We report here the first case of large granular lymphocytes (LGL) expansion following non-myeloablative allo-BMT for chronic myeloid leukemia. We characterized the morphologic, phenotypic and functional features of the LGL subset amplified in vivo14 months after allo-BMT. Our results indicate that LGL can mediate in vitro a cytolytic activity on tumor cells. In vivo, the timing of the LGL expansion was associated with a sustained complete molecular remission. These observations suggest that LGL are a subset with the properties of effector lymphocytes which may contribute to the graft-versus-tumor effect.
Bone Marrow Transplantation (2001) 28, 1157–1160.
Large granular lymphocytosis (LGL) was recognized as a distinct clinical entity more than 20 years ago, and this syndrome was characterized by lymphocytosis and tissue invasion.1 T-LGL proliferation follows a chronic course during which the major features are neutropenia, anemia, bone marrow infiltration, mild splenomegaly and multiple immune abnormalities. Several cases of T-LGL showed that these cells may contain perforin, a cytolytic molecule responsible for pore formation in target cells, suggesting a lytic potential.2 Recently, new approaches of allogeneic bone marrow transplantation (allo-BMT) using reduced conditioning regimens have been developed to obtain minimal procedure-related toxicity. The rationale for such strategies is to induce efficient and potent immunotherapy mediated by allogeneic T lymphocytes that can exert a direct cytolytic function on tumor cells. Persistently elevated numbers of LGL have been shown to be increased in a few cases after conventional allo-BMT with myeloablative conditioning regimens.3 We report here the first case of LGL expansion following non-myeloablative allo-BMT for chronic myeloid leukemia (CML). Our results indicate that expanded LGL can mediate in vitro a CD3-redirected cytolytic activity. Furthermore, in vivo the timing of LGL expansion was associated with a sustained complete molecular remission of this CML patient. These data suggest that LGL following allo-BMT are a subset with the properties of effector lymphocytes which may represent an important component of the graft-versus-tumor (GVT) effect.
A 59-year-old man diagnosed with CML in chronic phase was treated with a combination of fludarabine, busulfan and anti-T lymphocyte globulin (ATG).4 Time to transplantation from diagnosis was 38 months. Prior to allo-BMT, the patient received hydroxyurea and interferon-alpha with no cytogenetic response. The conditioning regimen included fludarabine 30 mg/m2 for 6 consecutive days, oral busulfan 4 mg/kg/day for 2 consecutive days and ATG (thymoglobuline; Imtix-Pasteur Merieux, Lyon, France) 2.5 mg/kg/day for 4 consecutive days. GVHD prophylaxis consisted of cyclosporin A (CsA) alone. The donor was his HLA-sibling brother. Patient and donor were CMV-seropositive. The bone marrow graft contained 1.1 × 106/kg CD34+ cells, 20.79 × 106/kg CD3+ cells, and 2.17 × 106/kg CD56+ cells. At day 31 post allo-BMT, the patient developed a grade III acute cutaneous graft-versus-host disease (GVHD) which rapidly responded to steroids in addition to CsA. Concomitant to GVHD and at day 71, he developed two episodes of blood CMV infection (as evidenced by positive blood antigenemia) which were successfully treated with ganciclovir (GCV). Three weeks following the second episode of CMV infection, neutropenia with relative lymphocytosis appeared and was attributed to GCV treatment. The patient remained stable until day 224, when he developed a severe pulmonary infection concomitant to a moderate oligoclonal IgG expansion on serum electrophoresis. Bacterial and viral investigations were negative and no etiology could be found to explain the IgG oligoclonal expansion. One year after allo-BMT, the patient was in hematological remission, but could not obtain a molecular remission as ascertained by polymerase chain reaction (PCR) analysis for the Philadelphia (Ph) chromosome. Fourteen months following allo-BMT, the patient was admitted for another episode of severe neutropenia with life-threatening septicemia. A bone marrow aspirate at this time showed an important infiltration with medium-sized abnormal lymphoid cells consistent with LGL. LGL or other abnormal cells could never be detected before this episode on different bone marrow aspirates performed regularly. In addition, the patient was in full donor chimerism at the time of LGL appearance. LGL were suspected to be responsible for this severe neutropenia. Therefore, the patient received steroids and 3 days of ATG (125 mg/day) which had little effect on the percentage of circulating LGL. Surprisingly, and simultaneously to the LGL infiltration, the patient became, for the first time since allo-BMT, negative for the Ph chromosome on PCR analysis. This case gives evidence that molecular remission in this patient overlaps to some extent with the LGL appearance. At the time of follow-up (18 months), complete molecular remission is still sustained. A summary of the most relevant clinicopathological features of this patient are shown in Table 1.
Characterization of LGL population
In serial peripheral blood and bone marrow samples abnormal lymphoid cells ranged from 16 to 70%. These cells were mainly large in size, showing round to indented nuclei with coarse chromatin and azurophilic cytoplasmic granules. We examined the distribution of Vβ segments in this LGL population using monoclonal antibodies directed against different Vβ subfamilies. LGL did not display an oligoclonal expansion using the currently available antibodies. Three-color immunophenotyping revealed that these cells were CD8- and CD57-positive. We confirmed that LGL were positive for CD2, CD3 and negative for CD16 and CD56. When focusing on early activation markers, we noticed that LGL were dimly positive for the IL-2 receptor CD25. CD8+CD57+ LGL displayed CD38, the β2 integrin CD11c, and adhesion molecule CD54, the late activation marker HLA-DR and were CD45RA-positive. CD28 expression was negative. These morphological and immunophenotypic features were consistent with LGL of T cell lineage.
The direct cytolytic capacity of effector lymphocytes ex vivo is primarily due to pore-forming proteins (perforin) contained within cytoplasmic granules and released into the vicinity of target cell membranes, thus triggering apoptosis. We assessed by flow cytometry the presence of perforin in the expanded LGL subset. We found a high amount of intracellular perforin in the LGL subset.
To directly assay the cytolytic capacity developed ex vivo by these CD8+CD57+ LGL cells, CD8+ T cells were sorted from peripheral blood and tested in anti-CD3 mAb redirected cytolytic assays against the murine Fc-receptor bearing mastocytoma P815 target cells. LGL cells from the patient exhibited a strong cytotoxic T lymphocyte (CTL) activity against P815 in the presence of anti-CD3, as compared to CD8+ T cells isolated from healthy volunteers (Figure 1a). When CD8− cells sorted from the patient were used as effector cells, they did not share the same lytic properties of CD8+CD57+ LGL (Figure 1a). CD8+ T cells from healthy volunteers exhibited very low, if any, cytolytic activity against P815 tumor cells. In contrast, LGL did not lyse K562 target cells (erythroleukemia with a deficiency of HLA class I molecules) (Figure 1b). Interestingly, only at a high E/T ratio, a moderate CTL activity against K562 target cells could be detected when non-LGL cells from the patient were used as effector cells. Hence, these data indicate that CD8+CD57+ LGL expanded following reduced or non-myeloablative allo-BMT can develop a lytic program upon cross-linking of the TCR–CD3 complex.
In this report, we show that a subset of T cells with morphologic and phenotypic features of LGL, developing after a reduced conditioning regimen can exert in vitro a cytolytic activity. The correlation between cytolytic function and LGL features was remarkably high, since only very weak or no lytic activity was found within the non-LGL fraction. Unfortunately, no initial leukemic cells were available to test the LGL subset lytic activity against the patient's tumor cells. Nevertheless, we observed a direct correlation between the timing in vivo of this LGL subset and the induction of complete molecular remission in this CML patient. Such coincidence is intriguing and raises the question of the major anti-leukemic role of CD8+CD57+ T-LGL cells expanded in vivo after allo-BMT. Chronic antigen-specific stimulation of CD8+ T cells due to viral infections like HIV or CMV has been associated with LGL expansion.5 The CMV infection encountered in this patient may have contributed to LGL expansion, but efficient treatment with ganciclovir and the long delay (11 months) between CMV infection and circulating LGL are not in favor of a close relationship between CMV infection and circulating LGL.
The complete molecular response encountered in our CML patient, occured while the patient did not experience any sign of GVHD. This observation illustrates that specific subsets of effector lymphocytes like LGL can mediate a potent GVT effect without the hazardous immediate and late complications of GVHD.
The striking difference between reduced or non-myeloablative and conventional conditioning regimens is related to the high immunosuppression of the host's immunity based on fludarabine and ATG. ATG results in depletion of lymphocytes and fludarabine and inhibits the mixed lymphocyte reaction in vitro. Thus, fludarabine-based conditioning with or without ATG may lead to a state of immune balance favorable to the emergence of specific T subsets, like LGL, with adverse effects against normal hematopoietic progenitors as evidenced by the different episodes of cytopenia encountered by our patient, but also a powerful anti-tumoral effect. Further studies are necessary to investigate the different non-myeloablative conditioning regimens including fludarabine and ATG and assess the role of post-transplant immunosuppressive therapies in such approaches in order to determine whether other agents or kinetics would allow us to elicit in vivo efficient specific cytotoxic effectors against tumor cells.
McKenna RW, Parkin J, Kersey JH et al. Chronic lymphoproliferative disorder with unusual clinical, morphologic, ultrastructural and membrane surface marker characteristics Am J Med 1977 62: 588–596
Oshimi K, Shinkai Y, Okumura K et al. Perforin gene expression in granular lymphocyte proliferative disorders Blood 1990 75: 704–708
Dolstra H, Preijers F, Van de Wiel-van Kemenade E et al. Expansion of CD8+CD57+ T cells after allogeneic BMT is related with a low incidence of relapse and with cytomegalovirus infection Br J Haematol 1995 90: 300–307
Mohty M, Faucher C, Vey N et al. High rate of secondary viral and bacterial infections in patients undergoing allogeneic bone marrow mini-transplantation Bone Marrow Transplant 2000 26: 251–255
Wang EC, Moss PA, Frodsham P et al. CD8highCD57+ T lymphocytes in normal, healthy individuals are oligoclonal and respond to human cytomegalovirus J Immunol 1995 155: 5046–5056
Mohamad Mohty was supported by a grant from the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC).
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Mohty, M., Faucher, C., Gaugler, B. et al. Large granular lymphocytes (LGL) following non-myeloablative allogeneic bone marrow transplantation: a case report. Bone Marrow Transplant 28, 1157–1160 (2001). https://doi.org/10.1038/sj.bmt.1703308
- anti-tumor activity
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