Anatolij Horuzsko

Tolerization of dendritic cells by HLA-G

The expression of HLA‐G at the fetal‐maternal interface during pregnancy and in transplanted tissue makes this a key molecule in the acceptance of a semiallogeneic fetus and allogeneic transplant. Dendritic cells (DC) play a critical role in the control of innate and adaptive immune responses. DC are present in maternal decidua, but must be kept under tight control. Here we describe the mechanism of tolerization of DC by HLA‐G through inhibitory receptor interactions. The HLA‐G‐ILT (immunoglobulin‐like transcript) interaction leads to development of tolerogenic DC with the induction of anergic and immunosuppressive T cells. Using human monocyte‐derived DC and ILT4‐transgenic mice, we show that (i) HLA‐G induces the development of tolerogenic DC with arrest maturation/activation of myeloid DC, (ii) HLA‐G‐modified DC induce differentiation of anergic and immunosuppressive CD4+ and CD8+ effector T cells, and (iii) the gene expression profile provides evidence that HLA‐G induces tolerogenic DC by disruption of the MHC class II presentation pathway. Ligation of ILT4 receptor on DC from transgenic mice diminished peptide presentation by MHC class II molecules and significantly prolonged allograft survival. These findings provide support that HLA‐G is an important tolerogenic molecule on DC for the acceptance of a semiallogeneic fetus and transplanted tissue/organ.

HLA-G inhibits the functions of murine dendritic cells via the PIR-B immune inhibitory receptor

Human histocompatibility leukocyte antigen (HLA)‐G is an MHC class Ib molecule that has been proposed to regulate immune responses during pregnancy. One of the possible mechanisms of that modulation is based on its interaction with immunoglobulin‐like transcript (ILT) receptors. In this study, we show that HLA‐G modifies the function of murine dendritic cells via interactions with the paired immunoglobulin‐like inhibitory receptor, a homologue of the human inhibitory receptor ILT4. Triggering of the immune inhibitory receptors resulted in prolongation of allogeneic graft survival. This demonstration forms the basis for exploring and exploiting HLA‐G molecules and immune inhibitory receptors in the development of a new therapeutic strategy to improve allogeneic graft survival.

Human ILT2 receptor associates with murine MHC class I molecules in vivo and impairs T cell function

Immunoglobulin‐like transcript 2 (ILT2/LILRB1/LIR1/CD85j) is an inhibitory receptor broadly expressed on leukocytes and antigen‐presenting cells that recognizes HLA‐class I and human cytomegalovirus UL18 proteins. The function of this receptor is to generate negative signals or to inhibit positive signals. Here, we demonstrate the model to study the function of ILT2 in vivo using a newly generated transgenic mouse expressing the human inhibitory receptor on T, B, NK, and NKT cells. ILT2 expression affects thymocyte development and targets the proximal TCR signaling pathway, resulting in long‐term survival or acceptance of skin allografts. The phenotype and constitutive tyrosine phosphorylation of ILT2 in transgenic mice illustrate the possible existence of a murine ligand. We report here that H‐2Db, a murine MHC class I molecule, associates with human ILT2 in vivo. This engagement with ILT2 directs effects on thymocyte development, negative regulation of TCR signaling, T cell activation, and alloimmune responses. Our finding provides support for an important inhibitory function of ILT2 in T cells in vivo and opens up strategies for targeting proximal TCR signaling for prevention of allograft rejection.

Mixed lymphocyte culture--evidence that pretransplant transfusion with platelets induces FcR and blocking antibody production similar to that induced by leukocyte transfusion.

Experimentation clinique: les transfusions de plaquettes purifiees peuvent induire un «facteur bloquant» efficace avant transplantation (renale)

Induction of HLA-G-specific human CD8+ T cell lines by stimulation across a polymorphism of HLA-G

THE NATURE of alloantigens seen by T lymphocytes and the role of peptide ligands in allorecognition are major factors in understanding the mechanisms of allograft acceptance. T cells of the maternal decidua have the capacity to develop immune responses against paternal HLA-G antigens. It has been suggested that paternal HLA-G antigens may elicit an immune response by T cells of the maternal decidua. Evidence for this comes from structural characteristics of HLA-G, polymorphisms in the gene, and thymic selection events restricted by HLA-G. Strong immune responses mediated by alloreactive and HLA-G-specific T cells under special pathologic circumstances may contribute to fetal loss. Our aim was to determine if human peripheral T cells can recognize HLA-G molecules as alloantigens in vitro. To address this question, the development of human cytotoxic T lymphocytes from 20 healthy individuals was examined by stimulation with myelogeneous leukemia K562 cells transfected with the HLA-G*01011 allele. HLA-G genotypes were determined by DNA-sequence analysis of the above individuals by PCR amplification of genomic DNA using HLA-G-specific primers, followed by fluorescent DNA sequencing using dye primer chemistry to increase our ability to detect heterozygotes. No CTL activity was obtained from individuals who were homozygous or heterozygous for the HLA-G*01011, HLA-G*01012, and HLAG*01013 alleles. Strong CTL responses were found in individuals with nucleotide variations in exon 3 that result in amino acid substitutions at positions 110 (Leu to Ile), 130 (Leu to Phe), and 178 (Met to Ile). Cytotoxic activity was blocked when target cells were preincubated with W6/32 mAb. HLA-G-specific CTL lines were established to examine whether alloreactive CTLs would recognize HLA-G on the surface of the antigen-processing defective cell line T2. These CTLs were able to kill T2 HLA-G transfectants with a specific lysis of 25% to 37%, indicating that some alloreactive HLA-G specific T cells are peptide independent. Our results demonstrate that amino acid substitutions in the peptide binding groove of HLA-G molecules can elicit HLA-G-specific T cell response.

Selective effect of noncytotoxic blocking alloantibodies produced after platelet transfusions on MLC, and mitogen and soluble antigen-induced responses of human lymphocytes

The transfusion of blood components (buffy coat and platelets) may induce characteristic alloimmune response or suppressive regulation that have, in certain cases, a beneficial effect on allograft survival. The blocking effect of the sera of donors immunized with platelets on mixed lymphocyte culture and on the response of lymphocytes to mitogen as well as soluble antigen (PPD, tetanus toxoid) stimulation was studied. Six sera from 7 volunteers displayed a strong and significant nonspecific MLC blocking effect that was detectable on the 10th day following the second platelet transfusion (PT). Incubation of isolated stimulator and effector cell population with this “blocking sera” showed that the latter are involved in the mediation of suppression in the MLC test. This inhibitory effect is associated with the serum IgG fraction lacking any correlation with either class I or class II specific cytotoxic antibodies. Selective blocking behavior was found on transformation activity induced by mitogens or soluble antigens. Thus, sera of platelet-transfused volunteers decreases the responsiveness of lymphocytes to phytohemagglutinin, while the response to concanavalin A, tetanus toxoid, and PPD was not suppressed. Separate treatment of T and B lymphocyte populations and monocytes with the blocking sera showed that only T and B lymphocytes are targets, and not the monocytes for the inhibition in the case of PHA-induced proliferation. Indirect evidence may support the notion that MLC-inhibiting and FcR-blocking antibodies may be analog products of a regulatory alloimmune response induced by leukocytes that are partially responsible for the beneficial transfusion effect in organ transplantation.

Non-cytotoxic blocking antibodies and suppressor cells induced by donor-specific transfusions in healthy volunteers and potential kidney transplant recipients

Donor-specific transfusion (DST)-induced immunosuppression plays a significant role in clinical and experimental transplantation. To clarify the mechanism of suppression on alloreactivity the suppressor cell induction and the non-cytotoxic blocking antibody production and importance was studied in 15 healthy volunteers and 3 kidney transplant recipients (KTR) after DST on mixed lymphocyte culture (MLC). Significant decrease of anti-donor MLC response was found in all of KTR and in 12 cases of the 15 transfused volunteers. Of 18 cases, 15 had blocking factors in their post-DST serum which strongly (52-91%) inhibited the MLC response. The IgG fractions isolated from the blocking sera were responsible for this inhibition. Eighty-one percent of non-cytotoxic blocking antibodies affected the responder cells in MLC and reacted with third party responder cells as well. Both buffy coat and platelet transfusions evoked production of the non-specific blocking antibodies. Our data strongly suggest that DST induces not only the differentiation of suppressor cells and production of anti-idiotypic antibodies, but also the appearance of non-specific, non-cytotoxic antibodies, which may participate in the cell-mediated immune suppression of the alloimmune reactivity.

Long-term related kidney graft survival in high-risk patients after monitored donor-specific transfusion protocol

Abstract Altogether 57 patients were included in the related kidney transplantation program. Forty‐four recipients were mixed lymphocyte culture (MLC) negative or slightly positive (SI < 7) against their mother/father donor, and most of them showed Fey RII [erythrocyte antibody inhibition (EAI)] blocking antibody in their sera as the consequence of previous random transfusion. Thirteen patients showed significantly high MLC reactivity against their prospective parent donor (SI < 7) and had no EAI blocking antibody in their sera. The latter group was immunized either by buffy coat or purified platelets obtained from their donor in general two or three times at biweekly intervals. The indication for transplantation of donor‐specific transfusion (DST)‐treated patients was based on the appearance of EAI antibody and a significant reduction in the MLC reactivity (9 patients). DST patients had 100% kidney graft survival for 5 years and the DST untreated ones 75 %. Suggested responsible factors for this observation are the haploidentity in HLA and the induction of suppressive immune regulation by DST.

Temporary Tolerance or Suppressive Regulation Induced By Non MHC Alloantigens in Transplantation and Pregnancy

A special type of tolerance or suppressive regulation will be reported, which can be characterized by the complementary participation of two alloantigen systems; the major histocompatibility complex, and either a minor histocompatibility or certain type of differentiation antigen (secondary, subordinated) alloantigen system of functional importance. There are representative experimental observations on this phenomena, from which one characteristic model, reported by Hutchinson and Morris (Hutchinson and Morris 1987) is explained. Prior to kidney transplantation between RTL incompatible rats recipients were transfused with blood obtained from various strains characterized by either matching or mismatching with the transfusion and organ donor or recipient strain in the minor histocompatibility system. The matching between transfusion and kidney donor and a mismatching between transfusion donor and organ recipient as regards minor histocompatibility alloantigen system has to be emphasized as a new requirement. No similar situation has been reported in human beings. However, the induction of tolerance by transfusion in certain models is a well established phenomena. The importance of class II antigen matching between transfusion donor and recipient were outlined and proved in series of clinical observations based on in vivo and in vitro parameters including cytotoxic antibody production, MLC, cytotoxic precursor cell function and kidney survival (Lagaay et al. 1989, Claas et al 1991, van Rood and Claas 1990, de Waal and van Twuyer 1991).

The innate immune receptor TREM-1 promotes liver injury and fibrosis

Inflammation occurs in all tissues in response to injury or stress and is the key process underlying hepatic fibrogenesis. Targeting chronic and uncontrolled inflammation is one strategy to prevent liver injury and fibrosis progression. Here, we demonstrate that triggering receptor expressed on myeloid cells 1 (TREM-1), an amplifier of inflammation, promotes liver disease by intensifying hepatic inflammation and fibrosis. In the liver, TREM-1 expression was limited to liver macrophages and monocytes and was highly upregulated on Kupffer cells, circulating monocytes, and monocyte-derived macrophages in a mouse model of chronic liver injury and fibrosis induced by carbon tetrachloride (CCl4) administration. TREM-1 signaling promoted proinflammatory cytokine production and mobilization of inflammatory cells to the site of injury. Deletion of Trem1 reduced liver injury, inflammatory cell infiltration, and fibrogenesis. Reconstitution of Trem1-deficient mice with Trem1-sufficient Kupffer cells restored the recruitment of inflammatory monocytes and the severity of liver injury. Markedly increased infiltration of liver fibrotic areas with TREM-1–positive Kupffer cells and monocytes/macrophages was found in patients with hepatic fibrosis. Our data support a role of TREM-1 in liver injury and hepatic fibrogenesis and suggest that TREM-1 is a master regulator of Kupffer cell activation, which escalates chronic liver inflammatory responses, activates hepatic stellate cells, and reveals a mechanism of promotion of liver fibrosis.