Primary infection with EBV during acute infectious mononucleosis (IM) is associated with a cytotoxic response against allogeneic target cells. C depletion with anti-CD3 (OKT3) and anti-CD8 (OKT8) mAb decreased the allogeneic cytolysis of two EBV-infected lymphoblastoid cell lines (LCL) by 96% and 89%, respectively. Complement depletion with the NK cell-specific mAb Leu-11b and NKH-1a resulted in only a slight decrease (less than 35%) in the lysis of these LCL. mAb inhibition studies with OKT3 and OKT8 inhibited the allogeneic lysis of two LCL by 87% and 82%, respectively. The alloreactive cytotoxic response was strongly inhibited by mAb specific for MHC class I determinants (W6/32, 65% inhibition and BBM.1, 58% inhibition). Acute IM lymphocytes lysed the allogeneic EBV-negative cell lines HSB2 (45%) and HTLV-1 T cell lines (16%). NK cell-depleted lymphocytes from an acute IM patient demonstrated preferential lysis of K562 transfected with human HLA-A2 (73%) compared with the K562 transfected control (20%). Cold target competition studies with allogeneic and autologous target and competitor LCL demonstrated no significant competitive inhibition between allogeneic and autologous cells. We interpret these results as evidence that 1) the acute IM-alloreactive cytotoxic response is mediated primarily by CTL; 2) these alloreactive CTL lyse allogeneic target cells irrespective of EBV antigenic expression; 3) MHC class I expression is sufficient for allogeneic recognition and lysis of target cells; 4) distinct effector CTL populations mediate lysis of autologous and allogeneic target cells; and 5) during acute IM, EBV infection results in the induction of both virus-specific and alloreactive CTL populations.

The CD8 Ag has long been used as a surface marker for the identification of cytotoxic and suppressor cells. Recently CD8-positive cells have been shown to release a soluble form of the CD8 Ag. We have devised a sandwich monoclonal enzyme immunoassay for the quantitation of this released CD8. Soluble CD8 was released in response to lymphocyte activation. In vitro, PHA or anti-CD3 mAb-mediated T cell activation led to release of CD8 into the culture supernatant. In vivo, serum from patients with EBV-induced infectious mononucleosis (IM), a disease associated with intense CD8+ T cell activation, demonstrated elevations in soluble CD8 (7939 U/ml, day 0) compared to serum from normal controls (289 U/ml). Levels of soluble CD8 correlated (r = 0.82, p less than 0.001) with the increased percentage of CD8+/HLA-DR+ (activated CD8+ T cells) observed in acute IM. Sequential analysis of serum during the course of IM shows that soluble CD8 levels parallel the decline in CD8+/HLA-DR+ cells that occurs with the resolution of the disease. These data suggest that released CD8 may be of value in monitoring the involvement of CD8+ T cells in response to a pathologic event. The functional role of the released CD8 molecule will require further investigation.

Epstein-Barr virus (EBV) induced infectious mononucleosis (IM) is characterized by the activation and expansion of T lymphocytes and the induction of cytotoxic responses able to mediate the lysis of EBV-uninfected, allogeneic MHC mismatched and EBV-infected autologous target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) were used to examine the nature of these cellular immune responses. Activated lymphocytes, as identified by HLA-DR expression, associated with EBV induced IM were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8+) T cells, helper/inducer (CD4+) T cells and natural killer (NK, CD16+) cells. CD8+ T cells were the primary activated population, representing 24% of the total lymphocyte population and 60-70% of the CD8+ T cell population. The activated CD4+ T cells and natural killer (NK) cells accounted for 7% and 4% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) and CD8 molecules demonstrated significantly (p<0.001) elevated levels in the sera of IM patients compared with normal controls. These elevated levels of serum IL-2R am CD8 molecules correlated, (r=0. 67 and r=0.82, respectively) with increased percentages of CD8/HLA-DR positive T cells (i.e., activated CD8 T cells). Increased levels of soluble cell surface molecules peaked during the acute phase and normalized as the patients progressed toward convalescence. Individual patients demonstrated strong correlations between the percentage of CD8/HLA-DR positive cells and soluble CD8 levels. The functional significance of the serum IL-2R and CD8 molecules is presently unknown. However, the strong correlative data between serum CD8, and to a lesser extent IL-2R, and CD8 T cell activation suggests that serum CD8 levels may provide a sensitive measure of CD8 T cell activation in systemic infections. The ability of freshly isolated acute IM PBMC to lyse allogeneic, EBV-infected lymphoblastoid cell lines (LCL), demonstrated the ability of acute IM effector cells to lyse MHC mismatched target cells. Effector cells from acute IM patients lysed allogeneic DM-LCL and AF-LCL target cells by 34% (n=7) and 23% (n=6), respectively, compared with 4% (n=5) and 0% (n=5), respectively, for normal controls. MAb-dependent complement depletion of CD3+ or CD8+ cells with anti-CD3 and anti-CD8 mAb decreased the non-MHC restricted cytolysis of LCL by 96% and 89%, respectively. In contrast, complement depletion with NK-cell specific mAbs Leu 11b and NKH-1, resulted in only a slight decrease (<35%) in the lysis of these LCL (46%). Depletion with anti-HLA-DR also significantly (p<0.001) decreased the lysis of LCL. Depletions with anti-CD4 demonstrated no decrease in LCL-lysis. MAbs OKT3 and OKT8 inhibited the non-MHC restricted cytolysis by 87% and 82%, respectively. We interpret these results as evidence that, 1) lysis of allogeneic cells is mediated primarily by CD3+, CD8+, HLA-DR+, cytotoxic T lymphocytes (CTL); and 2) these acute IM cytotoxic T cells utilize the T cell receptor and the CD8 antigen as an accessory molecule. An active role for target cell MHC class I molecules in the recognition and subsequent lysis of target cells is supported by a number of observations: 1) the MHC class I reactive mAbs W6/32 and BBM.1 significantly (p<0.05) inhibited the lysis of 63463-LCL by 65% and 57%, respectively; 2) acute IM effector T cells did not lyse the MHC class I negative Daudi cell line; 3) allogeneic MHC class I matched LCL mediated strong competitive inhibition (72% at 10:1 competitor to target cell ratio) vs 29% competitive inhibition for an allogeneic MHC class I mismatched LCL; and 4) NK-cell depleted effector cells from one patient mediated preferential lysis of the K562 cell line expressing MHC class I. HLA-A2 molecules. We interpret these results as evidence that target cell MHC class I molecules (or associated determinants) are the target antigen(s) for the allogeneic MHC cytotoxic response. The role of EBV in this acute allogeneic response was examined using target cell lines devoid of EBV genome. Acute IM CTL mediated lysis of the allogeneic HSB-2 T cell line (45%), and allogeneic HTLV-I transformed T cell lines (16%). The lysis of the HSB-2 T cell line was inhibited by anti-OKT3 (58% inhibition), W6/32 (53%) and BBM.1 (42%). Similarily, lysis of HTLV-I T cell lines was inhibited by W6/32 (69% inhibition), BBM.1 (69%) and OKT3 (38%). These data demonstrate that EBV antigenic expression is not required for allogeneic recognition and subsequent lysis of these allogeneic target cells. Effector cells from acute IM patients (n=5) were able to lyse their autologous EBV-infected LCL (mean lysis=21%), but were unable to lyse the EBV-uninfected autologous HTLV-I T cell line. These same effectors, however, were able to mediate lysis of both allogeneic B cell lines (21% lysis) and allogeneic T cell lines (8% lysis). These data are consistent with the observations by Strang et al. (1987a), who recently cloned virus specific/MHC-restricted CTL cloned from acute IM PBMC. These virus specific/MHC-restricted T cells presumably mediate the lysis of the autologous EBV-transformed B cell lines but not the autologous EBV-uninfected T cell lines. Whether the CTL which lyse the autologous EBV-transformed LCL are also responsible for the observed allogeneic reactivity was examined with cold target competition using autologous and allogeneic LCL. Lysis of autologous LCL was inhibited only by autologous competitor cells (64% inhibition compared with 24% for allogeneic LCL). Likewise, lysis of the allogeneic LCL was inhibited only by the allogeneic competitor cells (85% inhibition compared with 30% for autologous LCL). These data demonstrated no competition between allogeneic and autologous LCL and therefore support the concept that lysis of autologous LCL and allogeneic target cells is mediated by distinct effector populations. These data help us to understand the unusual immune response observed during acute IM. The strong allogeneic cytotoxic response is thought to represent polyclonal CD8 T cell activities induced by EBV-infected and transformed B cells which circulate in vivo. In addition, a population of CD8 CTL exist which mediate the lysis of autologous EBV-transformed B cells. These CTL likely represent virus-specific/MHC-restricted CTL and presumably play a major role in the control of EBV infections. The role, if any, of the markedly expanded alloreactive CTL population in the elimination of EBV infected and transformed B cells remains to be clarified.

Activated lymphocytes, as identified by HLA-DR expression, associated with acute Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM) were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8) T cells, natural killer (CD16) cells and helper (CD4) T cells. CD8 T cells were the primary activated population representing 24.5% of the total lymphocyte population. The activated CD4 T cells and natural killer cells accounted for 6.7% and 3.5% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) demonstrated significantly (p less than 0.001) elevated levels in the serum of acute IM patients compared with normal controls. Elevated levels of serum IL-2R were correlated (r = 0.67) with increased percentages of Leu 2a+/HLA-DR+T cells (i.e., activated CD8 T cells). Patients with X-linked lymphoproliferative syndrome and virus-associated hemophagocytic syndrome, two syndromes associated with severe acute EBV infections, demonstrated the most dramatic increase in serum IL-2R levels. These data demonstrate that EBV is associated with intense immune stimulation and that during acute IM activated lymphocytes, other than the CD8 T cells, may contribute to the immune response to EBV.