Atypical lymphocytosis (AL) is characterized by increased numbers of large reactive lymphoid cells identified on a Wright-stained peripheral smear and is most often seen in acute viral infection, especially herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), and varicella-zoster virus (VZV), but also pertussis, brucellosis, syphilis, toxoplasmosis, drug reactions, and lymphoid leukemia. The single most common clinical syndrome associated with AL is EBV-associated acute infectious mononucleosis (IM), in which patients typically present with fever, pharyngitis, and cervical lymphadenitis. In IM, EBV-infected B lymphocytes trigger an intense T cell-mediated immune response within oropharyngeal lymphoid tissues and increased atypical lymphocytes in the peripheral blood. Given the association of EBV with many forms of lymphoid malignancy, the AL in IM has been of enduring interest to pathologists, hematologists, virologists, and immunologists.
Previous phenotypic analyses of AL have been limited to EBV-associated IM. We present results of a comprehensive immunophenotypic analysis of peripheral blood lymphocyte subsets from 97 patients with AL, defined as an absolute lymphocyte count of >4000/μl with >10% atypical forms. Results from AL were compared with results obtained from 37 normal controls to identify AL-associated lymphocyte abnormalities. All cases of AL were examined so as to not only identify immunophenotypic abnormalities common to all cases of AL regardless of underlying etiology but also identify abnormalities specific for EBV-positive IM.
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- MATERIALS AND METHODS
- LITERATURE CITED
Several studies of peripheral blood lymphocytes in IM have demonstrated increased numbers of activated CD8-positive T lymphocytes as indicated by increased expression of the activation markers CD29, CD38, HLA-DR, and CD45RO (1–5). In the present study, a fivefold increase in CD3/CD8-positive suppressor/cytotoxic T cells was noted with a marked increase in HLA-DR and CD45RO positivity (only a slight increase in CD38), indicative of activated primed T cells (4). There was a marked increase (>20-fold) in CD95 expression by T cells in AL — another finding consistent with T cell activation. CD95-positive cytotoxic T cells induce Fas-mediated apoptosis when bound to CD95L-positive target cells (6).
CD28-positive T cells are activated by interaction with B7-expressing antigen-presenting cells. Although most reports indicate that CD28 is expressed by cytotoxic T cells (CTL) and not by suppressor T cells (7, 8), some reports indicate that CD8-positive CD28-negative T cells may be cytolytic effectors (9). Increased numbers of CD8/CD28-positive cytotoxic T cells have been reported in IM (10). CD8/CD28-positive T cells were increased in the present study, especially in the EBV-positive subgroup (EBV-positive subgroup, greater than sixfold; EBV-negative subgroup, less than threefold). However, CD28-negative CD8+ T cells were increased to an even greater extent (EBV-positive group, greater than eightfold; EBV-negative group, fivefold), a finding consistent with downregulation of CD28 expression on CD8-positive T cells reported in acute viral infection (11). Interestingly, when the larger lymphocytes are gated, there is a proportional increase in CD8/CD28-positive cytotoxic T cells, thus suggesting that the larger “atypical” T cells, unlike the smaller T cells, are cytotoxic T cells rather than suppressor T cells.
Expression of CD25, the p55 subunit of the IL-2 receptor, on CD8-positive cells has been shown to be decreased in IM (1, 5, 10, 12). In the present study, decreased CD25 expression was noted on both CD4 and CD8-positive T cells, but only in the EBV-positive subgroup. Despite the lack of CD25 expression by CD8-positive T cells in IM, these cells have been shown to be capable of responding to exogenous IL-2 in vitro through normal expression of the p70 subunit of the IL-2 receptor (12).
Although CD4-positive CD62L-negative suppressor-inducer T cells are reportedly decreased in IM (10), in the present study cells of this phenotype were significantly increased (two- to threefold). Decreased CD62L expression is also characteristic of interferon-γ-producing T effector memory (Tem) cells targeted to peripheral tissues (13). In chronic persistent EBV infection, CD62L-negative Tem and CD62L-positive Tcm (central memory) EBV-specific memory T cells are present (14). Perhaps the increase in CD62L-negative T cells in the peripheral blood reflects lymphocyte homing directed to the infected pharyngeal mucosa in early acute viral infection.
Some reports indicate that in addition to increased numbers of activated CD8-positive T cells, IM is characterized by increased numbers of activated CD4-positive T helper-inducer cells (1, 2, 4). Although in the present study CD4-positive T cells were not increased, there was a very significant increase in HLA-DR positivity, a finding consistent with CD4-positive T cell activation. Curiously, no similar increase in expression of activation markers CD25 and CD38 was detected on CD4-positive T cells.
Th2 cells promote B cell-mediated humoral immune responses rather than T cell-mediated cytotoxic T cell responses, and thus may impair T cell-mediated antiviral immunity. Although immunophenotypic differentiation of Th1 and Th2 cells is admittedly imprecise, several markers, including CD7, CD43, and CCR5, have been used in previous studies. CD7 negativity is characteristic of a Th0/Th2 T helper-inducer subset found in normal blood and increased in HIV infection, renal transplantation, and bone marrow transplantation (15, 16). In the present study, CD7-negative CD4-positive T cells were increased 2.7-fold. CD7 negativity has also been previously reported in a dimCD4-positive T cell population in IM (17). The proportional increase in CD4-positive CD43 (sialophorin)-negative T cells seen in AL is also consistent with an increase in Th2-type T cells (18). Perhaps Th2 cells in IM not only inhibit the T cell-mediated cytotoxic response to EBV, but also promote growth of EBV-infected B cells. On the other hand, in this study AL is also characterized by a proportional increase in CCR5-positive CD4-positive T cells. Since the chemokine receptor CCR5 is selectively expressed on human Th1 cells (19), the present data are consistent with an increase in Th1 cells — a result at odds with the previous results.
CD57 has been identified as a marker of CD8-positive effector CTL (11, 20–22). On the other hand, in one report CD8-positive CD57-positive cells were shown to inhibit generation of cytotoxic T cells to EBV-positive B lymphoblastoid cells (23). Within lymph nodes, CD57-positive T cells are present in B cell-rich reactive germinal centers and are increased in nodular lymphocyte-predominant Hodgkin lymphoma (24). The significantly decreased CD57 expression seen in the EBV-positive subgroup only is suggestive of an increased number of suppressor T cells in EBV-positive IM, complemented by the increase in CD4-positive CD62L-negative suppressor-inducer T cells.
A dual CD4/CD8-positive phenotype has been reported in a subset of cytotoxic T cell clones grown from patients with IM (2). However, significant numbers of dual positive T cells were not identified in the present study. The dual CD4/CD8-negative phenotype is characteristic of T cells that express the γδ T cell receptor, a population reportedly increased in IM (25, 26). In the present study, although they represent a minor population, a marked increase (greater than sevenfold) in γδ T cells was detected in the EBV-positive subgroup only. γδ-positive T cells, which preferentially localize to the alimentary tract, may be important in mucosal immunity (27, 28) and have been shown to respond to human EBV-infected B cells (29, 30). Perhaps circulating γδ-positive T cells, specifically increased in EBV-associated IM, are targeted to the EBV-infected pharyngeal mucosa and lymphoid tissues.
NK cells have been shown to be important in the early innate phase of the immune response to viral infection prior to initiation of an antigen-specific T cell-mediated response. Thus, it is not surprising to find that AL is characterized by a significant increase (threefold) in CD16/56-positive NK cells. An interesting new finding is that the NK lymphocytosis in EBV-positive IM is significantly higher than that seen in the EBV-negative subgroup of AL.
Two smaller previous studies identified a decreased number of B cells in IM (5, 31). In contrast, in the present study no significant change in absolute B cell count was noted. It is likely that the B cell count in early IM varies with time after infection and that lab results will be dependent upon sampling time. Although increased numbers of CD5-positive B cells have been reported in IM (32), we were unable to demonstrate an increase in this B cell subpopulation (data not shown). EBV transformation of B cells in vitro is accompanied by increased expression of the low-affinity IgE receptor CD23 (33), and increased CD23-positive B cells have been reported in IM (34). However, in the present study, a decrease in CD23-positive B cells was identified. Even if one assumes that circulating EBV-infected B cells do express CD23, these cells are very infrequent, with estimates ranging from 0.07–0.8% of the B cells (35, 36). It seems very unlikely that the low frequency of EBV-infected B cells in blood could lead to a detectable increase in CD23 expression by flow cytometry. EBV-infected B cells that express cytoplasmic immunoglobulin are CD23-negative (37). Since plasmacytoid cells are often seen in peripheral smears of IM, perhaps these cells represent CD23-negative EBV-infected B cells. Another factor contributing to the decreased CD23 expression in IM may be the rapid immune elimination of highly antigenic CD23-positive lymphoblastoid B cells that express the full complement of EBV latent genes with persistence of poorly immunogenic CD23-negative B cells expressing EBV LMP-2a only (38).
The present study was designed to further define the immunophenotype of AL and to identify immunophenotypic features that differentiate classic EBV-associated heterophile-positive IM from AL of other causes. Given the large number of cases examined, we have more carefully detailed previously described lymphocyte abnormalities in AL and have expanded the range of lymphocyte antigens examined. In addition, for the first time, we have identified eight specific immunophenotypic features that differ between EBV-positive and EBV-negative cases (Table 9). This information may prove useful in further understanding of the profound immunologic effects induced by primary EBV infection. Although it is unlikely that this information will lead to practical methods for diagnostic discrimination between EBV-positive and EBV-negative AL, it may allow for better discrimination between benign and malignant lymphocytosis.