Karen Piper

Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin's Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

In approximately 50% of patients with Hodgkins lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Patients with Wegener's granulomatosis demonstrate a relative deficiency and functional impairment of T-regulatory cells.

An increased proportion of CD4+ CD25+ T cells has been reported in Wegener’s granulomatosis (WG) and may represent an accumulation of regulatory T cells (Treg). CD25 is also expressed on recently activated effector T cells. We have determined the relative proportion of these subsets in a large patient cohort. The fraction of Treg in peripheral blood mononuclear cells from patients and healthy controls was determined by assessment of Foxp3 expression on CD4+ CD25+ T cells. The functional activity of Treg was determined by their ability to suppress proliferation and cytokine production in response to proteinase‐3. Although WG patients demonstrated an increased fraction of CD4+ CD25+ T cells, the percentage of Foxp3‐positive cells was decreased. In addition, the percentage of Treg was inversely related to the rate of disease relapse. CD4+ CD25hi T cells were able to suppress T‐cell proliferation to proteinase‐3 in healthy controls and anti‐neutrophil cytoplasm antibody (ANCA)‐ negative patients (at time of sampling) but not in ANCA‐positive patients. In patients with active disease, an increased proportion of CD4+ Foxp3+ cells was associated with a more rapid disease remission. Patients with WG demonstrate abnormalities in the number and function of Treg and this is most pronounced in those with most active disease. This information is of value in understanding the pathogenesis and potential treatment of this disease.

Selective accumulation of virus-specific CD8+ T cells with unique homing phenotype within the human bone marrow

The bone marrow plays a unique role within the immune system. We compared the phenotype and function of virus-specific CD8(+) T cells from matched samples of human peripheral blood and bone marrow. Analysis of virus-specific memory CD8(+) T cells showed widely divergent partition of antigen-specific populations between blood and bone marrow. T cells specific for Epstein-Barr virus (EBV) lytic antigens were enriched 3-fold in marrow compared with blood, whereas the response to EBV latent epitopes was equivalent between the 2 compartments. No difference in EBV viral load or expression of the EBV lytic protein was observed between blood and bone marrow. In direct contrast, although cytomegalo-virus (CMV)-specific T cells were the largest virus-specific population within peripheral blood, they were reduced by 60% within marrow. Bone marrow T cells were found to exhibit a unique CCR5(+)CXCR6(+)CXCR3(-) homing phenotype which has not been observed on T cells from other secondary lymphoid organs or peripheral organs. Expression of CCR5 and CXCR6 was higher on EBV-specific T cells within peripheral blood compared with CMV-specific populations. These observations identify a novel bone marrow homing phenotype for CD8(+) memory T cells, which necessitates a reevaluation of the magnitude of antigen-specific populations within the lymphoid system.

Functional HY-Specific CD8+ T Cells Are Found in a High Proportion of Women Following Pregnancy with a Male Fetus

Abstract Recent studies have demonstrated that fetal cells can be detected in the maternal circulation during virtually all human pregnancies. These fetal cells can engraft and may be isolated for many decades after pregnancy, leading to a state that may be maintained by the passage of pregnancy-associated progenitor cells. The clinical consequences of fetal cell microchimerism are unclear but may be potentially detrimental or valuable to the mother. One possibility is the generation of an alloreactive immune response by the mother to antigens expressed by the fetus; for example, the HY protein encoded by the Y chromosome. To test this we have screened a cohort of women with a range of parity histories within 8 yr of their last pregnancy for the presence of an HY-specific CD8+ T-cell response. Fluorescent HLA-peptide (HY) tetramers were used to stain short-term T-cell cultures from these women for analysis by flow cytometry. Responses were detected in 37% of women with a history of pregnancies that produced males, and this value rose to 50% in women with two or more pregnancies that produced males. HY-specific CD8+ T cells also could be detected directly in the peripheral blood of women with a history of at least two pregnancies that produced males. These HY-specific CD8+ T cells produced interferon gamma (IFNG) following peptide stimulation, demonstrating their functional capacity. In conclusion, our data indicate that alloreactive CD8+ T cells are generated frequently following normal pregnancy and retain functional capability for years following pregnancy.

Fetal-Specific CD8+ Cytotoxic T Cell Responses Develop during Normal Human Pregnancy and Exhibit Broad Functional Capacity

Tolerance of the semiallogeneic fetus presents a significant challenge to the maternal immune system during human pregnancy. T cells with specificity for fetal epitopes have been detected in women with a history of previous pregnancy, but it has been thought that such fetal-specific cells were generally deleted during pregnancy as a mechanism to maintain maternal tolerance of the fetus. We used MHC-peptide dextramer multimers containing an immunodominant peptide derived from HY to identify fetal-specific T cells in women who were pregnant with a male fetus. Fetal-specific CD8+ T lymphocytes were observed in half of all pregnancies and often became detectable from the first trimester. The fetal-specific immune response increased during pregnancy and persisted in the postnatal period. Fetal-specific cells demonstrated an effector memory phenotype and were broadly functional. They retained their ability to proliferate, secrete IFN-γ, and lyse target cells following recognition of naturally processed peptide on male cells. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy and that unlike reports from some murine models, fetal-specific T cells are not deleted during human pregnancy. This has broad implications for study of the natural physiology of pregnancy and for the understanding of pregnancy-related complications.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

Chronic lymphocytic leukaemia cells drive the global CD4+ T cell repertoire towards a regulatory phenotype and leads to the accumulation of CD4+ forkhead box P3+ T cells

Advanced chronic lymphocytic leukaemia (CLL) is associated with profound immunodeficiency, including changes in T regulatory cells (Tregs). We determined the pattern of expression of forkhead box P3 (FoxP3), CD25, CD27 and CD127 and showed that the frequency of CD4+FoxP3+ T cells was increased in CLL patients (12% versus 8% in controls). This increase was seen only in advanced disease, with selective expansion of FoxP3‐expressing cells in the CD4+CD25low population, whereas the number of CD4+CD25highFoxP3+ cells was unchanged. CD4+CD25low cells showed reduced expression of CD127 and increased CD27, and this regulatory phenotype was also seen on all CD4 T cells subsets in CLL patients, irrespective of CD25 or FoxP3 expression. Incubation of CD4+ T cells with primary CLL tumours led to a sixfold increase in the expression of FoxP3 in CD4+CD25‐ T cells. Patients undergoing treatment with fludarabine demonstrated a transient increase in the percentage of CD4+FoxP3+ T cells, but this reduced to normal levels post‐treatment. This work demonstrates that patients with CLL exhibit a systemic T cell dysregulation leading to the accumulation of CD4+FoxP3+ T cells. This appears to be driven by interaction with malignant cells, and increased understanding of the mechanisms that are involved could provide novel avenues for treatment.

DNA Fusion Vaccines Induce Epitope-Specific Cytotoxic CD8+ T Cells against Human Leukemia-Associated Minor Histocompatibility Antigens

The graft-versus-leukemia effect of allogeneic stem-cell transplantation is believed to be mediated by T-cell recognition of minor histocompatibility antigens on recipient cells. For minor histocompatibility antigens HA-1 and HA-2, normal cell expression is restricted to hemopoietic cells, and boosting the immune response to these antigens may potentiate graft-versus-leukemia effect without accompanying graft-versus-host disease. To increase efficacy, expansion of HA-1- or HA-2-specific CTL before transplantation is desirable. However, primary HA-1- or HA-2-specific CTL expanded in vitro are often of low avidity. An alternative approach is to prime specific CTL responses in vivo by vaccination. Clearly, donor vaccination must be safe and specific. We have developed DNA fusion vaccines able to induce high levels of epitope-specific CTL using linked CD4(+) T-cell help. The vaccines incorporate a domain of tetanus toxin (DOM) fused to a sequence encoding a candidate MHC class I binding peptide. This design generates antitumor CD8(+) T-cell responses and protective immunity in preclinical models. For clinical application, we constructed vaccines encoding HLA-A*0201-restricted peptides from human HA-1 and HA-2, which were fused to DOM, and tested their performance in HLA-A*0201-transgenic mice. Priming induced epitope-specific, IFNgamma-producing CD8(+) T cells with cytotoxic function boosted to high levels with electroporation. Strikingly, these mouse T cells efficiently killed human lymphoblastoid cell lines expressing endogenous HA-1 or HA-2. High avidity is indicated by the independence of cytolysis from CD8/MHC class I interaction. These safe epitope-specific vaccines offer a potential strategy to prime HA-1- or HA-2-specific CTL in transplant donors before adoptive transfer.

CD8+ T-cell immunity against cancer-testis antigens develops following allogeneic stem cell transplantation and reveals a potential mechanism for the graft-versus-leukemia effect

Background Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period. Design and Methods The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides. Results A cancer-testis antigen-specific CD8+ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8+ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow. Conclusions Functional cancer-testis antigen-specific CD8+ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.

Fetal microchimerism: the cellular and immunological legacy of pregnancy.

During pregnancy there is transplacental traffic of fetal cells into the maternal circulation. Remarkably, cells of fetal origin can then persist for decades in the mother and are detectable in the circulation and in a wide range of tissues. Maternal CD8 T cell responses directed against fetal antigens can also be detected following pregnancy. However, the impact that the persistence of allogenic cells of fetal origin and the maternal immune response towards them has on the mothers health remains unclear and is the subject of considerable investigation. The potentially harmful effects of fetal microchimerism include an association with autoimmune disease and recurrent miscarriage. Beneficial effects that have been explored include the contribution of persistent fetal cells to maternal tissue repair. A link between fetal microchimerism and cancer has also been proposed, with some results supporting a protective role and others, conversely, suggesting a role in tumour development. The phenomenon of fetal microchimerism thus provokes many questions and promises to offer further insights not only into the biology of pregnancy but fields such as autoimmunity, transplantation biology and oncology.