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B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by an accumulation of clonal malignant B cells. The intrinsic characteristics that permit this accumulation have been extensively studied and described. However, it is possible that proliferation and survival of this malignant clone is facilitated by a disruption in the interaction between B and T cells that normally regulate the immune system. In this study, using flow cytometry and cell culture techniques, marked abnormalities of the expression of certain key activation and interaction molecules on the peripheral blood T cells of patients with B-CLL were demonstrated. In particular, on comparison with normal controls, there was a marked reduction in the number of circulating T cells expressing CD25 (interleukin 2 receptor) (P = 0·007), CD28 (P = 0·01) and CD152 (CTLA-4) (P = 0·001). There was also a reduction in the number of circulating T cells expressing CD4 (P = 0·03), CD5 (P = 0·05) and CD11a (P = 0·01). There was no difference in the number expressing T-cell receptor αβ (P = 0·1), CD8 (P = 0·4), CD54 (P = 0·4) and CD154 (P = 0·5), and the only marker expressed on a greater number of circulating T cells in B-CLL patients was HLA-DR (P = 0·05). These results suggest that there is a profound T-cell dysregulation that may contribute to the survival of the malignant B cells in patients with B-CLL and to the related autoimmune phenomena of the disease.
B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by the accumulation of malignant B cells in lymphoid tissue, the bone marrow and the peripheral blood. The pathogenesis of B-CLL is poorly understood and is associated with a profound disturbance of immune regulation. In addition to the malignant B-cell clonal expansion, there is evidence that T-cell function is compromised. Morphological and functional abnormalities of the non-malignant T-cells have been confirmed in patients with B-CLL (Chiorazzi et al, 1979; Kay et al, 1979; Han et al, 1981; Kay, 1981; Foa et al, 1985; Ayanlar-Batuman et al, 1986; Totterman et al, 1989; Peller & Kaufman, 1991; Antica et al, 1993; Prieto et al, 1993; Dianzani et al, 1994; Rossi et al, 1996; Cantwell et al, 1997; Mu et al, 1997; Tinhofer et al, 1998; Hill et al, 1999). These abnormalities could theoretically be associated with an impaired ability to recognize and regulate normal and malignant B-cells.
In the normal immune response, T-cell activation is mediated by interactions between antigen presenting cells (APCs) such as B-cells and dendritic cells (DCs). These interactions involve a pathway of highly regulated events critical for specific activation and control of both B and T cells (Fig 1). A recent study has suggested that surface expression of CD154, the ligand for CD40, is reduced in the ‘normal’ T-cell compartment of patients with B-CLL (Cantwell et al, 1997). This finding, if confirmed, would lend support to a role for impaired T-cell function in the pathogenesis of B-CLL proliferation and associated autoimmunity. However, CD40 binding to CD154 is only one in a complex series of events in T-cell activation and interaction with B-cells.
When T cells encounter antigen in conjunction with APCs, cell–cell contact is first established with these APCs via leucocyte function associated molecule-1 (LFA-1) (CD11a) and intercellular adhesion molecule-1 (ICAM-1) (CD54) interaction, when cell binding is still non-specific and of low affinity as no antigen recognition has taken place. The LFA-1/ICAM-1 interaction is a major contributor to adhesion between T cells and other lymphoid cells (Makgoba et al, 1989; Figdo et al, 1990; Lub et al, 1995). The binding of LFA-1 (CD11a)/ICAM-1 (CD54) allows the cells to be brought into close enough contact for antigen recognition to take place via the T-cell receptor (TCR)/CD3 complex.
The immune response continues via signalling between HLA antigens and the TCR, a process that is facilitated by the CD3 complex and strengthened by CD4 (MHC class II) or CD8 (MHC class I) binding, thus triggering the T-cell activation cascade (Benjamini et al, 1996). Signalling through the TCR/CD3 complex leads to the initiation of key T-cell immune responses, including cytokine production and surface marker upregulation. The cell surface marker CD28 on the T cells interacts with the CD80/CD86 receptors on the B cells and upregulation of CD154 on the T cell leads to binding with its appropriate ligand, CD40, on the B-cell surface (Durie et al, 1994; Clark et al, 1996; Grewal & Flavell, 1996, 1997; Lenschow et al, 1996). Production of interleukin 2 (IL-2) is initiated and receptors for this cytokine (CD25/IL-2R) are constitutively expressed approximately 48 h post activation, facilitating recruitment of T cells and their continued activation and clonal expansion (Waldmann, 1986, 1991; Taniguchi & Minami, 1993). The final key stage in this cascade is the expression of CTLA-4 (CD152), believed to send a negative ‘off’ signal to the T cell and to control the immune response, either by terminating T-cell proliferation or by inducing apoptosis (Walunas et al, 1994; Krummel & Allison, 1995; Tivol et al, 1995; Schweitzer & Sharpe, 1998).
This study was therefore designed to examine, in vitro, T cells from the peripheral blood of patients with B-CLL, in five stages. The first was to confirm a previous report of reduced T-cell CD154 expression (Cantwell et al, 1997). The second was to examine expression of other key molecules involved in antigen recognition and T-cell activation: CD25 (IL-2R), CD28, CD152 (CTLA-4), TCRαβ, CD4, CD5, CD8 and HLA-DR. The third was to examine the expression of the adhesion molecules critical for cell to cell contact: LFA-1 (CD11a) and ICAM-1 (CD54). The fourth stage involved the removal of the malignant B-cell clone using magnetic bead separation and the subsequent stimulation of the remaining T cells for analysis of two key surface antigens, CD28 and CD152. The final stage involved depleting the B cells, stimulating with OKT3 and permeabilizing for analysis of internal CD25, CD28 and CD152.
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These results suggest that the circulating T-cell compartment in the peripheral blood is profoundly dysregulated in patients with B-CLL. Expression of key cell surface activation and interaction markers is markedly reduced after activation, with the exception of HLA-DR and CD154, the ligand for B-cell CD40. Each of the key markers discussed here plays a major role in stimulating not only the T cell on which it is expressed, but also in the activation of and interaction with many other key immune responders. The complete lack of expression of surface CD152 (CTLA-4) on the circulating T cells of half the patients with B-CLL, the persistence of its reduced expression after B-cell depletion and the increased intracellular expression in unstimulated cells are of interest. CD152 plays a pivotal role in immune regulation by effectively providing a negative feedback (‘switch-off’) signal to the T cell once an immune response has been initiated and completed (Walunas et al, 1994; Krummel et al, 1995; Tivol et al, 1995; Schweitzer & Sharpe, 1998).
The finding of increased expression intracellularly in unstimulated cells suggests that these T cells may be in a partial state of activation, yet are unable to effectively express the antigen or signal externally. Without the external expression, failure to ‘switch off’ the T-cell compartment could lead to the survival of a clone of T cells in this partial state of activation, able to make weak immune responses, possibly against self-antigen, yet unable to mount an effective response to known T-cell mitogens. CD152 knock-out mice display a clinical and pathological syndrome that is similar to that seen in the B-CLL patient (Tivol et al, 1995). This includes spontaneous lymphoproliferative disease with lymphocytic infiltrates in many tissues, splenomegaly, lymphadenopathy and higher rates of autoimmune phenomena than in CD152+ mice. Failure to complete a T-cell response or a continued partial response to malignant B-cell antigens could facilitate their proliferation in these mice and in human subjects. The lack of expression of CD152 may result in a failure to delete autoreactive T cells or prevent antigen-specific apoptosis of activated T cells. Anderson et al (2000) have recently shown that, if T cells are not fully activated or the TCR signal is weak, blockade of CD152 paradoxically inhibits immune responses. The lack of expression of CD152 demonstrated in the CLL T cells may equate to blockade in this respect.
Reduced expression of surface and intracellular CD28 after activation suggests a relative inability to interact effectively with any cell that expresses the CD28 ligands CD80 and CD86, including B cells, and thus an impaired ability to promote antigen presentation and processing. Cell–cell adhesion may also be impaired because of reduced expression of LFA-1 (CD11a). A reduced expression of surface CD25 after activation suggests a relative inability to respond to IL-2 and, thus, an impaired ability to control T-cell activation. However, an increased expression of intracellular CD25 in unstimulated T cells correlates with the finding of increased internal CD152. Both results imply that the T cells are in a partially activated state. Reduced expression of CD4 implies that the initial activation signal generated by MHC class II antigen presentation through the CD3/TCR/CD4 complex on the T-cell surface may be weakened, although reduction in expression of TCRαβ is not statistically significant. CD4 plays an important role in both adhesion between B and T cells and also in generating unique and rapid signals to the cell nucleus for activation (Benjamini et al, 1996). A reduction or absence of this antigen may impair or prevent transmission of signals for T-cell activation.
In contrast to our findings of reduced expression of these markers and to the findings of others (Cantwell et al, 1997), we have shown that surface CD154 (CD40L) is expressed equally on T cells in normal subjects and B-CLL patients. By using CD2 as the pan T-cell marker after activation instead of CD3, we may have included T cells that would not have been detected owing to CD3 receptor occupation by OKT3 or other anti-CD3 activators. OKT3 is believed to occupy and modulate the CD3 antigen and to either become endocytosed into the cell or block the receptor, preventing antibody binding and thus reducing the total number of T cells that will stain positively with CD3 (Sgro, 1995; Bonnefoy-Berard & Revillard, 1996; Reinke et al, 1997; Brusa et al, 1998).
We have also shown that HLA-DR is expressed on significantly more T cells in B-CLL patients than in normal controls. This anomaly of normal or increased expression of CD154 and HLA-DR compared with reduced expression of the other antigens in the activation pathway requires further investigation.
These abnormalities of cell surface antigen expression on B-CLL T cells are not altered by depletion of the malignant B-cell clone. This implies that either the effect of the malignant cells is long-lived or that the T-cell abnormality is a primary one. The former explanation is the more probable, suggesting a chronic but not necessarily irreversible dysfunction of the T-cell compartment. Studies are currently underway to address this question.