Neoplastic plasma cells generate an inflammatory environment within bone marrow and markedly alter the distribution of T cells between lymphoid compartments.

Monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) are characterised by the accumulation of malignant plasma cells within bone marrow and lead to a range of abnormalities in the peripheral blood T cell repertoire. We investigated the level of inflammatory chemokines within the bone marrow and blood of patients with MGUS and MM and related this to the pattern of chemokine receptor expression on T cells in both compartments.The expression of a wide range of chemokine ligands for CXCR3 and CCR4 was markedly increased within the bone marrow of patients with MGUS and MM compared to healthy donors. The most marked effects were seen for CCL4 and CXCL9 which were increased by 4 and 6 fold respectively in the bone marrow of patients with myeloma. The expression of CXCR3 and CCR4, the major TH1 and TH2-associated chemokine receptors, was increased substantially on T cells within the bone marrow of patients whereas the percentage of CXCR3-expressing T cells within blood was correspondingly decreased. The presence of even small numbers of neoplastic plasma cells or associated stroma can therefore generate an inflammatory chemokine tumour microenvironment. This leads to the selective recruitment or retention of specific T cell subsets which is likely to underlie many of the features regarding the peripheral T cell repertoire in myeloma and may also contribute to the immune suppression associated with this disease. This local inflammatory reaction may represent a tumour-specific immune response or may itself play an important role in tumour progression and as such may offers a potential novel target for therapeutic intervention.

Tolerability and Clinical Activity of Post-Transplantation Azacitidine in Patients Allografted for Acute Myeloid Leukemia Treated on the RICAZA Trial.

Disease relapse is the major causes of treatment failure after allogeneic stem cell transplantation (SCT) in patients with acute myeloid leukemia (AML). As well as demonstrating significant clinical activity in AML, azacitidine (AZA) upregulates putative tumor antigens, inducing a CD8(+) T cell response with the potential to augment a graft-versus-leukemia effect. We, therefore, studied the feasibility and clinical sequelae of the administration of AZA during the first year after transplantation in 51 patients with AML undergoing allogeneic SCT. Fourteen patients did not commence AZA either because of transplantation complications or withdrawal of consent. Thirty-seven patients commenced AZA at a median of 54 days (range, 40 to 194 days) after transplantation, which was well tolerated in the majority of patients. Thirty-one patients completed 3 or more cycles of AZA. Sixteen patients relapsed at a median time of 8 months after transplantation. No patient developed extensive chronic graft-versus-host disease. The induction of a post-transplantation CD8(+) T cell response to 1 or more tumor-specific peptides was studied in 28 patients. Induction of a CD8(+) T cell response was associated with a reduced risk of disease relapse (hazard ratio [HR], .30; 95% confidence interval [CI], .10 to .85; P = .02) and improved relapse-free survival (HR, .29; 95% CI, .10 to .83; P = .02) taking into account death as a competing risk. In conclusion, AZA is well tolerated after transplantation and appears to have the capacity to reduce the relapse risk in patients who demonstrate a CD8(+) T cell response to tumor antigens. These observations require confirmation in a prospective clinical trial.

Profile of maternal CD4 T-cell effector function during normal pregnancy and in women with a history of recurrent miscarriage.

The traditional paradigm suggests that during normal pregnancy maternal immunological tolerance of the allogenic fetus is association with a maternal T-lymphocyte shift from a Th1 to a Th2 phenotype, with the opposite effect reported in patients with recurrent miscarriage. However, studies on maternal peripheral blood are conflicting. In the present study, we characterized the maternal CD4 T-cell effector subsets, including the recently described Th17 subset, during normal pregnancy (cross-sectional cohort, n=71; longitudinal cohort, n=17) and contrasted this with women with recurrent miscarriage (n=24). Longitudinal analysis of peripheral blood from normal pregnancy demonstrated a fall in the percentage of Th17 cells between the first and second trimester (P≤0.05), but no significant changes were observed across gestation or the post-natal period in Th1 or Th2 subsets. In contrast, in women with a history of recurrent miscarriage, an elevated proportion of Th17 (0.314% compared with 0.097%; P=0.0009) and Th1 (12.4% compared with 5.3%; P=0.0002) cells was detected. The suggestion that Th17 cells may have a role in the normal events of implantation and early pregnancy requires further evaluation and mechanistic studies. The results of the present study, by conducting a careful longitudinal analysis, demonstrate that a peripheral Th1/Th2 shift is not a requirement for normal pregnancy. By contrast, the profound increase in Th1 and Th17 cells in women with recurrent miscarriage indicates that peripheral immunological dysfunction may be important in this group specifically, and these assays may be important in guiding therapeutic interventions in this group and warrant further investigation to determine whether they are predictive of outcome or responses to immunomodulatory therapy.

MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.

Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8(+) T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen-matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8(+) T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

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.

Suppression of antigen-specific T cell responses by the Kaposi's sarcoma-associated herpesvirus viral OX2 protein and its cellular orthologue, CD200.

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.

Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML).

Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic stem cell transplantation (SCT). Azacitidine (AZA) up-regulates the expression of tumor Ags on leukemic blasts in vitro and expands the numbers of immunomodulatory T regulatory cells (Tregs) in animal models. Reasoning that AZA might selectively augment a GVL effect, we studied the immunologic sequelae of AZA administration after allogeneic SCT. Twenty-seven patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia were treated with monthly courses of AZA, and CD8(+) T-cell responses to candidate tumor Ags and circulating Tregs were measured. AZA after transplantation was well tolerated, and its administration was associated with a low incidence of GVHD. Administration of AZA increased the number of Tregs within the first 3 months after transplantation compared with a control population (P = .0127). AZA administration also induced a cytotoxic CD8(+) T-cell response to several tumor Ags, including melanoma-associated Ag 1, B melanoma antigen 1, and Wilm tumor Ag 1. These data support the further examination of AZA after transplantation as a mechanism of augmenting a GVL effect without a concomitant increase in GVHD.

Cytomegalovirus sero positivity dramatically alters the maternal CD8+ T cell repertoire and leads to the accumulation of highly differentiated memory cells during human pregnancy.

BACKGROUND: Human pregnancy offers an immunological challenge for the immunocompetent women accommodating an allogenic fetus, while continuing to combat potentially infectious disease. Cytomegalovirus (CMV) infects the majority of the human population and establishes lifelong persistence, which can lead to the oligoclonal expansion of differentiated T cells. Primary CMV infection and, less commonly, secondary infection during pregnancy can cause fetal disease and morbidity. The balance between maternal immune competence and viral pathogenicity is thus delicately poised. Our objective was to investigate the influence of CMV serostatus on maternal CD8+ T-cell phenotype and cytokine profile in an apparently healthy cohort of pregnant women. Furthermore, we assessed if CMV serostatus modulated changes in CD8 T cells during gestation. METHODS: CD8+ T-cell phenotype was investigated in 87 pregnant women with samples obtained both during pregnancy [CMV immunoglobulin G (IgG) + n = 39, CMV IgG- n = 21] and in the early post-natal period (IgG+ n = 16, IgG- n = 11). Multiparameter flow cytometry was used to study T-cell phenotype and HLA-peptide tetramers identified CD8 T cells specific for CMV. Levels of 26 plasma cytokines, chemokines and chemokine receptors were assessed in a separate cohort of 20 women (IgG+ n = 10, IgG- n = 10) followed longitudinally during and after pregnancy. RESULTS: CMV seropositivity profoundly influenced the T cell repertoire and its dynamics during pregnancy. Naïve CD8+ T-cells (CCR7+CD45RA+) were reduced by 50% in CMV-seropositive women. The proportion of CD45RA effector cells was not increased in CMV-seropositive donors, although this population was more highly differentiated with reduced CD27 and CD28. However, there was a doubling in the proportion of CD45RA+ revertant memory cells (CCR7-CD45RA+) in seropositive donors. Moreover, seropositive women during late pregnancy demonstrated an accumulation of highly differentiated CMV-specific T-cells. T-cell activation independent of CMV was also seen in late pregnancy. No CMV-related changes in plasma cytokines, chemokines or their receptors were observed. CONCLUSIONS: Thus, CMV serostatus is a crucial consideration in studies of T cell memory and differentiation during pregnancy. The reduction in maternal naïve T cells in CMV-seropositive donors could have implications for the maternal response to infections during pregnancy. These findings shed light on the delicate balance between host, fetus and chronic infection during healthy pregnancy and will inform studies in relation to the importance of CMV on maternal and fetal health.

Dominant responses with conservation of T-cell receptor usage in the CD8+ T-cell recognition of a cancer testis antigen peptide presented through HLA-Cw7 in patients with multiple myeloma.

Cancer testis antigens exhibit physiological expression within germ cells and are frequently expressed in malignant tissue. Interestingly, immunological tolerance to cancer testis proteins does not appear to be established, and the expression of CTAg proteins within malignant cells can therefore lead to induction of cellular and humoral immunity. A considerable body of evidence now indicates that CD8-specific immunity plays an important role in the control of cancer cell growth, and a number of vaccine studies are in progress to boost CTAg-specific cellular immune responses. We have previously identified CTAg-specific immune responses in patients with multiple myeloma and reported that recognition of the MAGE-A1(289-298) peptide, which is described as being restricted by HLA-B*0702, was the most frequent response seen with our peptide panel. Here, we studied seven CD8+ T-cell clones specific for this peptide which were isolated from three patients with myeloma at several time-points. The affinity of peptide recognition was high with 50% maximal interferon-γ production observed at a peptide concentration of 10(-10) M and variation of only one order of magnitude between the affinities of the clones. Importantly, all the clones were able to recognise and kill multiple myeloma cell lines. Interestingly, one patient did not express HLA-B*0702, but three clones from this patient recognised the MAGE-A1(289-298) peptide on a lymphoblastoid cell line (LCLs) expressing HLA-Cw7, and we now show evidence that the MAGE-A1(289-298) peptide is expressed and recognised through Cw7. The T-cell receptor gene usage was determined in five clones and showed conserved features in both the α and the ß chain genes indicating correlation between T-cell receptor usage and peptide specificity of cancer testis antigen-specific T-cell clones.

Induction of a CD8+ T-cell response to the MAGE cancer testis antigen by combined treatment with azacitidine and sodium valproate in patients with acute myeloid leukemia and myelodysplasia.

Epigenetic therapies, including DNA methyltransferase and histone deacetylase inhibitors, represent important new treatment modalities in hematologic malignancies, but their mechanism of action remains unknown. We reasoned that up-regulation of epigenetically silenced tumor antigens may induce an immunologically mediated antitumor response and contribute to their clinical activity. In this study, we demonstrate that azacitidine (AZA) and sodium valproate (VPA) up-regulate expression of melanoma-associated antigens (MAGE antigens) on acute myeloid leukemia (AML) and myeloma cell lines. In separate studies, we observed that prior exposure to AZA/VPA increased recognition of myeloma cell lines by a MAGE-specific CD8(+) cytotoxic T-lymphocyte (CTL) clone. We therefore measured CTL responses to MAGE antigens in 21 patients with AML or myelodysplasia treated with AZA/VPA. CTL responses to MAGE antigens were documented in only 1 patient before therapy; however, treatment with AZA/VPA induced a CTL response in 10 patients. Eight of the 11 patients with circulating MAGE CTLs achieved a major clinical response after AZA/VPA therapy. This is the first demonstration of a MAGE-specific CTL response in AML. Furthermore, it appears that epigenetic therapies have the capacity to induce a CTL response to MAGE antigens in vivo that may contribute to their clinical activity in AML.

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-gamma-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.

Differential pattern of CD4+ and CD8+ T-cell immunity to MAGE-A1/A2/A3 in patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma.

The factors that determine progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma are unclear but may include the breakdown of immune surveillance. Cancer testis antigens (CTAgs) are expressed by the majority of myelomas and MGUS tumors and are a potential immune target. We have characterized CD4(+) and CD8(+) T-cell immune responses to MAGE-A1/A2/A3 in these patients. CD4(+) T-cell immunity to MAGE proteins is stronger and more frequent in MGUS compared with myeloma with a predominantly CD45RA(-)CCR7(-) effector memory profile and cytotoxicity against MAGE-positive cell lines. In contrast CD8(+) T-cell immune responses were present almost exclusively in patients with multiple myeloma, correlating with disease, with a CD45RA(+)CCR7(-) memory phenotype, localizing poorly to the bone marrow but were able to lyse myeloma cell lines in vitro. This suggests that the CD4(+) CTAg-specific immune response may play a role in controlling tumor growth, whereas the efficacy of the CD8(+) T-cell response appears to be limited in vivo. Despite this, patients with evidence of a CTAg-specific immune response had a 53% reduction in mortality over a median follow-up of 4 years. These findings have important implications for clinical approaches to CTAg-specific immunotherapy in patients with cancer.

Immunodeficiency and immunotherapy in multiple myeloma.

Multiple myeloma is a malignant tumour of plasma cells that remains incurable for the vast majority of patients, with a median survival of 2-3 years. It is characterized by the patchy accumulation of tumour cells within bone marrow leading to variable anaemia, bone destruction, hypercalcaemia, renal failure and infections. Immune dysfunction is an important feature of the disease and leads to infections that are both a major cause of morbidity and mortality and may promote tumour growth and resistance to chemotherapy. Numerous defects of the immune system have been described in multiple myeloma although the relative clinical importance of these remains elusive. There has been considerable interest in the identification of an autologous response against myeloma. Although T cells and humoral responses directed against myeloma-associated antigens have been described, it is uncertain if the immune system plays a role in preventing or controlling myeloma cell growth. There is increasing interest in the potential role of immunotherapy but the success of these interventions is likely to be modified by the immunologically hostile environment associated with multiple myeloma. This review attempts to summarize the current knowledge relating to the immune defects found in multiple myeloma.

CD8+ T cells specific for cancer germline gene antigens are found in many patients with multiple myeloma, and their frequency correlates with disease burden.

The expression of cancer germline antigens (CGAgs) is normally restricted to the testis but is also present in many types of malignant cells including plasma cells from patients with myeloma. Because T-cell immune responses to CGAg have been identified in patients with solid tumors, this may offer a novel target for immunotherapy in patients with myeloma. We have used 12 peptide epitopes from a range of CGAgs to screen for CGAg-specific T cells in blood from patients with multiple myeloma at various stages of their disease. T cells from 15 of 37 patients responded to one or more CGAg peptides and the magnitude of the CGAg-specific CD8+ T-cell response ranged between 0.0004% and 0.1% of the total CD8+ T-cell pool. Serial analyses showed that these immune responses were detectable in individual patients at multiple time points during the course of their disease. In patients undergoing treatment or in disease relapse, the magnitude of the CGAg-specific T-cell response was positively correlated with the level of paraprotein. Functional T cells specific for CGAgs are therefore present in a proportion of patients with multiple myeloma and offer the possibility of a novel approach for immunotherapy in this disease.