It is well accepted that naïve T cells mount a T cell response to any new antigen, whether pathogen-derived or an allogeneic pMHC complex; however, the contribution of memory cells to allo-HLA reactivity is less well defined. Several reports demonstrated alloreactivity of virus antigen-specific CD4+ (23
) and CD8+ (21
) T cell clones (reviewed in (45
), but none of these studies addressed this issue at the population level as we did here. Furthermore, depletion of naïve T cells has been proposed as a method of avoiding GvHD after allogeneic SCT (15
). We measured cytokine production at the single cell level by flow cytometry in allo-MHC primed T cells following short-term (6 hrs) restimulation with the same allogeneic PBMC and confirmed their alloreactive potential. However, the functional read-out may not accurately reflect the true number of alloresponding T cells. In these experiments we chose to analyze TNFα and IL-2 production after allogeneic or syngeneic secondary stimulations. It is possible that responder frequencies might be different when analyzing other effector functions. While these results do not therefore reflect overall allo-HLA responder T cell frequencies they do show that responder frequencies between naïve and memory T cells to allo-HLA stimuli are comparable.
Assays to demonstrate ex vivo
the alloreactive potential of T cells have made the assumption that such reactivity resided exclusively in the naïve pool (14
). We confirmed using ICD that such allo-pMHC reactivity can be identified in healthy donor T cells ex vivo
. Importantly, however, by combining such assays with the phenotypic identification of naïve, memory, and effector T cell subsets in a polychromatic flow cytometry approach, we demonstrate conclusively that predominant subsets reacting with pMHC targets are naïve CD4+ T cells and effector CD8+ T cells.
Since the DNA viruses are latently present in circulating B (EBV (47
)) and myeloid (CMV (48
)) cells, the analysis of allo-pMHC reactivity of memory T cells could be confounded by responses to viral antigens presented by the HLA disparate stimulators. However, activated T cells, which do not carry EBV or CMV, also elicited alloresponses, excluding the possibility that the responses were directed against these common DNA viruses.
The identification of alloreactivity in the memory pool raises the question whether cross-reactivity of viral antigen-specific CD4+ and CD8+ T cells occurred with mismatched virus antigen-free pMHC complexes. Most studies have only examined a few clones against a limited panel of allogeneic EBV-LCL (27
) or PBMC (20
) as APC. We earlier established that the chance of finding alloreactivity depended on the complexity of the responder cell population (Melenhorst et al
., unpublished observations) and the extent of HLA diversity of the T-APC panel. Activation marker-selected and expanded virus-specific T cell lines were analyzed for clonal composition upon restimulation with the cognate antigen (the immunodominant antigen from CMV, pp65, or a more complex source of antigen: EBV-LCL, which are known to express approximately eight EBV-encoded proteins (50
). To refine our selection of pure virus-specific T cells we sorted only the T cells that produced both TNFα and IFNγ in response to cognate or allo-antigen stimulation since the proportion of T cells producing both of these inflammatory cytokines in the absence of restimulation was negligible, whereas single cytokine producers were present in the unstimulated populations. TCRβ sequence analysis of CD4+ and CD8+ T cells responding to cognate antigen stimulation showed that the responder populations were highly oligoclonal, confirming previous reports (51
). The examination of the TCRβ sequences in the same T cell lines responding to an allogeneic target identified shared clonotypes with the cognate antigen-responsive population. To expand our findings with the clonotype analysis of virus-specific T cell lines reacting with allogeneic APC we subjected a third, CD4+ EBV-LCL-reactive T cell line to the same procedure. Here we also identified a shared clonotype between virus- and alloresponding cells (Fig. S3
). However, since the background production of cytokines by this cell line, i.e. in the absence of antigen stimulation, was substantial, we cannot formally prove the dual reactivity of the clone identified in both reactivities. Collectlively, our data do allow the conclusion that DNA virus antigen-specific T cells, which can make up 10% or more of the circulating post-HSCT T-cell population (51
) and in elderly donors (32
) are commonly reactive with unrelated pMHC complexes.
Our findings have clinical relevance. Firstly, the adoptive transfer of virus-specific T cell lines and clones given to treat reactivating viruses in (partially) matched recipients (53
) may risk sporadic but powerful alloreactions against the recipient. In our study we used T-APC and PBMC as a representative of GvHD targets because they were readily available. However it is possible that these targets are not as representative of GVHD targets as fibroblasts or cells from GVHD target tissues such as the skin, gastrointestinal tract, and the liver. Amir et al. (44
) have recently used an EBV-LCL, HLA-transduced erythroleukemic cell line, and PHA blasts. They demonstrated that a large proportion of virus antigen-specific T cell clones reacted with non-self peptide-MHC complexes. However, these findings should be interpreted with caution - in vitro reactivity of virus-specific T cells may not directly translate to the alloreactivity in the form of GvHD since other factors such as target antigen expression, homing, and in vivo expansion can affect the clinical outcome. Indeed the adoptive transfer of virus-specific T cell lines in the partial HLA-disparate setting (53
) has not resulted in GvHD (55
). Furthermore, a recent study by the Riddell group (56
) suggests that even minor histocompatibility antigen-specific T cells selected for reactivity with hematopoietic targets (EBV-transformed B cells) but non-reactivity with patient fibroblasts still exerted anti-lung reactivity in the patient, indicating that even fibroblasts may not express the relevant repertoire of target antigens of GvHD. Secondly, as others have demonstrated, cross-reactivity by viral antigen-specific T cell receptors with various non-self pMHC complexes (24
), implies that, when targeting an epitope presented in one particular HLA, (for example WT1RMF
presented in the context of HLA-A*0201), TCRs from multiple clones should be available for such therapeutic applications since, depending on the HLA make-up of the TCR donor, these complexes may recognize unrelated pMHC complexes. The same principles may apply to any TCR challenged with pMHC complexes not encountered during thymic selection in the original host (57
). Lastly, since alloreacting memory T cells with their rapid proliferation kinetic may kill their target within hours of engagement, they may contribute to the hyperacute GvHD mostly observed in partially matched SCT (58
). Though end-stage effector T cells are short-lived in vitro
), they arise from the memory pool and may continue to replicate (60
To conclude: T cell reactivity with unrelated pMHC complexes can originate from any post-thymic T cell population; alloreactivity is common, and mediated by the same TCR that recognizes viral and possibly self antigens.