Persistent viruses, like EBV and CMV, are normally controlled through cytotoxic T cell responses [1
] and complications can be cured through adoptive T-cell transfer [23
]. Adequate T-cell reconstitution after SCT is therefore crucial in preventing viral reactivation progressing to severe complications [9
We found that early T-cell reconstitution after SCT does not play a role in the onset of viral reactivation as numbers of CD4+
T cells during the first 3 months post SCT are similar in patients with or without EBV reactivation. Other studies have shown that the conditioning regime prior to SCT as well as in-vivo T-cell depletion around time of transplantation play an important role in the T-cell reconstitution rates after SCT [26
]. In-vivo T-cell depletion through alemtuzumab has been shown to delay both CD4+
T-cell reconstitution and ATG administration results in a delayed CD4+
T-cell reconstitution [27
]. Indeed, we did observe a significant difference in T-cell counts early after SCT between patients who received ATG and patients that did not (data not shown). During the time of average onset of viral reactivation (8 weeks after SCT), there was no significant difference in T-cell counts between patients that did or did not receive ATG (data not shown). However, we cannot exclude that viral reactivation was initiated already earlier after SCT and not detected yet, nor can we exclude differences in phenotypic markers that may explain divergent outcomes with respect to viral reactivation.
Generation of virus-specific CD8+
T cells is dependent on CD4+
T-cell help, as has been shown for several viruses such as HIV [28
] and CMV [7
]. We studied the role of EBV-specific CD4+
T cells in controlling EBV-reactivation after allogeneic stem cell transplantation. We applied a 12day expansion protocol, which has been shown to be very useful to measure low numbers of specific T cells in several situations [21
], as well as in SCT patients to investigate both the general as well as EBV-specific T-cell reconstitution. This technique also enabled us to compare reconstitution of CD4+
T cells using a similar qualitative assay. As read-out of functionality we choose IFNγ as this is the last cytokine to be lost upon exhaustion, and thus has the ability to reveal T cells still able to produce (this) cytokine. IFNγ is therefore the most robust cytokine and enabled us to measure the majority of cytokine-producing T cells without underestimation of specific T cells due to loss of IL-2 production as a result of exhaustion. As we grow the T cells in IL-2 supplemented medium however, we also skew the cells to a more Th1 phenotype, making the production of IL4 and IL10 less likely.
Although the study population is small, we showed- using the expansion assay- that non-specific functional T-cell reconstitution is hampered in patients with high-level EBV reactivations. In all 4 high-level reactivation patients no reconstitution was observed until 12 months post SCT. This is in line with findings by Annels et al. who suggested that pre-emptive intervention is necessary only in patients that lack an expansion of memory T cells during the initial phase of the reactivation [31
In our study, EBNA-1 specific T cells remain low throughout follow up in all patients and specifically EBNA-1 specific CD4+
T cells do not reconstitute to ‘normal’ values 12 months post SCT. In contrast, we detected EBNA-1 specific CD4+
T cells more readily in patients without or with a low-level viral reactivation suggesting that EBNA-1 specific T cells could play an important role in controlling the viral reactivation. Lack of adequate EBNA-1 specific T cell responses have been described to be associated with progression to EBV-related NHL [21
]. These findings are reflected in our PTLD patient, in whom few EBNA-1 specific CD8+
T cells were detectable throughout follow up also when high viral loads were present. In contrast, we observed 18% of CD8+
T cells producing IFNγ after BZLF-1 stimulation at the first time point following onset of PTLD. BZLF-1 specific T cells appear more readily in high-level reactivations and thus seem not capable of viral control. This is in line with a recent study using the 12 day expansion assay in PTLD. High CD8+
T-cell responses against BZLF-1 were measured that were shown to be dominant compared to EBNA-1 specific responses [32
]. Also Hislop et al. previously reported that BZLF-1 responses are very immunodominant [33
]. These data suggest that during reconstitution, outgrowth of EBNA-1 specific T cells is required to control EBV-reactivation.
Despite the small patient group, the combined measurement of general T-cell functionality together with EBV-specific functionality provides an extra tool in analyzing the T-cell reconstitution following SCT. Our data suggests that sufficient general functional T-cell reconstitution is necessary for a robust EBV-specific T-cell response and that although EBV-specific CD8+
could be detected in patients with EBV reactivations, their general T-cell reconstitution was severely hampered resulting in lack of viral control. Since all of the high-level reactivating patients were given αCD20 treatment upon viral loads exceeding 1000 copies/ml, we have no data on the percentage of high-level reactivating patients that would have been capable of clearing the reactivation themselves. However, our data do show that patients with high-level reactivations already have an altered functional and EBV-specific T-cell reconstitution prior to onset of viral complications and therapeutic intervention. A possible explanation for our findings could be that during the reconstitution process, where only a selective number of T cells will slowly reconstitute, T cells with lower avidity are activated by the viral reactivation. These cells will be of lower functionality and may not contain the reactivation. Because of the still low number of T cells, this will be reflected in both the total functional T-cell pool as well as the EBV-specific one. In addition, exhaustion may play a role in the face of high antigenic loads, leading to less ability of T cells to proliferate in vitro.
The importance of a robust T-cell reconstitution has been described before. A rapid reconstitution to at least 300 CD3+
T cells/μl blood was shown to distinguish between patients with viral control and patients at risk of reactivation [31
]. Also, for CMV it has been recently shown that a threshold number of absolute CMV specific T cells can be used to identify patients at risk of CMV disease [34
]. However, here we show that not so much the number of T cells is of importance but the functional capacity, i.e. the capability of IFNγ production following general stimulation, is impaired in individuals that develop a high-level EBV reactivation. We cannot exclude the possibility that in patients with an EBV-reactivation T cells have become more clonally exhausted and therefore may not have been able to proliferate in our in vitro system. We unfortunately did not measure markers of exhaustion.
Thus, both total functional and EBV-specific CD8+
T cells show a more rapid recovery compared to functional CD4+
T cells. EBV-specific CD8+
T-cell responses can be detected as early as 2 months post SCT, while EBV-specific CD4+
T cells responses remain low throughout follow up, even though both CD8+
T-cell responses are broadly targeted across all eight latent proteins [33
]. In addition, up to half of the currently defined CD4 epitopes are derived from EBNA1 and there is an equal distribution of epitope recognition among the early, immediate early and late lytic proteins [35
]. Interestingly, absolute CD4+
T-cell numbers do not reconstitute slower in our patients compared to CD8+
T cells. Why the EBV-specific CD4+
T cell response is not recovering to the extent the EBV-specific CD8+
T-cell response is recovering is unclear, but may lie in differences in ability of clonal expansion, especially in the situation of SCT or the repertoire of T cells involved. The fact that EBNA-1-specific CD4+
T cells did not normalize to healthy control levels one year post-SCT and were undetectable up to at least 8 months in the PTLD patient, however, do not rule out a role for EBV-specific CD4+
T cells in control of EBV-reactivations.