BCL6 is required for differentiation of Ig-like transcript 3-Fc-induced CD8+ T suppressor cells.

Ig-like transcript 3 (ILT3) is an inhibitory receptor expressed by tolerogenic dendritic cells. When human CD8(+) T cells are allostimulated in the presence of recombinant ILT3-Fc protein, they differentiate into antigenic specific T suppressor (Ts) cells that inhibit CD4 and CD8 T cell effector function both in vitro and in vivo. ILT3-Fc-induced CD8(+) Ts cells express high amounts of BCL6 that are crucial to their function. Knockdown of BCL6 from unprimed human T cells prevents their differentiation into Ts cells, whereas ex vivo overexpression of BCL6 converts CD8(+) T cells into Ts cells. NOD/SCID mice transplanted with human pancreatic islets and humanized by injection of human PBMCs tolerate the graft and develop BCL6(high) CD8(+) Ts cells when treated with ILT3-Fc before or after the onset of rejection. This indicates that ILT3-Fc acts through BCL6 and is a potent immunosuppressive agent for reversing the onset of allo- or possibly autoimmune attacks against pancreatic islets.

Ig-like transcript 3 regulates expression of proinflammatory cytokines and migration of activated T cells.

Ig-like transcript 3 (ILT3), an inhibitory receptor expressed by APC is involved in functional shaping of T cell responses toward a tolerant state. We have previously demonstrated that membrane (m) and soluble (s) ILT3 induce allogeneic tolerance to human islet cells in humanized NOD/SCID mice. Recombinant sILT3 induces the differentiation of CD8(+) T suppressor cells both in vivo and in vitro. To better understand the molecular mechanisms by which ILT3 suppresses immune responses, we have generated ILT3 knockdown (ILT3KD) dendritic cells (DC) and analyzed the phenotypic and functional characteristics of these cells. In this study, we report that silencing of ILT3 expression in DC (ILT3KD DC) increases TLR responsiveness to their specific ligands as reflected in increased synthesis and secretion of proinflammatory cytokines such as IL-1alpha, IL-1beta, and IL-6 and type I IFN. ILT3KD-DC also secretes more CXCL10 and CXCL11 chemokines in response to TLR ligation, thus accelerating T cell migration in diffusion chamber experiments. ILT3KD-DC elicit increased T cell proliferation and synthesis of proinflammatory cytokines IFN-gamma and IL-17A both in MLC and in culture with autologous DC pulsed with CMV protein. ILT3 signaling results in inhibition of NF-kappaB and, to a lesser extent, MAPK p38 pathways in DC. Our results suggest that ILT3 plays a critical role in the control of inflammation.

CD8+ T suppressor cells and the ILT3 master switch.

Similar to helper and cytotoxic T cells, CD8(+) T suppressor cells (Ts) acquire antigen specificity via direct interaction with antigen-presenting cells (APC). They induce the upregulation of the inhibitory receptor immunoglobulin-like transcript (ILT)3 on professional and nonprofessional APC, rendering these cells tolerogenic and able to induce the differentiation of further waves of regulatory and suppressor T cells. This review sums up evidence that ILT3 is the centerpiece of CD8(+) Ts-driven suppression and acts as a master switch in the regulation of CD8(+) and CD4(+) T-cell responses to antigens in transplantation, autoimmunity, allergy, and cancer.

CD8+ suppressor and cytotoxic T cells recognize the same human leukocyte antigen-A2 restricted cytomegalovirus peptide.

We explored the possibility that antigen-specific human CD8(+) T cells, which display cytotoxic or suppressor function, can recognize the same peptide epitope. Using the human leukocyte antigen-A0201 restricted immunodominant cytomegalovirus epitope pp65-NLVPMVATV for pulsing either mature/immunogenic or ILT3(high)ILT4(high) tolerogenic dendritic cells (DC), we generated cytotoxic and suppressor CD8(+) T-cell lines, respectively. Our data indicate that modulating the functional state of DC is crucial to the development of tolerogenic or immunogeneic peptide-specific vaccines.

Central role of ILT3 in the T suppressor cell cascade.

CD8+ T suppressor cells differentiate both in vivo and in vitro upon chronic exposure of responding T cells to allogeneic APC. These Ts are allospecific and exhibit their function interacting directly with priming APC which they render tolerogenic. Tolerogenicity of professional and non-professional human APC, such as dendritic cells and endothelial cells, respectively is due to the upregulation of the inhibitory receptors ILT3 and ILT4. ILT3 signals both intracellularly, inhibiting NF-kappaB activation, and transcription of costimulatory molecules, and extracellularly, inducing anergy and regulatory function in T cells with cognate specificity. Both membrane and soluble ILT3 are proteins with potent immunosuppressive activity which are of importance for treatment of rejection, autoimmunity and cancer.

Pancreas cancer and the role of soluble immunoglobulin-like transcript 3 (ILT3).

Attempts to ameliorate the poor prognosis of pancreatic cancer have been largely unsuccessful. Interventions to enhance patients' immune responses to malignancies have been also largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor immunoglobulin-like transcript 3 (ILT3) which may be responsible for such failures. Using a humanized severe combined immunodeficiency (SCID) mouse model, we demonstrate that soluble and membrane ILT3 induce CD8+ T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with carcinoma of the pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8+ T suppressor cells and impairs T cell responses in mixed lymphocyte culture. These responses are restored by anti-ILT3 mAb or by depletion of sILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggest that CD68+ tumor associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunobiotherapy, particularly in pancreatic cancer.

ILT3+ ILT4+ tolerogenic endothelial cells in transplantation.

T cells can recognize allogeneic major histocompatibility complex (MHC) antigens by two distinct routes: either directly as intact molecules or indirectly as processed peptides presented by syngeneic antigen-presenting cells (APC). The graft endothelium plays an important role in rejection eliciting and serving as a target of T cells activated via the direct and/or indirect allorecognition pathway. Recent evidence demonstrates, however, that endothelial cells are also endowed with the capacity to downregulate alloreactivity inducing tolerogenic responses. Similar to professional APC (such as dendritic cells), endothelial cells express high levels of inhibitory receptors (ILT3 and ILT4 in humans and PIR-B in rodents) and low levels of costimulatory and adhesion molecules upon interaction with allospecific CD8 T suppressor cells or exposure to inhibitory cytokines. Because of the importance of endothelial cells in the activation and control of T cell reactivity, understanding of their biology is crucial for the development of new strategies for induction of transplantation tolerance and treatment of cancer, chronic infection, and autoimmunity.

Pregnancy after liver transplantation: report of 8 new cases and review of the literature.

Improved survival and quality of life following liver transplantation are associated with an increased frequency of pregnancies in liver-transplanted women. We investigated the outcome, complications, and management of those pregnancies. We have reviewed the literature and report 8 pregnancies in 6 transplant recipients. Seven pregnancies were completed at 38+/-2 (mean+/-standard deviation) weeks. One miscarriage occurred at week 12. Newborns' weight averaged 2938+/-156 g. Main complications were preeclampsia (n=1) and reversible cholestasis (n=1). Among 285 pregnancies reported in literature, 78+/-20% were successful and the main complications were: preeclampsia (26+/-19%), hypertension (28+/-19%), reversible liver dysfunction (27+/-21%), cesarean delivery (23+/-10%), preterm birth (31+/-28%), small for gestational age infants (23+/-10%), rejection (10+/-7%). Gestational weeks were 36.7+/-1.3, perinatal mortality was 4+/-10%, malformation rate 3%. The rates of both abortions and complications (preeclampsia and/or hypertension) were inversely related to the time interval between transplantation and conception (p<0.05). Abortions occurred more often in recipients whose underlying disease was autoimmune cirrhosis than in recipients with inherited disorders. Rejection rate was approx. 10%, which appears higher than reported in a non-pregnant population after a comparable time interval from transplant (2-3%). Up to 28 months after delivery, maternal death was 5.5+/-7%. We conclude that: the time intervals between transplantation and conception as well as the original cause of liver failure influence the outcome and complications of pregnancies in liver recipients. However, neonatal survival is high, while malformations are relatively rare.

Immunosuppressive activity of recombinant ILT3.

Tolerogenic antigen presenting cells (APC) are characterized by high expression of the inhibitory receptors ILT3 and ILT4. We have engineered ILT3 and ILT4 cytoplasmic deletion mutants (ILT3delta and ILT4delta), which were transfected in the dendritic-like cell line KG1, to investigate ILT3 and ILT4's capacity to signal extracellularly. KG1.ILT3delta, similar to untruncated ILT3, inhibits T cell responses such as proliferation and cell-mediated cytotoxicity. In contrast, KG1.ILT4delta lost the suppressive activity of untruncated ILT4. This indicates that the inhibitory function of ILT4 relies entirely on the cytoplasmic region containing ITIM motifs. We further demonstrated that recombinant soluble ILT3 inhibits T helper and cytotoxic function while inducing the differentiation of CD8(+) Ts cells. Hence, Ts modulate APC function inducing inhibitory receptors, which in turn elicit the generation of Ts.

Stem cells, tissue engineering and organogenesis in transplantation.

Tissue engineering is an attempt to generate living tissues for surgical transplantation. In vitro and in vivo approaches have led to the production of vascular and cardiovascular components, bones, cartilages and gastrointestinal tissues. Organogenesis has a different aim, which is to create transplantable organs from embryonic tissue implanted into the recipient's omentum. This approach has been successful in creating kidneys and pancreases in animals. The use of stem cells in organogenesis and in tissue engineering has vastly enlarged the potential for clinical applications. The technique of nuclear transfer offers the possibility of creating cells, which are genetically identical to the host. Tissue engineering and organogenesis represent the future of transplantation in medicine. The progress in this field is of tremendous importance because it can produce a new generation of morphologically complex tissues and organs. In this review, the most relevant experiences in this area are summarized, including its perspectives for therapeutical applications.

Molecular characterization of allospecific T suppressor and tolerogenic dendritic cells: review.

T suppressor and regulatory cells have been shown to play an important role in the maintenance of central and peripheral tolerance thereby preventing allograft rejection, autoimmunity and allergy. We have previously described a distinct population of antigen-specific CD8(+)CD28(-) T suppressor cells (T(S)). These CD8(+)CD28(-) T(S) cells can be generated in vitro after multiple rounds of stimulation of human peripheral blood mononuclear cells (PBMCs) with either allogenic- or xenogeneic-donor APCs. CD8(+)CD28(-) T(S) cells are FOXP3+, MHC class I-restricted and tolerize both professional antigen presenting cells, such as dendritic cells (DC) and nonprofessional APC such as endothelial cells (EC) by up-regulating the cell surface expression of inhibitory receptors immunoglobulin-like transcript (ILT)-3 and ILT4 and down-regulating the expression of costimulatory molecules such as CD58 and CD86. Tolerized ILT3(high), ILT4(high) APC anergize CD4(+) T(H) cells and can induce the generation of antigen-specific CD4(+)CD25(+) T regulatory cells (T(R)) cells and CD8(+)CD28(-) T(S) cells. In this review, we present our recent studies on the molecular characterization of these antigen specific T suppressor cells and tolerogenic APC.

Diltiazem impairs maturation and functions of human dendritic cells.

The aim of this study was to define the effects of diltiazem, a calcium antagonist drug used in cardiology and in clinical transplantation, on the differentiation and maturation of human dendritic cells (DC). Herein, we demonstrate that diltiazem, in association with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), induces monocytes to differentiate into cells with many of the characteristic of DC. However, diltiazem-induced DC express high levels of mannose receptor and Fc gamma RII and, consequently, manifest a higher endocytic activity compared with GM-CSF+IL-4-induced DC. Importantly, diltiazem-induced DCs have an impaired responsiveness to lipopolysaccharide and CD40 ligand because they produce decreased levels of IL-12 and reveal a reduced ability to stimulate alloreactive T-cell responses as well as in inducing interferon-gamma producing Th1 cells. These effects may contribute to a decreased DC-dependent T-cell activation and may help to explain the immunoregulatory function of diltiazem and its effectiveness in preventing transplant rejection.

Overlap between molecular markers expressed by naturally occurring CD4+CD25+ regulatory T cells and antigen specific CD4+CD25+ and CD8+CD28- T suppressor cells.

Alloantigen specific CD8+CD28- T suppressor (TS) cells differ from naturally occurring CD4+CD25+ T-regulatory (natural TR) cells not only by their phenotype but also by their mechanism of action. Natural TR have been extensively studied, leading to the identification of characteristic "molecular markers" such as Forkhead box P3 (FOXP3), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). We have investigated the expression of these genes in alloantigen specific TS and CD4+CD25+ T regulatory (TR) cells and found that they are expressed at levels similar to those observed in natural TR. Furthermore, similar to natural CD4+CD25+ TR, antigen-specific CD8+CD28-CD62L+ TS cells have more suppressive capacity than CD8+CD28-CD62L- TS cells. In spite of these similarities, natural TR are not antigen-specific and inhibit other T cells by T cell-to-T cell interaction, whereas TS are antigen-specific and exert their inhibitory function by interacting with antigen-presenting cells and render them tolerogenic to other T cells. The molecular characterization of TS cells may contribute to a better understanding of mechanisms involved in inhibition of immune responses in autoimmunity, transplantation, and chronic viral infection.

Interleukin-10 induces the upregulation of the inhibitory receptor ILT4 in monocytes from HIV positive individuals.

A characteristic of human immunodeficiency virus infected individuals is an impairment of immune responses, which can result in opportunistic infections. Elevated levels of interleukin-10 (IL-10), produced by virally infected monocytes, are found in the sera of HIV infected individuals. Such elevated levels have been associated with the impaired function of CD4(+) and CD8(+) T cells, and antigen presenting cells (APC), such as monocytes. IL-10 has been reported to upregulate the cell surface expression of the inhibitory receptors ILT3 and ILT4 on monocytes and dendritic cells. This study demonstrates that the decreased antigen presenting ability of monocytes in HIV(+) individuals is in part due to the upregulation of ILT4 on the monocytes caused by the elevated serum IL-10 levels seen in these individuals.

Tailoring of immunosuppression in renal and liver allograft recipients displaying donor specific T-suppressor cells.

Although transplantation tolerance cannot be yet reliably achieved in humans, there is evidence that active immunosuppression contributes to the maintenance of quiescence. However, the mechanism that underlies quiescence and the precise identity of regulatory cells are not completely understood. We have demonstrated that allograft recipients who remain rejection-free display allospecific T-suppressor cells (Ts). Ts express the CD8(+) CD28(-) phenotype, recognize major histocompatibility complex (MHC) class I antigens, and suppress the up-regulation of costimulatory molecules induced by CD40 ligation of donor antigen presenting cells. The presence of Ts is inversely correlated with T cell alloreactivity to donor MHC peptides, alloantibody production, and rejection. Monitoring of Ts has been successfully used in our studies for tailoring immunosuppression in kidney and liver allograft recipients.

Regulatory CD8+CD28- T cells in heart transplant recipients.

Human regulatory CD8+CD28- T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific, and results in Th anergy. To study the population of CD8+CD28- T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients and 12 normal controls. A significant expansion of CD8+CD28- T cells was found in transplant recipients as compared with normal individuals (p = 0.005). Expression of CD38, human leukocyte antigen-DR, and perforin positive cells within the CD8+CD28- subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28- T cells from patients without rejection as compared with patients in rejection (p = 0.005), indicating that the memory-like CD8+CD28-CD27+ T-cell subset comprises regulatory cells, which play a protective role for the graft. CD8+CD28- T cells isolated from transplant patients did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes that occur in patients with heart transplants and opens new avenues for the induction of specific immunosuppression in transplantation.

The concept of "partial" clinical tolerance.

The status of "partial" tolerance to organ allografts versus the status of complete tolerance is the main topic of this paper. Progress made in immunosuppression, particularly by use of various lymphocyte depleting agents for "induction therapy", seems to favor the subsequent development of T cells with suppressor/regulatory properties. The effective deletion of alloreactive T helper and cytotoxic cells in conjunction with the expansion of antigen-specific suppressor (CD8 + CD28 - FOXP3+) and regulatory (CD4 + CD25+ FOXP3+) T cells creates a milieu in which the graft is well tolerated under an "umbrella" of low dosage immunosuppression. The most effective induction treatment is Campath-1H, although ATGAM at high dosage is also widely used. Total lymphoid irradiation (TLI) is another very effective pretreatment strategy in spite of the risks which are associated with it. The induction of "partial" tolerance is a step in the right direction for exploring strategies that may lead to the induction of complete tolerance. It is safe for the patients and can prolong significantly the function of the graft, preventing the onset of chronic rejection.

Generation and function of antigen-specific suppressor and regulatory T cells.

The identification and characterization of regulatory and suppressor T cells that control immune responsiveness to self and non-self antigens has become the focus of innumerable studies. There are two broad categories of naturally occurring and induced CD4(+)CD25(+) regulatory T cells. Naturally occurring T(R) are antigen non-specific and interact directly with other T cells inhibiting their activation. Induced T(R) are either CD4(+)CD25(+) or CD8(+), produce immunosuppressive cytokines such as IL-10, act directly on other T cells or APC and are antigen specific in some but not in all systems. Finally, a distinct subset of T suppressor cells, characterized by their CD8(+)CD28(-) phenotype have been shown to be antigen-specific, recognizing HLA class I/peptide complexes. T(S) act directly on APC inducing the up-regulation of inhibitory receptors ILT3 and ILT4, which render the APC tolerogenic. Tolerized APC, which expresses high ILT3 and ILT4, trigger the generation of antigen-specific CD4(+) T(R) propagating antigen-specific suppression. Up-regulation of ILT3 and ILT4 appears to be a general characteristic of tolerogenic DC since it is also induced by use of vit D3, IL-10 and/or IFN-alpha. The clinical relevance of these inhibitory receptors is in the maintenance of transplantation tolerance as well as in progression of AIDS has been demonstrated.

Rat CD8+ FOXP3+ T suppressor cells mediate tolerance to allogeneic heart transplants, inducing PIR-B in APC and rendering the graft invulnerable to rejection.

Human CD8+ FOXP3+ T suppressor cells (TS) were previously shown to induce the expression of the inhibitory receptors, Immunoglobulin-like transcript (ILT) 3 and ILT4 on dendritic and endothelial cells, rendering them tolerogenic to allogeneic T cells. We have demonstrated the importance of CD8+ TS in a rat model of heart allo-transplantation. Tolerance was induced in ACI recipients by multiple transfusions of UVB-irradiated blood from Lewis heart donors. CD8+ T cells from tolerant ACI rats expressed FOXP3, transferred tolerance to naive secondary hosts and induced the upregulation of the inhibitory receptor, paired immunoglobulin-like receptor (PIR)-B, an ILT4 orthologue, in Lewis dendritic cells (DC) and heart endothelial cells (EC). When long-term surviving Lewis heart allografts with PIR-B+ EC were retransplanted from a primary to a secondary ACI recipient they did not elicit rejection. This study focuses attention on the need to develop agents that act directly on graft EC in order to achieve tolerance.

Anti-HLA antibodies in heart transplantation.

We have analyzed the relationship between the development of transplant-related coronary artery disease (TRCAD) and the following potential risk factors: (a). number of HLA mismatches between recipient and donor; (b). production of anti-HLA antibodies; (c). growth of lymphocytes infiltrating the graft; and (d). frequency of biopsy proven episodes of acute rejection. The study population consisted of 285 adult heart allograft recipients who were monitored over a period of two years or more. The results demonstrate a significant correlation between TRCAD, generation of anti-HLA class II antibodies and potential of lymphocytes infiltrating the graft to proliferate ex-vivo in medium containing IL-2. Humoral and cellular immune responses to HLA-DR antigens expressed by the graft seem to underlie the development of TRCAD.