Gwen Wilkie

Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells

BACKGROUND Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disease (PTLD) is a common, often fatal, complication of bone-marrow and solid-organ transplantation. Since tumour growth results from inadequate T-cell control of latent EBV, new immunotherapeutic approaches to treatment are being pioneered. METHODS In a phase 1/2 trial, eight patients with progressive PTLD unresponsive to conventional treatment were given one to six infusions of partly HLA-matched allogeneic EBV-specific cytotoxic T lymphocytes (CTLs) from a frozen bank of CTLs derived from healthy blood donors. FINDINGS Of the five patients who completed treatment, three had complete remission and two had no clinical response. One patient partly responded after two infusions. No graft-versus-host disease or allo-specific antibodies were detected, and graft function improved in three cases. Tumour responses were mainly seen in those with early, localised, polyclonal disease. EBV load in peripheral blood fell to undetectable levels in all patients who responded to treatment, but was more variable in those who did not. INTERPRETATION Treatment of EBV-associated PTLD with partly HLA-matched CTLs grown from unrelated donors is effective. Spontaneous remission is very unlikely to account for tumour regression in our patients; however, a larger, controlled trial is needed to assess this treatment further. The frozen bank of allogeneic CTLs is less prohibitively labour intensive and expensive for wide scale use than treatment with autologous CTLs. Such banks could be established to treat other infectious and neoplastic diseases in many patients.

Complete regression of posttransplant lymphoproliferative disease using partially HLA-matched Epstein Barr virus-specific cytotoxic T cells.

BACKGROUND Adoptive immunotherapy with autologous and donor-derived cytotoxic T lymphocytes (CTL) has recently been used to treat Epstein Barr virus (EBV)-positive posttransplant lymphoproliferative disease (PTLD). METHODS AND RESULTS We report complete regression of EBV-positive PTLD in an 18-month-old small bowel and liver transplant recipient after one infusion of partially human leukocyte antigen (HLA)-matched EBV-specific CTL grown ex vivo from an EBV seropositive unrelated blood donor. No infusion-related toxicity or evidence of graft-versus-host disease was observed. The tumor showed signs of regression within 1 week and EBV load in peripheral blood dropped to undetectable levels. Limiting dilution analyses (LDA) detected no EBV-specific CTL precursor (CTLp) cells before the infusion, and high numbers of CTLp at 4 hr and 24 hr post-CTL infusion. There was a reversal of the CD4/8 ratio in peripheral blood and an increase in HLA-DR positive CD8 cells. The patient has been in complete remission for 24 months. CONCLUSION If this success is repeated in more PTLD patients, then stored CTL could be used for antiviral and antitumor therapies in immunocompromised patients.

Establishment and characterization of a bank of cytotoxic T lymphocytes for immunotherapy of epstein-barr virus-associated diseases.

Adoptive immunotherapy using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) generated ex vivo can be an effective treatment of EBV-positive posttransplantation lymphoproliferative disease (PTLD). We describe the establishment of a cryopreserved repository of allogeneic virus-specific CTL lines, to our knowledge the first of its kind in the world. CTL lines were grown by weekly stimulation with autologous EBV immortalized lymphoblastoid cell lines (LCLs) from 96 EBV-seropositive blood donors. Analysis of 60 CTL lines grown continuously for 7 to 10 weeks showed an average proportional weekly increase in cell numbers of 1.4, with an overall increase ranging from 1.1 to 83.4. The greatest increase occurred during the early culture period. After four rounds of stimulation, killing of autologous LCLs was generally high (mean 48%); however, most lines required 9 or 10 stimulations to reduce the killing of nonspecific targets. Overall, 79% of CTLs generated showed acceptable levels of specific killing. Phenotypically, the CTL lines consisted of TCR&agr;β+, CD8+ T cells (medians 97% and 90% respectively) with a minority population of CD4+ T cells (median 2%). Most cells expressed the activation and differentiation markers, HLA-DR, CD26, CD45RO, CD69, and CD150. Favorable results have been obtained in an open trial using partially HLA-matched, allogeneic CTLs from this bank to treat PTLD patients. This now represents a single resource that can provide therapeutic CTLs rapidly on a countrywide basis, superseding the time-consuming, expensive practice of generating autologous CTLs from each patient requiring treatment. Additionally, other patient groups, such as those with EBV-positive Hodgkin disease, may benefit from CTL treatment.

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology.

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.

Detection of Theileria lestoquardi (hirci) in Ticks, Sheep, and Goats Using the Polymerase Chain Reaction a

ABSTRACT: Theileria lestoquardi (=T. hirci) is a protozoan parasite of sheep and goats that is morphologically and biologically similar to T. annulata, the causative agent of bovine tropical theileriosis. Both parasites are transmitted by ixodid ticks of the genus Hyalomma. However, because of their morphological similarity, they cannot be distinguished in the salivary glands of infected ticks by traditional staining methods such as Feulgen or Methyl green‐pyronin. Thus a need has arisen for sensitive and specific diagnostic tests that will distinguish between the two species in the vector tick, allowing the epidemiology of both diseases to be clearly defined. A contribution to this has been the development of a polymerase chain reaction using specific primers which amplify, only in T. lestoquardi‐infected ticks, a 785 bp fragment of the gene that codes for a 30 kD merozoite surface protein. The sensitivity of this test and its application to the detection of T. lestoquardi in infected H. anatolicum anatolicum ticks, in the blood of three species of domestic ruminants and in cell cultures established in mononuclear cells of sheep and goats is also discussed.

Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders

Adoptive immunotherapy using autologous Epstein‐Barr virus (EBV)‐specific cytotoxic T‐lymphocytes (auto‐CTL) can regress posttransplant lymphoproliferative disorders (PTLD). Widespread applicability of auto‐CTL remains constrained. Generation is time‐consuming, and auto‐CTL cannot be established in patients treated with the B‐cell depleting antibody rituximab. By contrast, pregenerated allogeneic CTL (allo‐CTL) offers immediate accessibility. Allo‐CTL has previously shown efficacy in “early” polyclonal‐ PTLD. We treated three patients with aggressive, advanced monoclonal‐PTLD following solid‐organ transplantation. All were refractory to at least three prior therapies. Despite HLA disparity, there was negligible toxicity, with early in vivo antiviral efficacy and reconstitution of EBV peptide‐specific immunity. Two patients attained complete remission (CR). One remains in CR 17 months following therapy, coincident with persistence of donor‐derived tumor targeted EBV‐specific CTL; the other died of non‐PTLD related pathology. In the third patient, autopsy demonstrated homing of allo‐CTL at the tumor site. Larger prospective studies of EBV‐specific allo‐CTL in PTLD are warranted.

Bovine Cells Infected in Vivo with Theileria Annulata Express CD11b, the C3bi Complement Receptor

Forsyth, L.M.G., Jackson, L.A., Wilkie, G., Sanderson, A., Brown, C.G.D. and Preston, P.M., 1997. Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor. Veterinary Research Communications, 21 (4), 249-263Bovine cells from cattle infected with Theileria annulata were phenotyped with monoclonal antibodies recognizing bovine leukocyte antigens. Macroschizont-infected, transformed cell lines prepared from peripheral blood mononuclear cells of cattle, infected with sporozoites, were assessed by flow cytometry; parasitized cells in tissues from infected cattle were examined by immunocytochemical techniques. Co-expression of markers for different cell lineages by the cell lines precluded a definite conclusion as to their phenotypic origins. For, while the pattern of leukocyte antigens expressed by these in vivo-derived schizont-infected cells, which included CD11b, was indicative of a myeloid origin, the possibility that they were NK cells could not be excluded. The monoclonal antibody (MAb) IL-A15, which recognizes CD11b, reacted with a high proportion of parasitized cells in sections of tissues from infected cattle at all stages of acute disease. Mononuclear cells infected with parasites at all stages of differentiation, from macroschizont to microschizont, expressed CD11b. Such parasitized cells occurred throughout the lymphoid tissues, being found in the thymus, spleen and lymph nodes, particularly the prescapular node draining the site of infection, the hepatic, mesenteric and precrural nodes, as well as in the reticulo-endothelial tissue of the liver, kidney, lung, abomasum, adrenal and pituitary glands. These observations provided the first evidence for a myeloid origin for the parasitized T. annulata cells found in infected bovine tissues and blood and suggested a mechanism whereby schizonts could transfer from cell to cell during mechanical infection with schizont-infected cells.

Treatment of Epstein-Barr-virus-associated primary CNS B cell lymphoma with allogeneic T-cell immunotherapy and stem-cell transplantation

344 http://oncology.thelancet.com Vol 6 May 2005 An 8-year-old girl presented in October, 2003, with a 3-week history of headaches, vomiting, diplopia secondary to palsies in the IV and VI cranial nerves, dysarthria, and unsteady gait. Over the following 2 weeks, bulbar palsy progressed, and she developed quadraparesis and obtunded consciousness that needed intensive care with assisted ventilation. The patient did not have lymphadenopathy or hepatosplenomegaly. Gadolinium-enhanced cranial MRI showed moderate hydrocephalus and several cerebral and cerebellar ring-like lesions with no central liquefaction (figure 1A,B). Urgent right frontal external ventricular drainage was done. Cerebrospinal fluid taken during the operation showed no white cells, presence of normal proteins, and normal glucose concentrations; the fluid was negative on PCR, microscopy, and culture for bacteria and fungi. 40 380 copies/mL of Epstein-Barr virus (EBV) DNA were detected by PCR of cerebrospinal fluid, and 6 316 copies/mL were found in peripheral blood. Histological analysis of a biopsy sample of the right parietal brain obtained by image guidance showed an infiltrate of CD79a-positive CD30-positive ALK-1-negative, activated B lymphocytes with pleomorphic nuclei and prominent nucleoli. Cells expressed EBV latent membrane protein 1 (LMP1), EBV encoded small RNA (EBER, shown by in-situ hybridisation) and EBV DNA (shown by PCR). Chest and abdominal CT, and bonemarrow biopsy sample were unremarkable, confirming primary CNS EBV-associated, B-cell lymphoma. The patient had had recurrent bacterial respiratory-tract infections during childhood. Furthermore, at age 5 years she was admitted to hospital for 3 weeks because of severe chickenpox, and was absent from school for Lancet Oncol 2005; 6: 344-46

Mechanism(s) of attenuation of Theileria annulata vaccine cell lines.

Summary Attenuated vaccines are an important means of controlling Theileria annulata infection of cattle. Production is by prolonged cultivation of macroschizont‐infected cells. The mechanism of attenuation remains unclear. There are three general nonmutually exclusive possibilities: Selection of avirulent subpopulations, genome rearrangements and alterations in gene expression. Several groups, including ours, have provided evidence that the population structure usually tends to simplify during attenuation. Our data on the T. annulata (Ta) Ankara cell line show that attenuation is not necessarily accompanied by the population becoming clonal. We have been unable to detect large DNA rearrangements. Evidence for alterations in host and parasite gene expression during attenuation is available. With respect to the host we have shown that attenuation is accompanied by loss of expression of parasite induced matrix metalloproteinases (MMPs). However, in different lines different protease activities are involved. In the T. annulata Ode line we have shown that 8 activities (including MMP9) are downregulated and that this correlates with a loss of metastatic behaviour. This has previously been shown in vitro using reconstituted basement membrane (Matrigel™) and is demonstrated in vivo using scid mice in this study. Thus part of the pathology, namely the ability to disseminate, mediated by host MMPs, is lost upon attenuation. Re‐isolation experiments have shown that the reduction/loss of MMP is a stable transferable trait. A logical extension is that loss of MMP activity (and virulence in general) must be at the most fundamental level a genetic trait of the parasite. Evidence for loss of parasite gene expression is implied by the loss of the ability to differentiate into merozoites on attenuation. Specific evidence for loss of parasite gene expression has been obtained using differential RNA display. We view virulence as a multifactorial phenomenon involving interacting subpopulations of cells and attenuation is a threshold effect whereby the number of virulence factors is reduced below a critical level. On this basis there will be many different ways to achieve attenuation.

Theileria lestoquardi and T. annulata in Cattle, Sheep, and Goats: In Vitro and in Vivo Studies a

ABSTRACT: Theileria annulata, causing bovine tropical theileriosis, and T. lestoquardi (syn T. hirci), the agent of malignant ovine theileriosis, are both transmitted by ticks of the genus Hyalomma. Their distribution is thus very similar and, should these parasites infect more than one ruminant species, the difficulty in interpreting epidemiological studies is magnified considerably. A pilot series of experiments was thus conducted in which cattle, sheep and goats were infected with sporozoites of a single stock of each of T. annulata and T. lestoquardi from a laboratory colony of H.a.anatolicum. Reciprocal cross‐immunity and serological studies and in vitro culture isolations in mononuclear cells of each ruminant species illustrated both the similarity of these organisms and their differences. The importance of these findings in discriminating parasites in epidemiological studies and the control of these diseases with cell culture vaccines is emphasized.