Linda Barber

Dominant influence of HLA-B in mediating the potential co-evolution of HIV and hla

The extreme polymorphism in the human leukocyte antigen (HLA) class I region of the human genome is suggested to provide an advantage in pathogen defence mediated by CD8+ T cells. HLA class I molecules present pathogen-derived peptides on the surface of infected cells for recognition by CD8+ T cells. However, the relative contributions of HLA-A and -B alleles have not been evaluated. We performed a comprehensive analysis of the class I restricted CD8+ T-cell responses against human immunodeficiency virus (HIV-1), immune control of which is dependent upon virus-specific CD8+ T-cell activity. In 375 HIV-1-infected study subjects from southern Africa, a significantly greater number of CD8+ T-cell responses are HLA-B-restricted, compared to HLA-A (2.5-fold; P = 0.0033). Here we show that variation in viral set-point, in absolute CD4 count and, by inference, in rate of disease progression in the cohort, is strongly associated with particular HLA-B but not HLA-A allele expression (P < 0.0001 and P = 0.91, respectively). Moreover, substantially greater selection pressure is imposed on HIV-1 by HLA-B alleles than by HLA-A (4.4-fold, P = 0.0003). These data indicate that the principal focus of HIV-specific activity is at the HLA-B locus. Furthermore, HLA-B gene frequencies in the population are those likely to be most influenced by HIV disease, consistent with the observation that B alleles evolve more rapidly than A alleles. The dominant involvement of HLA-B in influencing HIV disease outcome is of specific relevance to the direction of HIV research and to vaccine design.

IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome.

Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3+ CD4+ IL‐17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS (P < 0·01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4+CD25high FoxP3+ regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease (P < 0·005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis (P < 0·01). Tregs from MDS patients suppressed interferon‐γ (IFN‐γ) secretion by effector CD4+ T cells but had no effect on interleukin (IL)‐17 production. In addition, the serum levels of IL‐7, IL‐12, RANTES and IFN‐γ are significantly elevated in low risk MDS, while inhibitory factors, such as IL‐10 and soluble IL‐2 receptor, are significantly higher in high risk disease. The ‘unfavourable’ Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.

Xenogeneic Graft-versus-Host-Disease in NOD-scid IL-2Rγnull Mice Display a T-Effector Memory Phenotype

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; “Hu-PBMC mice”) are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγnull), notably the NOD-scid IL-2Rγnull (NSG) and BALB/c-Rag2 null IL-2Rγnull (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45+ compartment and higher engraftment levels of CD3+ T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (TEM) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting TEM-cell driven GvHD.

Acute myeloid leukaemia cells secrete a soluble factor that inhibits T and NK cell proliferation but not cytolytic function - implications for the adoptive immunotherapy of leukaemia

Evidence of an immune mediated graft‐versus‐leukaemia effect has led to the belief that T and NK cell based adoptive immunotherapy can constitute effective treatment for relapsed leukaemias. However, work on solid tumours has shown this strategy may be hampered, by an immune escape mechanism in which tumour secreted immunosuppressive factors compromise T and NK cell function. Indeed, acute myeloid leukaemia (AML) cells secrete immunosuppressive factors that block the synthesis of Th1 type cytokines in T cells. We demonstrate here that this immunosuppression, mediated by both HL60 AML cell line and primary AML blasts, inhibits T and NK cell proliferation but not cytolytic activity.

Detection of vimentin-specific autoreactive CD8+ T cells in cardiac transplant patients.

Background. Evidence is emerging that autoimmunity can play a role in allograft rejection. Reports have described the presence of autoantibodies in transplant patients and CD4+ autoreactive T cells in rodent models of allograft rejection. The objective of this study was to seek evidence of CD8+ T-cell–mediated autoimmunity in the transplant setting. The authors have previously observed autoimmunity to the non-polymorphic cytoskeletal protein vimentin in cardiac transplant patients. In this study, vimentin antibody-positive patients were screened for the presence of vimentin-specific self-major histocompatibility complex class I-restricted CD8+ T cells. Methods. Two peptide sequences from vimentin that bound HLA-A*0201 were identified and fluorochrome-labeled A*0201 tetramers with each peptide were constructed to screen for vimentin-specific T cells. Results. Tetramer-binding CD8+ T cells were detected in peripheral blood lymphocytes from two of six patients after expansion by in vitro stimulation with peptide. Tetramer-binding T cells produced interferon-γ in an antigen-specific fashion. No autoreactive T cells specific for vimentin were detected after peptide stimulation of T cells from eight healthy A*0201-positive volunteers. Conclusions. This finding is the first evidence of CD8+ T-cell–mediated autoimmunity in human transplant patients.

Delayed attainment of full donor chimaerism following alemtuzumab-based reduced-intensity conditioning haematopoeitic stem cell transplantation for acute myeloid leukaemia and myelodysplastic syndromes is associated with improved outcomes

This prospective study evaluated the kinetics of lymphoid (CD3) engraftment in 110 patients with acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) after allogeneic transplantation and conditioning with fludarabine, busulphan and alemtuzumab, using ciclosporin for post‐transplant immunosuppression. Declining donor CD3 chimaerism beyond day+100 was treated with pre‐emptive donor lymphocyte infusion (pDLI). The median age of patients was 53·0 years (range: 19–72 years), and the median follow‐up was 690 d (range:168–1470 d). Patients achieving full CD3 donor chimaerism (FDC, n = 46) by day+100 had a significantly inferior disease‐free survival (DFS) and overall survival (OS) compared to patients with mixed donor chimaerism (MDC, n = 59). Twenty patients had stable MDC and did not require pDLI. Patients attaining early FDC had a higher transplant‐related mortality compared to those who maintained stable levels of MDC (P = 0·02), with no difference between the FDC and pDLI groups (P = 0·07). There was no difference in relapse between all three groups (P = 0·21). On multivariate analysis, only CD3 chimaerism status at day+100 and disease status at transplantation had a significant effect on DFS and OS. In patients with AML/MDS undergoing alemetuzumab based‐RIC HSCT, prolonged MDC beyond day+100 is associated with an improved OS. Future studies need to be directed towards establishing the underlying factors that dictate T‐cell engraftment, expansion and homing post‐transplantation.

Investigation of Peptide Involvement in T Cell Allorecognition Using Recombinant HLA Class I Multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-γ upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.

Relative Resistance of Human CD4+ Memory T Cells to Suppression by CD4+CD25+ Regulatory T Cells

Successful expansion of functional CD4+CD25+ regulatory T cells (Treg) ex vivo under good manufacturing practice conditions has made Treg‐cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, Treg cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4+CD25− T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti‐CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that Treg cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human Treg‐cell therapy, it is important to define the effectiveness of Treg cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded Treg cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that Treg cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of Treg cells.

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

Graft-versus-host disease (GVHD) remains the downside of the graft-versus-leukemia effect following allogeneic stem cell transplantation. This investigation of 25 patients with myeloid malignancies treated with reduced intensity conditioning and with GVHD prophylaxis, has focused on cell populations and their correlation with outcome. Interesting imbalances of effector and regulatory CD4+ T cells were detected, which, if confirmed in larger cohorts, should provide insight into how the immunological storms accompanying allogeneic stem cell transplantation can be harnessed and weathered. Background A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care. Design and Methods Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease. Results Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO+ CD27−) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25high Foxp3+) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease. Conclusions Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.

Human CD80/IL2 lentivirus‐transduced acute myeloid leukaemia (AML) cells promote natural killer (NK) cell activation and cytolytic activity: implications for a phase I clinical study

Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti‐leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)‐transduced AML cells stimulate in‐vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2‐expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA‐DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up‐regulation of the cytolytic marker CD107a was detected following autologous culture with LV‐CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46·6% to 90·4%) and allogeneic AML cells (from 11·8% to 20·1%) following in‐vitro stimulation by CD80/IL2‐expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40‐fold (from 0·4% to 22·5%). These studies demonstrated that vaccination of patients with CD80/IL2‐transduced AML cells could provide a powerful strategy for T/NK cell‐mediated stimulation of anti‐leukaemic immunological responses.