The role of CD4+ T cells in B cell maturation, immunoglobin class-switching, and licensing of antigen-presenting cells to promote the expansion of functional CD8+ cytotoxic T lymphocytes is well established. Less is known about the role of CD4+ T cells in the direct control of infection. Here, we demonstrate that highly differentiated CD4+ T cells, specific for CMV pp65, mediate antiviral effector functions. Specifically, we find that: (a) CD27− memory CD4+ T cells can degranulate in response to cognate antigen; (b) a greater proportion of CD4+ T cells contains granzyme A, granzyme B, and perforin as they mature; (c) CD4+ T cells from an individual in which degranulation occurred in a subset of cells with a high frequency of perforin killed target cells bearing the cognate CMV pp65-derived MHC class II–restricted epitope; and (d) the frequency of CD4+ T cells that degranulate and produce MIP-1β increases with maturational status. In addition, we sorted activated CD4+ T cells into degranulating and nondegranulating populations and showed that degranulating CD4+ T cells were not clonotypically unique.
Although our data show epitope-specific killing of B-LCLs by CD4+ T cells from subject 7, the subject in whom the majority of the surface mobilization of CD107a occurred in a population of CD4+ T cells with a high frequency of perforin expression, our data do not show a direct link between surface mobilization of CD107a, perforin, and granzyme content and killing. In fact, it is possible that perforin-independent pathways may be involved in CD4+ T cell killing that we have shown in three individuals. Although we looked for other pathways and could not provide evidence that they were operative, it is still possible that the expression of FAS ligand was below our ability to detect, or a TNF-α–dependent mechanism was responsible for the observed killing.
Although we cannot say with certainty what the mechanism of CMV antigen-specific killing is, we have shown ample evidence of ex vivo killing. We have shown killing with three different individuals using three different peptide epitopes. In each case killing was shown with repeated assays. In subject 7, we demonstrated killing twice with purified CD4+ T cells and five different times with concentrations of PBMCs ranging from 1 to 4E06/ml. In the two individuals infected with HIV, CMV epitope-specific killing was shown in two and three different assays, respectively. In all of our assays, the ratio of peptide-loaded B-LCLs to nonloaded B-LCLs was determined at either three or four different time points. In addition, to assure that the observed killing was not artifactual, we loaded autologous B-LCLs with an irrelevant peptide and failed to show killing using PBMCs from subject 7. In almost all cases, and at least once for each person in which killing was demonstrated, we ran parallel control incubations using the same antigen-loaded and -unloaded stained autologous B-LCLs that were used in the killing assays. In these assays no effector cells were added. This assured that there was not a significant difference in the rate of growth between B-LCLs caused by either the peptide used or by staining with CMTMR or CSFE. In all of the above-mentioned incubations, when incubations were performed under the appropriate circumstances, killing was observed. We did not observe killing in all cases. We tried to show killing using cells from subjects 1 and 6. In subject 1, using 1E06 CD4+ T cells prepared by either positive or negative selection, we failed to show killing in two different assays. In subject 6, using either 1E06 PBMCs or 1E06 CD4+ T cells prepared by negative selection, we also failed to see any evidence of killing in two different assays.
Several reports have tried to describe functional capacity using changes in the expression of surface markers (26
). These reports have used a combination of reversible and irreversible surface markers. Many of these markers have well-established functional roles themselves. We followed three easily measured surface markers: CD45RO, the isoform of CD45 associated with the transition from naive to memory cell, CD27, a costimulatory marker irreversibly lost with exposure to cognate epitope, and CD57, a marker of replicative incompetence. As others have suggested, our data show frequent production of IFN-γ, and TNF-α, and decreased IL-2 frequencies as CD4+
T cells mature (49
). Similarly we find that CD4+
T cells show an increased frequency of degranulation and MIP-1β production with maturation. Nonetheless, ascribing all functionality to maturation appears to be an over simplification. The amount of perforin present in CD4+
T cells varies greatly in CD57+
T cells. Similarly, it seems clear from the data presented in that degranulation occurs at different frequencies in response to different pathogens, even with similar levels of expression of CD27 and CD57. Whether these differences are due to different milieus at initial encounter of cognate epitope, or to changes that occur during chronic stimulation, remains to be determined.
The delay in the development of cytotoxic function and MIP-1β production to the stage at which CD4+
T cells acquire an effector phenotype is attractive teleologically. Such a delay in the development of these functions would serve to protect MHC class II–presenting cells in lymphoid tissue from damage during periods when CD4+
T cell help is required to develop and maintain an effective immune response. At the same time, effector CD4+
T cells could contribute to the control of pathogens in peripheral tissues. Indeed, in many ways the functional progression that we demonstrate here during CMV-specific CD4+
T cell differentiation is similar to that seen in CD8+
CTL development (37
), even though effector CD4+
T cells in the periphery should still be able to supply help through the CD154–CD40 pathway. The persistent expression of CD154/40L, even in cells that are CD57+
, suggests the importance of this ligand in CD4+
T cell function. Although much speculation is possible, we can certainly conclude that the pattern of response described here is more complicated than has been previously suggested for most virus-specific CD4+
T cells (51
The clonotypic data indicate a more varied CD4+
clonal response to CMV than suggested previously by others (53
). Rather than one dominant clonotype, we see a more balanced response with at least three or four prevalent clonotypes in each epitope-specific CD4+
T cell population. There are several possible reasons for this difference in the apparent number of antigen-responsive clones. First, sorting antigen-responsive CD4+
T cells by CD107a and CD154 readouts may identify lower frequency clones than previously reported. It is also possible that the selection of subjects with a high response frequency to a specific epitope could select for more polyclonal MHC class II–restricted CD4+
T cell populations.
Overall, our data indicate that as pp65-specific CD4+ T cell clonotypes progress from CD27+ memory to a terminally differentiated effector memory phenotype, they acquire greater effector, and specifically cytotoxic, potential. The role of these CD4+ T cells in providing help is unclear. The relatively low frequency of IL-2–producing CD4+ T cells compared with IFN-γ, TNF-α, and MIP-1β production suggests that the primary role of CMV pp65-specific CD4+ T cells during chronic infection is not one of supplying CD4+ T cell help. The role of the CD40L–CD95 pathway in these cells remains unclear. Our findings do not apply to all CD4+ T cells specific to every virus () and may not necessarily apply to other CMV proteins. However, our data support a model where certain unique antiviral effector functions of virus-specific CD4+ T cells are elaborated after an appropriate maturational state has been achieved. It remains to be determined whether the expression of these functions is dependent on key characteristics of the pathogen to which the CD4+ T cells are targeted, or the immunologic milieu in which those T cells become initially or subsequently stimulated during the course of the infection.