J. Alero Thomas

Analysis of the T-cell micro-environment in Epstein–Barr virus-related post-transplantation B lymphoproliferative disease

Epstein–Barr virus (EBV) post‐transplantation B lymphoproliferative disease (BLPD) may undergo regression after immunosuppression withdrawal and restoration of EBV‐specific cytotoxic T‐cell (CTL) activity in the immunocompromised allografted host. The presence of morphologically normal T cells in the BLPD micro‐environment may influence tumour behaviour in vivo. In this immunopathological study, the phenotype and the number of T cells and other immunoregulatory cells have been investigated in seven primary and four recurrent BLPD biopsies from nine solid organ transplant recipients. BLPD with either viral lymphadenopathic or polymorphic lymphoma appearances was found to contain sizeable T‐cell populations, mainly of memory/helper (TCRα/β+, CD3+, CD4+, CD45RO+) type. Cytotoxic (TCRα/β+, CD3, CD8+, Tia‐1+) T cells were strikingly low in all samples. Low CD28 and CD25 expression suggested that secondary signals for functional and sustained T‐cell activation may be deficient in these tumours. No close correlation was found between the degree of T‐cell infiltration and clinical outcome, although appreciably higher numbers of CD8+ T cells were detected in three BLPD tumours showing prolonged clinical remission after treatment. While some level of EBV‐specific T‐cell function may be present in untreated BLPD, the overall findings of this study suggest that the nature of T‐cell infiltrates may reflect a response to immunosuppressive therapy rather than to EBV infection per se. The possibility that a local EBV‐specific T‐cell response is generated in BLPD undergoing regression after treatment needs to be investigated.

Immunodeficiency-related lymphoproliferative disorders: prospective data from the United Kingdom Children's Cancer Study Group Registry.

Summary. Clinical data and biological samples were prospectively collected in 42 children with lymphoproliferative disease (LPD) secondary to organ/bone marrow transplant‐related immunosuppression (30: 11 liver, 10 heart/lung, 8 kidney and 1 bone marrow), other drug‐induced immunosuppression (2), congenital immunodeficiency (8) or human immunodeficiency virus (HIV)‐related immune dysfunction (2). Ages ranged from 10 months to 17 years and there were 15 girls. Pathology was centrally reviewed and showed polymorphic features in 5 cases, monomorphic in 23, mixed pattern in 5 patients and 9 other types. Using the Revised European–American Classification of Lymphoid Neoplasms, 5 were B lymphoblastoid, 24 were high‐grade B and 14 were other subtypes. Using the Pittsburgh classification, 9 were lymphadenopathic, 10 were systemic, 25 were lymphomatous and, with the Murphy grouping for non‐Hodgkins lymphoma (NHL), 10 were localized and 32 non‐localized. Twenty‐four out of 38 evaluable cases were Epstein–Barr virus positive. Thirty‐five patients were evaluable for clonality; 24 were monoclonal and 11 were polyclonal. Reduced immunosuppression in solid organ transplant patients resulted in resolution of disease in 14/24, which was sustained in 11. Nineteen patients received chemotherapy, 14/18 evaluable responded, which was sustained in 8 cases. Seven out of 29 solid organ transplant and 10/13 other immune‐deficient patients died. In the largest group of patients, solid organ transplants, no significant clinical or biological characteristics that predicted outcome were identified. In the transplant group close monitoring of response during reduction in immunosuppression is essential and the early use of B NHL chemotherapy may be effective.

Biogenesis of secretory organelles during B cell differentiation

The differentiation of B cells into Ig‐secreting plasma cells requires the expansion of secretory organelles to cope with the increased cargo load. To evaluate the timeline of this process, we have quantitated the kinetics of secretory organelle expansion relative to Ig secretion and examined regulatory components of secretory transport following in vitro activation of human B lymphocytes. Unstimulated B cells contain minimal endomembranes. After activation, ER membrane induction appears as tightly packed spherical structures of 0.5–1 μm diameter concentrated in a juxtanuclear position. When the cells differentiate into plasmablasts, there is dramatic cell‐size increase, but the ER remains concentrated close to the nucleus and only later fills the entire cell. In sharp contrast, previous studies in other cell types have found that the ER expands in synchrony with increasing cell size during interphase, by extension of ER tubules under the PM. In this study, the Golgi remains consistently as a single juxtanuclear structure but linearly expands sixfold in volume during B cell activation. Furthermore, following active cell proliferation, ER exit sites proliferate rapidly, increasing almost fourfold in number, in parallel with a sharp increase in Ig secretion. These findings demonstrate that the control of organelle biogenesis and expansion in primary human B cells are differentially regulated by cargo flux caused by Ig synthesis.

Expansion in scid mice of Epstein-Barr virus-associated post- transplantation lymphoproliferative disease biopsy material

Post-transplant lymphoproliferative disease (PTLD) biopsy material is rarely available in adequate quantity for research. Therefore, the present study was designed to expand biopsy material in scid mice. Epstein-Barr virus (EBV)+ve PTLD samples from five transplant patients were established in scid mice. PCR analysis of immunoglobulin gene rearrangements demonstrated that four of the five biopsies (80%) gave rise to scid tumours which represented the original tumour cell clones. Immunophenotyping showed that these four biopsies (and all scid tumours) expressed all EBV latent genes and a B lymphoblast phenotype; <or=26% T cells were found in the biopsy material whereas scid tumours showed a paucity of T lymphocytes. RT-PCR analysis revealed expression of IL-2, -4, -6, -10 and IFN-gamma in all tumour material, suggesting key roles for these factors in tumour growth. The results show that EBV+ve PTLD material can be expanded in scid mice giving rise to quantities of homogeneous malignant tissue sufficient for research studies.