We show here that cross-reactive T cells specific to a previously encountered virus could be major contributors to the overzealous CD8+ T cell response that defines IM. In 5 out of 8 patients, influenza M1–specific CD8+ T cells participated in EBV-induced lymphoproliferation. Of these 5 patients, 2 had dramatically skewed M1-specific TCR repertoires and increased levels of clearly identifiable, tetramer-defined, cross-reactive CD8+ T cells capable of recognizing the 2 dissimilar epitopes influenza M1 and EBV-BMLF1. Based on our ability to culture these cross-reactive cells from 3 out of 8 healthy donors with previous exposure to both viruses, these cross-reactive cells are maintained in memory and their functional responses to either antigen can include cytotoxicity and the production of MIP-1β, IFN-γ, and TNF-α. Cross-reactive T cells may play a major role in the development of IM, and the diversity of their functions may contribute to the severity of the syndrome. These studies examined only one cross-reactive population while it is likely that infection with EBV, a virus with the potential to encode hundreds of epitopes, could reactivate many memory T cell populations yet undefined. As demonstrated here, the identification of cross-reactive T cells can be complicated by their ability to recognize alternative peptides having little sequence similarity to their native ligand, their strict growth requirements in vitro, and the sensitivity of the different techniques used to detect them. These are all challenges for future elucidations of individual cross-reactive T cell responses and their potential impact on the outcome of IM.
Our work suggests that acute EBV infection activated influenza M1–specific CD8+
T cells. These cross-reactive M1-specific T cells are most likely memory cells for the following reasons: (a) by this age, everyone is immune to influenza A virus; (b) almost all HLA-A2+
individuals develop an M1-specific response; and (c) costaining with M1- and BMLF1-loaded tetramers showed that these cross-reactive cells brightly stained with the M1-loaded tetramer, suggestive of a high avidity interaction typical of antigen-specific memory. The activation of cross-reactive memory cells coincident with the development of IM disease pathology is highly analogous to the examples of heterologous immunity we have observed in mouse models, where memory T cell responses to a prior infection with an unrelated virus altered the host’s immune response to a subsequent infection and caused a marked deviation in disease course (34
It has recently been shown that acute HIV infection can up-regulate the expression of activation markers such as CD38, HLA-DR, and Ki67 on memory cells specific to influenza A virus, EBV, and CMV, but the role of the TCR in this activation was not determined (35
). Our work suggests that acute EBV infection can activate influenza-specific memory cells through a TCR-dependent mechanism. The expansion of M1-specific memory cells was evident in only 5 of 8 patients with IM despite the fact that all probably had memory to M1 and all would have been influenced by any cytokine-mediated, or bystander, activation. When possible, we also looked for the expansion of a second memory T cell population, specific for CMV-pp65. Only 2 IM patients proved to be CMV seropositive, E1155 and recent enrollee E1238. The frequency of pp65-specific T cells in patients E1155 and E1238 was low during massive, EBV-induced lymphoproliferation. At day 0, E1155 and E1238 had a pp65-specific T cell frequency of 0.2% and 0.7% respectively while, by 41–50 days after presentation, those frequencies climbed to 0.6% and 1.1% respectively. These data would suggest that the pp65-specific memory populations of these 2 patients did not contain T cell clones cross-reactive with EBV and were therefore initially diluted out by the extensive proliferation of EBV-specific T cells.
For 2 of the 5 patients with higher M1 frequencies (E1101 and E1178), we determined that BMLF1 was at least 1 of the EBV-derived antigens recognized by cross-reactive T cells. Although both patients shared this particular pattern of cross-reactivity, their responses remained unique. Vβ17+
subclones using Jβ2.3 preferentially expanded in patient E1101 while those using Jβ1.2 preferentially expanded in patient E1178. Thus, the M1-specific TCR repertoire of both patients was notably skewed from that known to be conserved among healthy HLA-A2+
individuals (Y.N. Naumov et al., unpublished observations). These data are further suggestive of antigen-driven clonal expansions because a bystander activation mechanism would drive the expansion of all clones and would maintain the conserved repertoire organization. The fact that cross-reactive T cells specific for M1 and BMLF1 were observed in 2 but not all patients is probably reflective of these clonal differences, known as the private specificity of each individual TCR repertoire. In support of this, cross-reactive T cell responses involving lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VV) showed that only 50% of VV-challenged LCMV-immune mice mounted a strong response to a specific LCMV epitope, NP205–213
, and this was a function of the private specificity of the LCMV memory TCR repertoire of each mouse (22
). The existence of T cell responses unique to the individual adds to the complexity in resolving the importance of cross-reactive responses in human infections.
Previous studies showed that the extent of T cell activation and proliferation correlated with the severity of IM (7
). We suggest that the magnitude of the T cell response represents the combined efforts of cross-reactive memory and primary T cells. Our limited clinical data indicate that the number of cross-reactive cells specific for M1 and BMLF1 may correlate with disease severity, but further investigation is necessary to confirm this observation. The severity of IM might be influenced by both the number of activated cross-reactive cells and their effectiveness at clearing virus. There is recent evidence for the exacerbation of human disease by the activation of cross-reactive influenza-specific memory CD8+
T cells during acute HCV infection. Like EBV, acute HCV infection is often asymptomatic, but when clinical symptoms manifest, they are likely caused by the immune response. HCV encodes an epitope, NS31073–1081
, that can activate influenza NA231–239
–specific memory cells (14
). However, unlike EBV-BMLF1 and influenza M1, which are only 33% similar in sequence, HCV-NS3 and influenza NA are 78% similar in sequence. In congruence with our observations during EBV infection, the activation of these cross-reactive NA-specific memory cells enhanced the magnitude of the CD8+
T cell response to HCV and resulted in severe disease pathology (37
). Despite strength in numbers, these HCV-specific T cells were unable to sufficiently clear the virus, and the patients developed persistent HCV infections (37
). Cross-reactive cells may be inefficient at interacting with infected host cells due to a low avidity for the alternative ligand, but they may still produce pathology-generating cytokines. Their presence may interfere with the primary T cell response by preventing access to the infected cells or changing patterns of T cell immunodominance, thereby prolonging resolution of the infection.
Our work in animal models has suggested that cross-reactive T cells can induce pathological conditions despite their ability to clear virus. LCMV-specific memory cells lower the titer of VV delivered intranasally but in doing so also alter the disease pathology from pulmonary edema to bronchiolitis obliterans (34
). The cytokines, notably IFN-γ, secreted by these activated memory cells may have played a major role in the development of this immunopathology. In the present study, we showed that cross-reactive cells specific to M1 and BMLF1 secreted several cytokines in a hierarchal pattern whereby most secreted MIP-1β, fewer secreted IFN-γ, and even fewer secreted TNF-α. Similar functional hierarchies were observed in studies with freshly isolated HIV- or CMV-specific CD8+
T cells from healthy donors with these persistent infections as well as during the primary immune response to EBV in IM patients (40
). The mechanism behind T cell functional heterogeneity has been extensively studied by varying the quantity of TCRs engaged and by varying the quality of ligands used to engage them (28
). Our work presents an opportunity to apply this knowledge to T cell cross-reactivity involving 2 natural viral epitopes in order to understand how cross-reactive T cells mediate the development and severity of IM. Interestingly, both MIP-1β and TNF-α levels are known to be elevated in the serum and tonsils of IM patients compared with healthy controls (46
). MIP-1β can be readily secreted because it is preformed and stored within human CD8+
T cells (48
). Even a low avidity interaction may stimulate its release from the cell. This is logical because MIP-1β broadens the immune response by recruiting other immune cells to the site of infection (49
). The increase in number of activated immune cells may enhance IM severity. Fewer of the cross-reactive cells secreted TNF-α following peptide stimulation, suggesting that a higher avidity interaction may be required to initiate its production. However, an overall increase in the number of responding T cells capable of secreting TNF-α at the site of infection could be harmful to the host and promote the clinical symptoms of IM (50
). In fact, the high production of TNF-α, possibly by cross-reactive memory cells, has been implicated in the immunopathogenesis associated with dengue hemorrhagic fever (52
). Thus, depending on the cross-reactive specificity pattern and private specificity of the TCR repertoire, cross-reactive memory T cells activated by EBV may function and modulate the disease outcome of each individual very differently. Identifying these cross-reactive patterns will be a challenge for the future.
In conclusion, our data suggest that cross-reactive memory T cells participate in the massive lymphoproliferation that characterizes EBV-associated IM and may influence disease severity. For the purposes of this study, we focused on detection and activation of M1-specific memory cells, but EBV, a virus with a large genome that encodes numerous different proteins, has the potential to generate epitopes reactive with many different memory T cell populations. The cross-reactive pattern that emerges is influenced both by an individual’s unique history of infection and by the private specificity of the TCR repertoire responding to each of those infections. Overall, this demonstration of cross-reactivity involving 2 immunodominant epitopes derived from 2 of the most common human viruses among people that share the most common MHC class I haplotype in North America highlights the potential importance of cross-reactive T cells in human disease states.