Mark W. Lowdell

A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease

Huntingtons disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. We examined the peripheral immune system and found widespread evidence of innate immune activation detectable in plasma throughout the course of HD. Interleukin 6 levels were increased in HD gene carriers with a mean of 16 years before the predicted onset of clinical symptoms. To our knowledge, this is the earliest plasma abnormality identified in HD. Monocytes from HD subjects expressed mutant huntingtin and were pathologically hyperactive in response to stimulation, suggesting that the mutant protein triggers a cell-autonomous immune activation. A similar pattern was seen in macrophages and microglia from HD mouse models, and the cerebrospinal fluid and striatum of HD patients exhibited abnormal immune activation, suggesting that immune dysfunction plays a role in brain pathology. Collectively, our data suggest parallel central nervous system and peripheral pathogenic pathways of immune activation in HD.

Stem-cell-based, tissue engineered tracheal replacement in a child: A 2-year follow-up study

BACKGROUND Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor β to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

Directly Selected Cytomegalovirus-Reactive Donor T Cells Confer Rapid and Safe Systemic Reconstitution of Virus-Specific Immunity Following Stem Cell Transplantation

BACKGROUND Adoptive transfer of virus-specific T cells may accelerate reconstitution of antigen-specific immunity and limit the morbidity and mortality of viral infections following allogeneic hematopoietic stem cell transplantation. The logistics of producing virus-specific T cells has, however, limited the application of cellular therapies, particularly following the introduction of more-recent regulatory stipulations. METHODS We investigated the ability of cytomegalovirus-specific T cells, directly isolated from donor leucapheresates on the basis of interferon γ secretion, to restore antiviral immunity in a group of 25 patients following related-donor transplantation in a single-arm phase I-II study. Selected cells were administered early following transplantation, either after the detection of cytomegalovirus DNA by polymerase chain reaction-based surveillance or prophylactically between day 40 and day 50. RESULTS Cell selection was successful in all cases, yielding a product biased towards CD4(+) over CD8(+) T cells. The target cell dose of 1 × 10(4) CD3(+) T cells/kg of recipient weight contained a median of 2840 cytomegalovirus-specific CD4(+) cells/kg and 630 cytomegalovirus-specific CD8(+) cells/kg, with a median purity of 43.9% interferon γ-secreting cells. Expansions of both CD4(+) and CD8(+) cytomegalovirus-specific T cells were observed in vivo within days of adoptive transfer. These cells were predominantly terminally differentiated effector-memory cells and showed the same T cell receptor variable β chain (TCRBV) -restriction as the infused cells. They offered protection from reinfection in the majority of patients. CONCLUSIONS These data indicate that application of cytomegalovirus-specific T cells generated by direct selection using γ-capture is both feasible and effective in a clinical environment. These simple in vitro methodologies should allow more widespread application of virus-specific T cell immunotherapies.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells.

Summary. Although it has been known for more than 40 years that allogeneic immune responses cure leukaemias after bone marrow transplantation, autologous leukaemia‐specific immunity remains controversial and its impact upon survival has not been established. Here we have tested 25 patients with de novo acute leukaemias, while in remission at completion of their anti‐leukaemia therapy, for evidence of autologous cytolytic immunity to their leukaemic cells taken and cryopreserved at disease presentation. We have measured this degree of cell‐mediated cytotoxicity in vitro and termed it ‘leukaemia cytolytic activity’ (LCA). Patients whose disease ultimately relapsed had significantly lower LCA than those who remained in remission beyond 2 years (P < 0·001); the absence of LCA when in remission predicted subsequent relapse within 2 years with a sensitivity of 100% and specificity of 77%. LCA was mediated in vitro by CD56+/CD8α+/CD3– natural killer cells. We propose that it is this immune response, rather than the chemotherapy per se, which is responsible for continued remission and that measurement of LCA in patients at completion of therapy may be used as an indicator of risk of subsequent relapse. Patients lacking this response will require further treatment, either with an allogeneic donor transplant or an alternative immunotherapeutic strategy.

The role of Vδ2-negative γδ T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation

Reactivation of cytomegalovirus (CMV) remains a serious complication after allogeneic stem cell transplantation, but the role of γδ T cells is undefined. We have studied the immune reconstitution of Vδ2negative (Vδ2neg) γδ T cells, including Vδ1 and Vδ3 subsets and Vδ2positive (Vδ2pos) γδ T cells in 40 patients during the first 24 months after stem cell transplantation. Significant long-term expansions of Vδ2neg but not Vδ2pos γδ T cells were observed during CMV reactivation early after transplantation, suggesting direct involvement of γδ T cells in anti-CMV immune responses. Similarly, significantly higher numbers of Vδ2neg γδ T cells were detected in CMV-seropositive healthy persons compared with seronegative donors; the absolute numbers of Vδ2pos cells were not significantly different. The expansion of Vδ2neg γδ T cells appeared to be CMV-related because it was absent in CMV-negative/Epstein-Barr virus-positive patients. T-cell receptor-δ chain determining region 3 spectratyping of Vδ2neg γδ T cells in healthy subjects and patients showed restricted clonality. Polyclonal Vδ2neg cell lines generated from CMV-seropositive healthy donors and from a recipient of a graft from a CMV-positive donor lysed CMV-infected targets in all cases. Our study shows new evidence for role of γδ T cells in the immune response to CMV reactivation in transplantation recipients.

Selective removal of alloreactive cells from haematopoietic stem cell grafts: graft engineering for GVHD prophylaxis.

One of the main goals in allogeneic bone marrow transplantation is the abrogation of graft-versus-host disease with the preservation of antileukaemia and antiviral activity. We have established a novel system for the selective removal of alloreactive lymphocytes from donor grafts while retaining an effective allogeneic response to third-party stimulator cells. Initial feasibility studies were done with unrelated HLA-mismatched pairs and then extended into the matched setting. Mononuclear cells from HLA-matched donors were cocultured with irradiated recipient cells prestimulated with cytokines (γ-IFN and TNF-α) in a modified mixed lymphocyte culture (MLC). Alloreactive donor lymphocytes were identified by expression of CD69, an early activation marker and selectively removed by paramagnetic bead sorting. The remaining ‘non-alloreactive’ lymphocytes were tested in proliferative assays against the original matched recipient and to a third-party donor. A mean depletion of proliferative capacity to 11.5 ± 9.9% of the original matched recipient response was achieved while the residual third-party response was largely preserved at 77.8 ± 20.9% which should translate into improved immune reconstitution and preservation of antiviral activity. The non-alloreactive lymphocytes could also possess functional antileukaemia activity. Moreover, the alloreactive cells are easily recoverable in this selective T cell depletion strategy for cryopreservation and ready for immediate access as therapeutic donor lymphocyte infusions in cases of frank relapse post transplant.

Identification of both myeloid CD11c+ and lymphoid CD11c− dendritic cell subsets in cord blood

Dendritic cells (DCs) are the most potent antigen‐presenting cells described to date. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have recently been described as being exclusively of the immature CD11c− lymphoid DC subset. Using an alternative method of enrichment, based on a negative selection system, both lymphoid (HLA‐DR+ CD123+++ CD11c− CD33−) and myeloid (HLA‐DR++ CD123+ CD11c+ CD33+) DCs were identified in CB. Although the majority of CB DCs showed a lymphoid phenotype, a significant number of CD11c+ myeloid DCs (25·6% ± 14·5%, n = 13) were also present. Other markers, such as CD80 and CD83, were negative in both subsets. Analyses of the allostimulatory capacity of both subsets showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the inverted CD11c+/CD11c− ratio observed in CB DCs (1:3) with respect to adult blood DCs (3:1) remains to be explained.

Temporal dynamics of CD69 expression on lymphoid cells

Lymphocyte activation remains an area of intense interest to immunologists and cell biologists and the dynamics of expression of surface molecules during the process are widely studied. The CD69 C-type lectin is reportedly the earliest activation antigen on lymphocytes and can be detected within hours of mitogenic stimulation. Recently reports have described differential activation dynamics with respect to different antigenic or mitogenic stimuli. This study has investigated the dynamics of CD69 expression over time after mitogenic, allogeneic, cytokine and target cell mediated activation of T-cell and NK cell subsets. It is apparent that the dynamics of CD69 expression differ with respect to the cell type and the method of stimulation. Mitogenic stimulation resulted in the most rapid expression of CD69 on both T- and NK cells while alloantigen stimulation induced a far slower response. Target cell stimulation of NK cells gave paradoxical results in that the CD69 + ve subset increased as a proportion of the total NK cells but did not increase in number. This was due to the selective binding of CD69 - ve NK cells to the target cells and their subsequent loss from the lymphoid gate. We confirmed this by showing that CD69 + ve NK cells do not lyse K562 target cells. This observation demonstrates the caution needed in the analysis of flow cytometric data based solely upon relative proportions of cells within discrete subsets.

Functional Impairment of Cytomegalovirus Specific CD8 T Cells Predicts High‐Level Replication After Renal Transplantation

Human cytomegalovirus (HCMV) remains an important cause of morbidity after allotransplantation, causing a range of direct effects including hepatitis, pneumonitis, enteritis and retinitis. A dominant risk factor for HCMV disease is high level viral replication in blood but it remains unexplained why only a subset of patients develop such diseases. In this detailed study of 25 renal transplant recipients, we show that functional impairment of HCMV specific CD8 T cells in the production of interferon gamma was associated with a 14‐fold increased risk of progression to high level replication. The CD8 T‐cell impairment persisted during the period of high level replication and was more prominent in patients above 40 years of age (odds ratio = 1.37, p = 0.01) and was also evident in dialysis patients. Threshold levels of functional impairment were associated with an increased risk of future HCMV replication and there was a direct relationship between the functional capacity of HCMV ppUL83 CD8 T cells and HCMV load (R2= 0.83). These results help to explain why a subset of seropositive individuals develop HCMV replication and are at risk of end‐organ disease and may facilitate the early identification of individuals who would benefit from targeted anti‐HCMV therapy after renal transplantation.