14
THE ROLE OF T-CELLS IN
CHRONIC B-LYMPHOCYTIC LEUKEMIA
 
AD DONNENBERG AND VS DONNENBERG
 
ABSTRACT
 
    Chronic B-cell lymphocytic leukemia (B-CLL) has a unique natural history characterized by a long period during which a clonal B-cell lymphocytosis may be the only sign. The events involved in the maintenance of this state and the evolution of the disease process are poorly understood. In addition to the increased number of small resting B-cells, which characterizes early stages of disease, a small population of large atypical lymphocytes is a frequent finding.  We have determined that these cells include a population of activated T-lymphocytes which account for 10-60% of the large lymphocytes. In this review, we postulate an autoimmune-like short circuit in the interactions between T-cells and the nascent B-CLL clone, which confers a great selective advantage on the clone. According to this interpretation, its continuous expansion culminates, over time, in frank disease manifestations. The hypothesis that helper T-cells play a key role in the evolution of B-CLL suggests a series of experiments exploring T-cell/B-CLL interactions, and if correct identifies a new target for therapeutic intervention.
 
INTRODUCTION
 
Natural History of B-CLL
 
    Chronic B-cell lymphocytic leukemia is the most common leukemia in the United States with an annual incidence estimated at 3 cases per 100,000 persons (1). In persons over age 60 the annual incidence exceeds 20 cases per 100,000 (2). Although B-CLL has traditionally been characterized as a disease of the elderly, the ability to detect clonal B-cell expansion by flow cytometry has provided data which are causing us to revise our view of its natural history.  CLL often begins insidiously in mid-life; in the initial stages clonal B-cell lymphocytosis may be the only finding. Because of this clinically inapparent onset, diagnosis is often incidental and the early natural history has been difficult to study. Despite (or perhaps because of) its indolent course, B-CLL is presently considered incurable (3). As the lymphocytosis progresses over the course of years, anemia, thrombocytopenia and increased susceptibility to infection become significant disease manifestations. Less commonly, B-CLL is marked by a progression to prolymphocytic leukemia or other B-cell malignancies.  Prolymphocytic leukemias almost always arise from the B-CLL clone, whereas about half of the large B-cell lymphomas, acute lymphoblastic leukemias and multiple myelomas developing after B-CLL are genetically unrelated (4). Autoimmune disorders and unrelated neoplasms may also complicate the course of advanced B-CLL. Although B-CLL is a clonal proliferative disorder, chromosomal abnormalities are varied, can only be demonstrated in about half of B-CLL cases, and are less frequent in early disease (5). Prognosis is still best assessed by a relatively simple clinical staging procedure, although chromosomal abnormalities, bone marrow histology, immunophenotype and lymphocyte count doubling time have also been reported as predictors of disease progression. The Rai classification (6) recognizes 5 stages (0-IV) based on hemogram and physical examination (Table 1). This has been condensed into 3 categories which differ with respect to survival: good prognosis (Rai Stage 0), intermediate prognosis (stages I and II) and poor prognosis (stages III and IV). The routine use of flow cytometry to investigate the cause of persistent elevated lymphocyte counts has resulted in the occasional detection of clonal B-cell populations (by clonal excess of kappa or lambda light chains) in the absence of marrow lymphocytosis, suggesting a category preceding Rai stage 0.
 
 
B-CLL displays functional and phenotypic characteristics
of mantle zone B-cells
 
    Many similarities between B-CLL cells and normal mantle zone B-cells have been documented (7, 8). The small clonal B-lymphocytes of B-CLL usually express low levels of membrane immunoglobulin (IgM, IgD or both) and CD5, a glycoprotein present on follicular B-cells, a subset of peripheral B-cells, and almost all normal T-lymphocytes (see below). In addition, most B-CLL cells express HLA-DR, CD19, CD20, CD21, CD24, and CD37, which are also present on normal mature B-cells. Most B-CLL also express CD23 which has received attention as a potential B-cell autocrine growth factor (9). Most do not express CD10 (common acute lymphocytic leukemia antigen) or other early markers found in other B-cell neoplasms. Additional markers commonly expressed on B-CLL include CD27, CD39 and NuB1. KiB3, 7F7, and CD25 may be absent or present. Taken together, these populations bear a marked resemblance to cellular subsets in the mantle zone (10). In addition, expression of bcl-2 is high in B-CLL cells and in normal tonsillar follicular mantle zone B-cells, but not in germinal center B-cells or normal peripheral CD5+ B-cells (11). Thus, for the majority of early B-CLL cases, which do not express chromosomal abnormalities or markers discordant with respect to lineage or differentiation, their “normal” phenotype contrasts strikingly with the remarkable tumor mass regularly attained by the B-CLL clone. As an example, consider a Rai stage 0 patient (without demonstrable lymph node, spleen or liver involvement) in whom a typical B-cell count of 50,000/mL multiplied by the blood volume (5L) yields a total of 2.5x10 11 clonal B-cells. This estimate is conservative since it fails to take into account tumor in the bone marrow, lymph nodes and spleen.
 
REGULATION OF B-CELL GROWTH AND
DIFFERENTIATION
 
    T-B lymphocyte interactions regulate the proliferation of mature B-lymphocytes and the synthesis of antibody directed against T-dependent antigens.  It is now recognized that this process involves a lively multiway conversation between CD4+ helper T cells (T h ), B-cells and a variety of professional and part time antigen presenting cells (APC) (reviewed in 12). The molecular mediators of such cellular conversations include an array of signaling molecules, adhesion molecules, growth factors and their receptors. Because B-CLL growth and differentiation appear to stop short at a stage equivalent to that of the resting mantle zone B-cell, it is useful to review the events leading to this stage. Recent studies have focused on two ligand-receptor interactions which appear to be proximate to the initiation of APC/T-cell and T-cell/B-cell interactions, respectively (reviewed in 13). Th cells recognize processed antigen presented in the context of MHC class II (HLA DR) via the T cell antigen receptor (TCR). Although there are a variety of costimuli which can augment this process, the critical event which prevents Th anergy and favors activation and cytokine release appears to be the interaction of B7.1/B7.2 (induced or upregulated on the APC) with CD28 (constitutively expressed on the Th cell).  B-cells themselves can serve as APC, and in murine systems, favor the generation of Th-2 over Th-1 (38). Once activated through their TCR, T-cells transiently express CD40 ligand (CD40L) which upon contact with CD40 (constitutively expressed on B-cells) induces the expression of growth factor receptor (IL-4R) and CD80 (B7.1) in resting B-cells.
 
Th-1 versus Th-2 in freshly isolated human cell
 
    Helper T-cells have been classified as Th-0, Th-1 or Th-2 on the basis of cytokine production patterns. Th-1 is equated with the production of IL-2 and IFN-g (limited B-cell help, DTH, macrophage activation, cytotoxicity) whereas Th-2 produce IL-4, IL-5, IL-9 and IL-10 (B-cell help). Th-0 cells, which in some schemes are thought to represent a common precursor of Th-1 and Th-2, produce IL-2, IL-4, IL-5, GM-CSF and IFN-g. In mouse and in man, characterization of Th-subsets has been accomplished by the analysis of long term T-cell lines and clones. In short term bulk activated cells, these characteristic cytokine patterns are easily obscured by the heterogeneity of responsive cells and the presence of cells of transitional phenotypes. Recently a population of short term, bulk activated T-cells, defined on the basis of surface marker expression (CD30+, CD25+, CD45RO+, CD4+, CD3+), was identified as the major source of T-helper activity in a pokeweed mitogen activated B-cell system (14). The spectrum of cytokine production (IL-5high, IFN-g high, IL-2low) did not fit the classical Th-1 Th-2 pattern, resembling more closely the Th-0 cell. Recent advances in flow cytometry permit the detection of multiple cytokines produced in single cells. Studies in normal and helm-inth-infected subjects indicate that CD27 can be used to resolve populations of CD4+ T cells which produce IL-4 plus IFN-g, versus IL-4 or IL-5 or IFN-g alone (15). Such methods may help clarify the role of activated T cells in B-CLL (see below).
 
IMMUNOPATHOPHYSIOLOGY OF B-CLL
 
Significance of CD5+ expression
 
    CD5 is a monomeric glycoprotein expressed on virtually all T-cells and on a subset of B-cells. Although the function of CD5 in B-cells has not been fully determined, it associates with the B-cell receptor complex and serves as a substrate for receptor-induced tyrosine kinase activity. Thus, CD5+ is thought to have the potential to moderate signals delivered by the B-cell receptor (44).  Its natural ligand, the C type lectin CD72 (45) is constitutively expressed on B-cells, giving rise to the possibility of homotypic interactions between B-cells.  Antibodies directed against CD5 can be costimulatory in culture, although it is unclear to what extent this mimics interactions with the natural ligand. The relative scarcity of CD5+ peripheral B-cells in healthy adults (<30% of normal peripheral B-cells are CD5+), their abundance in cord blood and lymph node follicles as well as their association with autoimmune diseases and B-cell malignancies have lead to conflicting interpretations concerning the significance of CD5 expression on B-cells. CD5+ B-cells have been proposed to comprise a separate B-cell lineage (16), or alternatively a stage of maturation (17) or a subpopulation with suppresser activity (18). CD5+ B-cells have been characterized as being particularly responsive to IL-5; their repertoire of V H and VL usage has been reported to be restricted and prone to expression of autoantibodies primarily restricted to the IgM isotype. The functional significance of CD5 expression is particularly important in CLL, since the great majority of B-CLL clones are CD5+. However, the results of a recent report indicate that functional assumptions based on the expression of the CD5+ phenotype must be viewed with caution (19). In contrast to CD5+ cells in the lymph node, which are sIgM+, CD5+ B-cells isolated from the peripheral blood of healthy subjects express levels of IgG and IgA comparable to their CD5 negative counterparts. Further, CD5 negative cells rapidly and transiently became CD5+ upon exposure to PMA and anti-CD3 activated T-cells.  In cell separation experiments, the CD5-B-cells, which became CD5+ in culture, produced the greatest amounts of immunoglobulin. CD5+ and negative B-cells produced comparable levels of autoantibody. Taken together with the reports cited above, it is clear that CD5 can be expressed on B-cells displaying a broad spectrum of activities and ranging widely in maturational status.  According to this interpretation, CD5 expression on B-cells may be analogous to the expression of certain T-cell “activation markers.” For example, CD38 is constitutively expressed on late thymocytes but is also inducible during activation of mature memory T-cells. Like CD5, its expression is not restricted to cycling cells and in fact is detected on a proportion of small resting T-cells as well. In B-CLL and normal mantle zone B-cells, the expression of CD5 on predominantly small resting cells may reflect their history of activation and proliferation. Thus CD5 may reveal more about the recent experience of a B-cell than it does about its pedigree or maturational state. Accordingly, it is not surprising that a variety of pathologic processes involving B-cell proliferation or activation have been associated with CD5 expression.
 
The specificity of B-CLL surface immunoglobulin
 
    B-CLL hybridomas have been successfully made by a number of laboratories (20, 21). Productive hybridomas have been isolated at high frequency using a variety of murine myeloma fusion partners. Virtually all primary hybridomas produced IgM monoclonal antibody (MAb) which were remarkably similar between patients. Many were broadly reactive, using a restricted
set of Ig variable region heavy and light chains encoded by germline or minimally mutated germline genes (22), and shared cross reactive idiotypes with antibodies associated with rheumatoid arthritis. In a very detailed analysis of the complete heavy and light chain sequences of seven B-CLL hybridomas, three displaying polyreactive binding used V H 4 family members and three which displayed rheumatoid factor activity expressed V H 1 family genes (23).  Kipps has suggested that normal B-cells expressing such autoantibodies may be perpetually stimulated, thereby increasing the opportunity for malignant transformation into CLL, or alternatively that anti-self-reactivity may enhance the survival of a B-cell clone a transformation event (39). In the discussion below we will entertain the hypothesis that these polyreactive autoantibodies may interact with signaling and/or adhesion molecules on T-lymphocytes.
 
T-cell expansion in B-CLL
 
    Normally B-cells comprise a relatively small proportion of circulating lymphocytes. Their clonal expansion in B-CLL raises their proportion such that T-cells appear as a minor subpopulation. However, examination of absolute T-lymphocyte counts (CD3+ cells/mm 3 ) revealed an increase in T-cells as well (1.9-fold higher than control subjects in a series of 10 Rai stage 0 patients and controls, p = 0.004, Student's t-test.). This highly significant increase in T-cells was masked by the overwhelming increase in B-cells when proportion rather than absolute number was considered. We (24) and others (25) have examined expression of activation markers on T cells from patients with B-CLL. One of the most intriguing findings in our study of stage 0 B-CLL patients was that 10-60% of cells falling within the high forward scatter/low side scatter region (which encompasses large atypical lymphocytes and also lymphocyte clusters) were actually CD3+ T-cells. Because of these unique light scatter properties, we were able to compare cells falling within this region with small lymphocytes from the same individuals. Large atypical T-cells did not comprise a discernible population in young healthy control subjects and were only occasionally detected as a minor population in control subjects who were age-matched to the patients. We compared T-cell differentiation and activation markers on large/atypical- and small-lymphocyte light scatter populations within 10 individuals with Rai stage 0 B-CLL, and between the total lymphocyte scatter population of 10 healthy control subjects. No differences were observed between markers on the small lymphocyte population of the B-CLL group and the total lymphocytes of the healthy controls. In contrast, most CD4+ large atypical T-cells were CD45RO+ (77% vs. 27%, large vs. small lymphocyte). Significant subsets within the CD4+ population were CD45RO+/CD29high (77% vs. 25%, Figure 1) and CD25+/CD38dim (32% vs. 16%). Quantitative cytokine determinations in isolated large lymphocytes from one CLL patient (Table 2) revealed a robust T-helper response. The observed increase in IL-2 production (23-fold over control response) has recently been attributed to the effect of B-cells as APC and could be duplicated when T-cells from healthy subjects were exposed to normal B-cell APC (26).
 
 
 
Anti-T Cell Agents and B-CLL Therapy
 
    The unique natural history of B-CLL makes its treatment problematic.  Early disease (Rai stage 0, Binet stage A) is not treated until it progresses.  Advanced disease and rapidly progressing early disease are treated, traditionally with alkylating agents or combination chemotherapy, and more recently, with deoxypurine analogs. The deoxypurine analogues fludarabine, 2-chlorodeoxyadenosine, and pentostatin are gaining acceptance as first line agents (27). The purine analogs have potent effects against normal T cells as well as leukemic B-cells, owing to the high activity of adenosine deaminase in lymphocytes.  Cyclophosphamide, prednisone, and methotrexate, three other agents used in CLL regimens, exert immunosuppressive effects on T as well as B lymphocytes. In fact, one of the limiting toxicities of fludarabine/prednisone combination therapy has been opportunistic infections associated with prolonged severe depression of CD4+ T cell counts (28). Opportunistic infection and depressed CD4 counts were also seen in patients treated for 16 weeks with the humanized anti-CD52 antibody Campath 1-H (40). Campath 1-H targets CD52 on both T and B lymphocytes. Finally, Cyclosporine (CsA), a T-cell immunosuppressive agent with poorly characterized anti-B-cell activity, has been successfully used to treat autoimmune disorders such as pure red cell aplasia occurring in conjunction with B-CLL (41). We know of three cases in which treatment with CsA (unusually combination with glucocorticoids) resulted not only in an improvement in erythropoiesis, but in a marked decrease in the number of circulating leukemic B-cells as well. An example from our own center is shown in Figure 2. Although several other clinical anecdotes reporting beneficial effects of CsA have been published, its use as a primary therapeutic agent for CLL is far from accepted. In a recent series of 5 patients prospectively treated with CsA, a Belgian center reported a marginal anti-tumor response and an unacceptable worsening of treatment related immunosuppression (42). That the benefits of anti-T cell directed CsA therapy were short lived (B-cell counts increased on discontinuation of CsA, Figure 2), may imply an inhibitory as well as stimulatory role for T-cells.
 
    It should be noted that regardless of the regimen, chemotherapy rarely, if ever, results in the cure of B-CLL. With the exception of bone marrow transplantation, which has resulted in elimination of the malignant clone as evidenced by negative PCR (43), but has limited followup, even the best of today’s therapies are ultimately ineffective. Interestingly, BMT provides not only the advantage of dose intensive therapy, but of complete ablation of the T as well as B-cell compartment.
 
    Taken together, these data strongly support the notion that agents with anti-T-cell activity may indeed interrupt T-cell/B-cell interactions which support the growth of the B-cell neoplasm. They also sound cautionary notes: 1) that anti-T-cell agents which are relatively well tolerated in the treatment of other conditions may have unacceptable toxicity in CLL; one of the major consequences of the extensive outgrowth of the neoplastic B-cell clone may be interference with T-cell regenerative capacity following massive insults, such as those delivered by T-cell toxic agents, 2) That nonspecific ablation of the T-cell compartment may remove anti-leukemic as well as stimulatory populations.  The identification of targets which interfere with the delivery of exogenous growth and survival signals to the B-CLL clone, without causing undue injury to the T-cell compartment may be particularly useful in the development of new therapies.
 
 
Potential Roles of T-cells in B-CLL
 
    From the evidence presented above, it is clear that T cells play an, as yet, undefined role in B-CLL. The three most obvious answers to the question “What are activated T-cells doing in a B-cell leukemia?” are: 1) They are mediators of immune surveillance, doing their best to keep a B-cell malignancy in check; 2) They are passively swept along by a dysregulated B-cell neoplasm
that acts as both antigen presenter (26) and cytokine source (30); or 3) As in normal T/B interactions, helper T-cells are in the driver's seat, promoting B-CLL growth by providing the contact dependent and cytokine mediated signals described above. The first hypothesis is of potential importance, because if correct, it provides an avenue for therapeutic intervention through augmentation of a preexisting anti-tumor response. Likewise, the notion that T-cells are polyclonally activated by the B-cell neoplasm could explain antibody mediated autoimmune processes, such as anemia and thrombocytopenia which frequently develop in B-CLL. These are otherwise difficult to explain, since the specificity’s of the autoantibodies are different from that of the B-CLL.  Indiscriminate T-cell activation could have the disastrous consequence of inducing antibody expression in previously silent autoreactive B-cell clones.  Finally, the third answer opens the possibility that B-CLL itself is an autoimmune process, resulting from a short circuit in the normal lines of T-B communication.
 
B-CLL AS A SHORT CIRCUIT IN T-CELL/B-CELL
INTERACTION: A HYPOTHESIS
 
    Any attempt to reconcile this hypothesis with the observed natural history of B-CLL must take into account the clonal nature of the disease and the failure of B-CLL cells to progress beyond the sIg low state. Since every member of the B-CLL clone expresses surface immunoglobulin of the same specificity, it is conceivable that this binding specificity itself confers the selective
advantage that results in clonal outgrowth. Of course, it must also be recognized that any B-cell mutation resulting in growth autonomy would also show clonal immunoglobulin expression, providing that the transforming event occurred subsequent to Ig gene rearrangement. As reviewed above, the majority of stage 0 B-CLL are without evident chromosomal abnormalities; when abnormalities are observed, they are diverse and are an indicator of poor prognosis.  Entertaining the unconventional hypothesis that the CLL B-cell starts off life as a “normal” B-cell with an inopportune specificity, we can postulate at least three experimentally testable mechanisms that could lead to a privileged relationship with helper T-cells.
 
    From least to most involved they are:
 
    1) Preferential binding of CLL B-cells to activated T-cells (Figure 3):  B-CLL sIg binds to a surface determinant expressed on activated, but not resting, T-cells. T-cells activated by irrelevant antigens are physically captured by B-CLL cells through immunoglobulin mediated binding in conjunction with other low affinity membrane-membrane interactions. Once bound, B-CLL cells are stimulated by interaction with CD40L and cytokines (eg, IL-4, IL-5), expressed by the activated T-cell. B-CLL cells, in turn, back-stimulate the T-cells with equally nonspecific signals (eg, B7). Yellin and colleagues have demonstrated that the cell line Jurkat D1.1, which constitutively expresses CD40L, induces expression of CD80 (B7.1) on resting normal and CLL B-cells (31). These findings support our short circuit hypothesis, since upregulation of CD80 (CD28 ligand) on B-cells renders them competent to present antigen to T-cells. In addition to driving proliferation, activated T-cells can also provide anti-apoptotic signals to B-cells through CD40L (32) and IFN-g (37).  According to this scenario, any event initiating T-cell activation (eg, immunization, infection), would trigger a sympathetic increase in B-CLL cells as well.  This hypothesis is not strictly dependent on sIg specificity as the preferential binding to activated T cells could be mediated by the coordinated expression of conventional adhesion molecules on the B-CLL clone.
 
    2) Mitogenic hypothesis (Figure 4): B-CLL sIg provides an activation signal to resting T-lymphocytes by binding public domains of the TCR (ie, like a superantigen), or more likely, to structures which provide costimulatory signals (eg, CD2, CD28). This interaction increases the probability that a random T-cell in proximity to a B-CLL cell will undergo activation. Since CD40L expression on the T-cell is typically rapid and short lived, this would give the B-CLL cell the advantage of being in the right place at the right time when T-activation is initiated. As in hypothesis 1 above, once activated, the B-CLL cell would initiate a bi-directional paracrine loop by back-stimulating the activated T-cell or its resting neighbors.
 
    3) Paired clone hypothesis (Figure 5): This scenario is a special case of hypothesis 2, in which a particular T-cell clone or clones recognize the B-CLL idiotype as antigen (or vice versa). Since B-cells can also act as APCs, and favor selection of Th-2 over Th-1 (38), it is likely that the B-CLL specific T-cell would provide back-stimulatory signals to the B-CLL APC. This would provide another entry point into the T-Cell/B-CLL paracrine loop. The recent finding of skewed TCR Vb expression in B-CLL provides evidence in favor of this hypothesis (33), although our own results on a series of 5 patients revealed that the majority of the presumably clonally expanded T-cells were CD8+ rather than CD4+ (34). This hypothesis bears some analogy with the emerging picture of the etiology of a particular type of gastric lymphoma of the mucosa-associated lymphoid tissue (MALT) in which both clonal T and B lymphocytes have been detected (35). In some instances the origin of MALT lymphoma has been attributed to an aberrant T cell-dependent B-cell response to antigens present on an infectious agent, Helicobacter pylori (see Chapter 8) (36).
 
    The hypotheses presented above are not necessarily mutually exclusive with those that envision a protective role for T-cells, nor those that predict other autoimmune consequences of aberrant T cell activation. What they have in common is the idea of a self-perpetuating bi-directional stimulatory short circuit between T- and B-lymphocytes that gives the B-CLL clone a survival advantage but does not allow it to progress beyond the sIg low stage of development. It is not difficult to imagine how, over the course of months, years or decades, chronic stimulation of a single B-cell clone results in the emergence of mutant lines with less stringent growth requirements. If T cells do in fact play such a role in the natural history of B-CLL, it may be possible to disrupt the disease process by targeting T cells or the signals that they provide.
 
 
 
 
ACKNOWLEDGMENTS

    This work was supported by grant RO 1 CA44887 from the National Institutes of Health. Dr. Donnenberg is the recipient of a Carter-Wallace fellowship for AIDS research. The authors would like to thank Drs. Arnold Meisler, Margaret Ragni, Delynne Myers, Witold B. Rybka and Edward D. Ball for obtaining patient specimens and clinical data, and Tim Patton, E. Michael
Meyer and Deborah Griffin for their expert technical assistance.
 

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