We have analyzed the phenotype, cytokine profile, and mitotic history (telomere length) of monoclonal T-cell expansions in 5 CD3 ؉ T-cell large granular lymphocyte (TLGL) leukemia patients by fluorescence activated cell sorting (FACS) and single-cell polymerase chain reaction (PCR). We confirm that the common phenotype of TLGL leukemia is CD3 ؉ CD8 ؉ CD45RA ؉ CD27 ؊ CD94 ؉ (CD57 ؉ ). Interestingly, the C-type lectin-like type killer cell receptor CD94 was invariably associated with the activating form of its signaltransducing molecule NKG2. Furthermore, when judged by criteria such as interferon gamma (IFN-␥)/tumor necrosis factor (TNF) production, expression of granzyme, FasL, and NKG2D, the TLGL cells had all the features of a cytotoxic effector T cell. Telomere shortening in TLGL cells was in the normal range for CD8 ؉ T cells, indicating that they had not divided significantly more than chronically stimulated CD8 ؉ T cells in healthy individuals. In 25 of 27 controls, cells with a TLGL phenotype occurred at low (1%-3%) frequencies. However, in the other 2 individuals (ages 28-36 years), large stable (> 3 years) monoclonal expansions of CD3 ؉ CD8 ؉ CD45RA ؉ CD27 ؊ CD57 ؉ CD94 ؉ NKG2C ؉ were found which rendered these controls phenotypically indistinguishable from TLGL leukemia patients. We believe that the TLGL clonopathy, rather than being IntroductionLymphoproliferative disorders of large granular lymphocytes (LGLs) are classified as lymphoid neoplasms. 1,2 They are divided into 2 distinct entities: approximately 10% to 20% are of the natural killer (NK) lineage (CD3 Ϫ CD16 ϩ ), whereas the others consist of mature CD3 ϩ T cells expressing either an ␣␤ or a ␥␦ T-cell receptor (TCR). NK-LGL leukemia is by far the more aggressive form. Most patients have more than 10 ϫ 10 9 /L LGLs in their blood and a significant percentage dies within 2 months after diagnosis. 3 By contrast, CD3 ϩ T-cell LGL (TLGL) leukemia that affects mainly elderly patients progresses in a much milder way. Lymphocytosis is usually modest 4 and many patients may even remain asymptomatic. 3,[5][6][7] Moreover, clinical complications consist primarily of neutropenia or autoimmune features. 8 Therefore, it remains under debate whether this T-cell clonopathy of variable significance 9 should be considered as a malignancy or rather as a secondary reactive phenomenon. 6 Apart from the low malignancy of TLGL leukemia, there are more indications that the clonal expansion is the result of a perhaps rather common dysregulation of CD8 ϩ T cells. In fact, large oligoclonal expansions of CD8 ϩ T cells are quite frequent in elderly people. 10 Such clones that may be maintained by cytokines rather than by antigen 11 are of a phenotype similar to that of normal CD8 ϩ effector T cells of which the clonal size is still controlled through apoptosis. The latter is also true for the TLGL clone. In the majority of patients, these cells are CD8 ϩ CD45RA ϩ CD27 Ϫ , 6,7,12,13 express Fas and FasL,14,15 and may or may not express CD57. 6,7 Therefore, they share m...

A subset of CD8 + T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8 + T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7 + memory cells and CCR7 -effector cells. Since NKG2A can only be induced on naive CD8 + T cells while CD94 -memory cells are refractory, it is likely that commitment to the CD94:NKG2A + subset occurs during the first encounter with antigen. CCR7 + CD94:NKG2A + T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-c. These cells occur as a separate CD94:NKG2A + T cell lineage with a distinct TCR repertoire that differs from that of the other CD8 + CD94 -T cells activated in situ.

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A subset of effector/memory CD8 + T cells expresses natural killer cell receptors (NKR). Expression of inhibitory NKR at that stage of T cell differentiation is poorly understood. Interestingly, recent studies in mice indicated that transgenic expression of an inhibitory NKR induced the accumulation of memory T cells by inhibiting activation-induced cell death (AICD). To further understand the role of inhibitory NKR on T cells, we characterized the subset of human peripheral T cells expressing the inhibitory NKR, CD158b, and studied the modulation of antigen-driven T cell expansion by an endogenous inhibitory NKR. We found that CD158b expression was confined to a population of CD8 + TCRab + effector T cells as defined by a CD45RA + CCR7 -phenotype and high constitutive expression of granzyme B1. Few cells expressed the activating form CD158j in the absence of CD158b. Functionally, engagement of CD158b by MHC ligands diminished early TCR signaling, as well as AICD. However, the reduced AICD did not rescue cells for proliferation, since T cell expansion in the presence of CD158b triggering was impaired. Expression of inhibitory NKR on effector CD8 + T cells may explain in part the poor replicative capacity of T cells at that stage of differentiation.

We have studied the alterations in CD45R phenotypes of CD4+CD45RA–RO+ T cells in recipients of T cell‐depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re‐expression of CD45RA by former CD4+CD45RA– T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA+ T cells through the thymic pathway. We found that CD4+CD45RA–RO+ recipient T cells could re‐express CD45RA but never reverted to a genuine CD4+CD45RA+RO– naive phenotype. Even 5 years after transplantation, they still co‐expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower (∼ 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4+CD45RA–RO+ T cells may re‐express CD45high isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co‐expression of CD45RO. Therefore, it is likely that the long‐lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.