enhancing doses of HLA‐G induced a Th1‐type cytokine response. HLA‐G purified from first‐trimester placenta does not affect allo‐proliferative responses nor does it alter the percentage of CD4+ or CD8+ T cells in MLCs. These findings support a potential role for HLA‐G‐mediated suppression of allo‐CTL formation in normal pregnancies. In addition, the effects observed at lower concentrations of HLA‐G may have interesting implications for the condition of pre‐eclampsia in which concentrations of this HLA class I molecule are reduced.

Immunological Applications of Stem Cells in Type 1 Diabetes

Current approaches aiming to cure type 1 diabetes (T1D) have made a negligible number of patients insulin-independent. In this review, we revisit the role of stem cell (SC)-based applications in curing T1D. The optimal therapeutic approach for T1D should ideally preserve the remaining β-cells, restore β-cell function, and protect the replaced insulin-producing cells from autoimmunity. SCs possess immunological and regenerative properties that could be harnessed to improve the treatment of T1D; indeed, SCs may reestablish peripheral tolerance toward β-cells through reshaping of the immune response and inhibition of autoreactive T-cell function. Furthermore, SC-derived insulin-producing cells are capable of engrafting and reversing hyperglycemia in mice. Bone marrow mesenchymal SCs display a hypoimmunogenic phenotype as well as a broad range of immunomodulatory capabilities, they have been shown to cure newly diabetic nonobese diabetic (NOD) mice, and they are currently undergoing evaluation in two clinical trials. Cord blood SCs have been shown to facilitate the generation of regulatory T cells, thereby reverting hyperglycemia in NOD mice. T1D patients treated with cord blood SCs also did not show any adverse reaction in the absence of major effects on glycometabolic control. Although hematopoietic SCs rarely revert hyperglycemia in NOD mice, they exhibit profound immunomodulatory properties in humans; newly hyperglycemic T1D patients have been successfully reverted to normoglycemia with autologous nonmyeloablative hematopoietic SC transplantation. Finally, embryonic SCs also offer exciting prospects because they are able to generate glucose-responsive insulin-producing cells. Easy enthusiasm should be mitigated mainly because of the potential oncogenicity of SCs.

Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor‐specific antigens

The Human MHC Project aims at comprehensive cataloging of peptides presented within the context of different human leukocyte antigens (HLA) expressed by cells of various tissue origins, both in health and in disease. Of major interest are peptides presented on cancer cells, which include peptides derived from tumor antigens that are of interest for immunotherapy. Here, HLA‐restricted tumor‐specific antigens were identified by transfecting human breast, ovarian and prostate tumor cell lines with truncated genes of HLA‐A2 and HLA‐B7. Soluble HLA secreted by these cell lines were purified by affinity chromatography and analyzed by nano‐capillary electrospray ionization‐tandem mass spectrometry. Typically, a large peptide pool was recovered and sequenced including peptides derived from MAGE‐B2 and mucin and other new tumor‐derived antigens that may serve as potential candidates for immunotherapy.

Interaction of papain-digested HLA class I molecules with human alloreactive cytotoxic T lymphocytes (CTL)

Acute immunological rejection events of transplanted allogeneie organs are strongly dependent on T cell reactivity against foreign MHC products. The recognition requirements of alloreactive cytotoxic T cells arc of particular interest for finding approaches to modulating allorcactivity. The role of the allogeneic MHC molecule itself and/or an associated peptide in the interaction with the T cell receptor is still, however, unclear. Our studies have focused on the interactions of papain‐digested HLA class I molecules with alloreactivc CD8+ CTL. These polypeptidcs, consisting of the polymorphic α1 and α2 and the monomorphic α3 domains, were used in both soluble and immobilized form to study their functional effects on anti‐HLA‐A2 reactive CTL. Purified polypeptides were of molecular mass 32–34 kD. HLA‐A2 polypcptides (0–55 μg/ml) in soluble form induced half‐maximal reduction of CTL cytotoxicity. These concentrations were quantitatively comparable to the effective doses of intact HLA class I molecules, which contain the hydrophobic transmembrane domain and the intracytoplasmic tail. In addition, specific activation requirements of these CTL were investigated in a serine esterasc release assay. Maximal degranulation was observed after 2 h of antigen contact. Purified HLA class I molecules allospccifically activated the anti‐HLA‐A2 CTL to degranulate serine esterase, when immobilized on plastic microtitre plates. Thus, polypeptidcs containing the polymorphic αl and α2 domains of human class I molecules potentially modulate the cytotoxic T cell response. This might have implications for the reduction or prevention of allograft rejection in recipients of foreign organs.

ES-Cell Derived Hematopoietic Cells Induce Transplantation Tolerance

Background Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells. Methodology/Principal Findings Here, we derived CD45+ HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts. Conclusions Our data show, for the first time, the efficacy of ES-derived CD45+ HPCs to engraft in allogenic recipients without the use of immunosuppressive agents, there by protecting cardiac allografts from rejection.

CHARACTERIZATION OF SOLUBLE HLA MOLECULES IN SWEAT AND QUANTITATIVE HLA DIFFERENCES IN SERUM OF HEALTHY INDIVIDUALS

Soluble class I molecules were immunoprecipitated from human sweat and serum using the BB7.7 monoclonal antibody (mAb) coupled to immunomagnetic beads. Molecules were analysed biochemically on SDS‐PAGE gels and finally by 1D‐isoelectric‐focusing (IEF). Serum‐ and sweat‐HLA IEF‐band patterns of the same individual were fully identical, showing that HLA excreted in sweat possess polymorphic structures like those in serum. Quantitatively, we used a highly sensitive competitive enzyme‐linked immunosorbent (ELISA) assay to determine soluble class I concentrations. The first group was that of non‐HLA‐A9 and ‐Bw62 sera, which were found to contain HLA levels with a mean concentration of 0.82 ± 0.63 μg/ml (n= 44). However, sera that were HLA‐A23 or −24 (splits of HLA‐A9) contained higher levels, with a mean of 3.2 ± 0.94 μg/ml (n= 20). Similarly, HLA‐Bw62 individuals had a higher mean of 2.05 ± 0.65 μg/ml (n= 10). The difference of the HLA‐A9 group to the first group was statistically highly significant, P < 0.0001, and that of the HLA‐Bw62 to the first was also significant, P < 0.004. Individuals who were both HLA‐A9 and ‐Bw62 (n = 5) did not express significantly higher levels than those who only had one of these specificities. Sweat HLA levels had a mean of 0.42 ± 0.4 μg/ml (n= 10). These results show for the first time that soluble class I peptides are excreted in relatively high concentrations in sweat and possess polymorphic structures identical to those of serum HLA and that serum HLA levels are allotype dependent.

Diversity of the human monocyte/macrophage system as detected by monoclonal antibodies.

Four monoclonal antibodies against the human monocyte/macrophage system, termed KI‐M1, KI‐M6, KI‐M7, and KI‐M8, are described with regard to their immunohistochemical tissue distribution pattern and their subcellular reactive sites. The differences found applying these analyses are also reflected by the various molecular weights of the recognized antigens. Based on these data it is proposed that the monocyte/macrophage system can be divided into the phagocytosing compartment on one hand and the immune accessory compartment on the other hand; the latter constitutes the interdigitating reticulum cells, the indeterminate dendritic cells, and the Langerhans cells, as well as the follicular dendritic cells (dendritic reticulum cells) as the accessory cells for T‐ and B‐cell immune response, respectively.

Oral feeding of an immunodominant MHC donor-derived synthetic class I peptide prolongs graft survival of heterotopic cardiac allografts in a high-responder rat strain combination

The efficacy of two synthetic major histocompatibility complex (MHC)‐derived DA (RT1.Aa) 25‐mer peptides (residues 56–80 and 96–120) to modulate alloreactivity was tested in Lewis (RT1.A1) responder animals. The DA peptide 56–80, but not peptide 96–120, induced delayedtype hypersensitivity (DTH). DTH was significantly reduced by oral feeding of peptide 56–80, P = 0.004. In addition, oral feeding of this peptide in combination with a short course of cyclosporin A (CsA) prolonged graft survival of 60% of heterotope transplanted DA cardiac allografts in Lewis recipient rats. Long‐term survivors developed low levels of allo‐antibodies against donor tissue as compared to rejecting animals and increased levels of interleukin‐4 (IL‐4) within the allograft. Similarly, IL‐4‐secreting splenocytes were identified by flow cytometry in these animals, indicating a Th2‐type cytokine pattern. However, graft survival was particularly limited to cardiac allografts because donor‐type skin grafts were acutely rejected in tolerant animals. It is interesting that residue alignment of peptide 56–80 to the motif of the RT1.A1 molecule showed a preferred class I motif within this sequence, suggesting indirect presentation of this peptide to recipient T cells. Thus, peptide 56–80 appears to represent a dominant epitope that can be exploited for establishing tolerance in this transplantation strain combination. J. Leukoc. Biol. 67: 793–800; 2000.

Dynamic HoxB4-regulatory network during embryonic stem cell differentiation to hematopoietic cells

Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies to treat hematologic diseases. To date, HoxB4 remains the most effective transcription factor (TF) the overexpression of which in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop more efficient protocols for in vitro derivation of HSCs. In the present study, we performed global gene-expression profiling and ChIP coupled with deep sequencing at 4 stages of the HoxB4-mediated ESC differentiation toward HSCs. Joint analyses of ChIP/deep sequencing and gene-expression profiling unveiled several global features of the HoxB4 regulatory network. First, it is highly dynamic and gradually expands during the differentiation process. Second, HoxB4 functions as a master regulator of hematopoiesis by regulating multiple hematopoietic TFs and chromatin-modification enzymes. Third, HoxB4 acts in different combinations with 4 other hematopoietic TFs (Fli1, Meis1, Runx1, and Scl) to regulate distinct sets of pathways. Finally, the results of our study suggest that down-regulation of mitochondria and lysosomal genes by HoxB4 plays a role in the impaired lymphoid lineage development from ESC-derived HSCs.

Efficacy, safety and tolerability of recombinant factor VIII (REFACTO) in patients with haemophilia A: interim data from a postmarketing surveillance study in Germany and Austria.

Summary.  An open‐label, multicentre, postmarketing surveillance study conducted in Germany and Austria with recombinant factor VIII (REFACTO®) has enrolled 217 patients (mean age 26.3 years) from 38 haemophilia centres during the first 4.8 years. Most patients (188/217; 86.6%) had severe to moderately severe haemophilia A, of whom 153 completed sufficient diary information for the main efficacy analysis. These 153 patients experienced a median of 6.6 (interquartile range 1.4–18.6) bleeding episodes per year. Patients treated with prophylaxis experienced a median of 4.4 (1.1–9.3) bleeds per year, while patients treated on‐demand experienced a median of 22.8 (11.3–29.0) bleeds per year. Overall, most physicians (41/43 [95.3%]) were ‘very satisfied’ or ‘satisfied’ with the efficacy of REFACTO in the treatment of bleeding episodes. A total of 137 non‐serious adverse events have been reported in 52/217 patients (24.0%) to date. In addition, 129 serious adverse events in 87 patients (40%) were reported, including 41 cases of ‘less than expected therapeutic effect’ (LETE). Of these, 39 LETE cases were reported in one centre; however, patients in this centre experienced considerably fewer bleeding episodes per year than patients outside this centre. Overall, six patients (2.8%) have developed de novo inhibitors, three of which were considered high titre. Four of these patients were at high risk (0–50 exposure days [ED]) of inhibitor formation, one was at intermediate risk (51–100 ED) and one was at low risk (>100 ED). These results emphasize the benefit of postmarketing surveillance and, overall, this study confirms the efficacy, safety and tolerability of REFACTO in the treatment of patients with haemophilia A.