Peter Lloyd Amlot

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.

Follicular dendritic cells share a membrane-bound protein with fibroblasts.

The expression of a fibroblast antigen (AS02) on a proportion of CD21+ follicular dendritic cells (FDCs) provides evidence in support of their fibroblastic reticular origin. This antigen is expressed on the membrane of tissue fibroblasts but is absent from lymphocytes, macrophages or granulocytes. The distribution of AS02 in conjunction with other FDC markers (DRC‐1, RFD3, CD23, IgM, and vitronectin) showed six types of FDCs. AS02 is present in the outer layers of primary and secondary follicles, but gradually decreases and disappears in the centre of germinal centres. In contrast, there is a progressive up‐regulation of the other FDC markers. AS02 is re‐expressed in involuting FDCs. Intermediate forms from fibroblastic to dendritic appearance are also apparent and occasionally FDC processes contain collagen type I and IV fibres, a characteristic feature of fibroblasts. In pathological follicles the normal differentiation pattern is disrupted, with persistence of the fibroblast marker, possibly due to altered interactions between FDCs and disrupted lymphocytic patterns. These findings provide new evidence for a local differentiation pathway of fibroblasts to mature FDCs. Copyright

Inhibition of alloresponsive naive and memory T cells by CD7 and CD25 antibodies and by cyclosporine.

Human naive and memory T cells can be isolated from each other by their CD45RA and CD45RO expression, respectively. This enables the assessment of their differential sensitivities to immunosuppressive agents for the first time. We have investigated the ability of cyclosporine or CD7 and CD25 antibodies to selectively block alloantigen stimulated naive and memory T cells in vitro. CD7 antibodies blocked the proliferation of naive (P less than 0.025) but not memory T cells in a primary MLR. CD25 antibody inhibited both naive and memory subsets but a significantly greater effect was found on the memory T cells (P less than 0.005). The constitutive CD7 and CD25 antigen expression on resting naive or memory T cells was related to the inhibitory activities of these antibodies on both subsets. Accordingly, naive T cells expressed more CD7 antigen than memory cells while memory T cells displayed low levels of CD25 antigen that was absent from naive populations before activation. Cyclosporine, like CD25 antibody, inhibited both subsets in a primary MLR but had a greater effect on memory cells (P less than 0.02). Memory T cells, therefore, are more dependent than naive cells on IL-2 for proliferation. There was great individual variation in the ability of CsA to block the MLR. The simultaneous addition of CD25 or CD7 antibody together with CsA, however, enhanced the MLR inhibition as the effects of all three were additive. This suggested interference by these agents at different points during T cell activation. Thus, in CsA sensitive individuals, one-tenth of the optimal CsA concentration together with CD25 antibody maintained maximum immunosuppression in vitro. These results demonstrate the possibility of using CD7 and CD25 antibodies for selective inhibition of naive or memory T cells and also the possibility of augmenting the inhibition of and reducing the CsA concentration required for clinically effective immunosuppression.

THE EFFECT OF A CHIMERIC MOUSE-HUMAN CD7 ANTIBODY ON HUMAN T, NATURAL KILLER, AND LYMPHOKINE-ACTIVATED KILLER CELL ACTIVITY IN VITRO

A chimeric CD7 antibody has been constructed with mouse variable and human constant regions and is currently being assessed in the prophylaxis of renal graft rejection. In this study we have investigated if this antibody or its murine parental form inhibits the function of a number of immune effector mechanisms involved in host defense against infection and/or malignancy. Most memory T cells and all natural killer cells express the CD7 antigen and could therefore be affected by CD7 antibody. Murine and chimeric CD7 antibodies significantly inhibit the alloproliferation of naive (65 +/- 4% and 66 +/- 8%, respectively) but not memory T cells (86 +/- 2% and 98 +/- 4%, respectively) in a primary mixed lymphocyte reaction relative to the negative control CD10 antibody (P less than 0.001). The memory T cell proliferative response to recall antigen is also largely unaffected by murine and chimeric CD7 antibodies relative to the negative control antibody (91 +/- 12% and 103 +/- 10%, respectively). The CD7 antigen is almost completely modulated from the surface of NK cells after incubation for 24 hr with either the murine or chimeric CD7, but not the CD10, negative control. The modulation of CD7 antigen by antibody, however, does not affect the cytotoxic function of either the NK or lymphokine-activated killer cells significantly. Preincubation with the chimeric antibody however, consistently showed a small inhibition relative to the negative control of 75-80% in NK assays and to 80-90% in LAK assays. These data suggest that both murine and chimeric CD7 antibodies may have a selective effect on alloproliferation but may largely spare a major component of the hosts innate immunity as well as memory T cell proliferation to previously encountered antigens.

CD7 Monoclonal Antibodies

It is now a decade since the prototype CD7 monoclonal antibody (Mab) 3A1 was described (Haynes, Eisenbarth, and Fauci, 1979). At this time ten years ago it was first noted that 3A1+ cells respond more vigorously upon stimulation with lectins than 3A1− ones, suggesting that this Mab identifies a large functionally distinct set of T cells. Mab 3A1 and two other Mabs, 4A and CL1-3, were designated as the CD7 cluster at the First Leucocyte Typing Workshop. Since then many CD7 Mabs have been produced, some of which are listed in Table 18.1.