We here show that memory CD4+ T cells undergo a state of antigen unresponsiveness during the fall of antigen. We demonstrate that when antigen reaches to certain low levels, B cells capture antigen via their antigen receptors and induce unresponsiveness in CD4+ T cells. We provide evidence that long-lived quiescent memory T cells become activated upon re-infection with the virus or an in vivo challenge with antigen and a TLR-9 ligand, CpG. We suggest that this might be a mechanism adopted by memory CD4+ T cells for long-term survival in the absence of antigen.
We have previously shown that clonal CD4+
T cells become hyporesponsive by presentation of low levels of antigen31-33, 53
. Furthermore, we have shown that different forms of peptide-MHC complexes including short-lived peptide–MHC and low densities of long-lived agonist peptides induce T cell unresponsiveness through engagement of ~1000 TCR, as opposed to T cell activation that requires the engagement of over 4000 TCR31, 32, 53
. Interestingly, only memory CD4+
T cells, and not activated or naïve CD4+
T cells were susceptible to become hyporesponsive upon encounter with low densities of peptide-MHC ligand. These observations led us to suggest that all forms of ligands that engage less than optimal numbers of TCR induce anergy or hyporesponsiveness in T cell clones or CD4+
memory T cells54
. As such, the quantity of antigen or the density of peptide-MHC ligand presented to memory T cells can be regarded as a signal to activate or tolerize. In this study, one can associate high density of ligand with the presence of infection and low density of ligand with the resolution of infection.
Our original studies showing that low densities of agonist peptide induce anergy in CD4 memory T cells were established in HLA-DR1 transgenic mouse populated with heterogeneous CD4+
T cells as identified by HA306-318
/DR1 tetramer staining31
. A parallel comparison with TCR transgenic T cells adoptively transferred to recipient mice confirmed similar memory T cell sensitivity and responsiveness to the tolerogenic peptide treatments, giving us confidence that our experiments presented here are representative of the memory T cells in polyclonal systems55-58
. Furthermore, the experiments presented here are performed with only 2.0×105
Tg T cells transferred into the recipient mice, which is five times lower than the original system34
Here we provide strong evidence that the resolution of an infection as characterized by availability of low levels of antigen, would also associate with CD4+ memory T cells that are hyporesponsive. Indeed, our experiments provide direct evidence that B cells bearing specific antigen receptors are the APC that perform this task effectively. Using CpG or Vaccinia viral infection as models to study the immune response, we observed that purified B cells from the infected or immunized mice could induce hyporesponsive memory T cells. Those experiments showed that antigen capture was indeed 1,000-10,000 folds more efficient when OVA antigen was targeted to HEL-specific BCR transgenic B cells, providing a direct evidence for the role of BCR in capturing of antigen at very levels (10-5-10-8 pmol), making it highly unlikely for any other APC to capture the antigen.
Since transferring B cells from infected mice generated hyporesponsive memory CD4+
T cells in the recipient mice, it was possible that this state of rest could develop spontaneously in memory CD4+
T cells in mice that had recovered from infection. Indeed, we observed that memory T cells began to become hyporesponsive after the contraction phase in the absence of any external interference. When either Vaccinia-OVA, or OVA/CpG were used as mimics of infection, OVA specific CD4+
T cells contracted in numbers and became unresponsive to antigen once the effector phase ended. Several studies have already demonstrated that CD4+
T cells increase in number during the effector phase and decline over time after gaining memory characteristics following the resolution of infections, consistent with our observation11, 15, 59
. Our findings are also in agreement with the reports that memory T cells adopt a resting state59
because of programmed metabolic switches that control glycolysis and/or fatty acid oxidation60-62
. While those studies indicated that memory T cells are in resting state, we have demonstrated here that CD4+
memory T cells undergo a resting state initiated by B cells and triggered by certain low levels of antigen during the resolution of infection. It is noteworthy that low level of antigen presented by B cells is concurrent with low-level expression of danger signals as well. This resting state is a transient condition and may be reversed by antigen and IL-2, a condition that is met during the re-emergence of an infection due to inflammatory conditions. We demonstrate that a second viral infection even after nearly 14 months post infection is stimulatory to the memory populations that are otherwise fully unresponsive to a peptide alone challenge in vitro
. Similarly, IL-2 plus peptide, or CpG plus antigen administered in vivo
recalled vigorous responsiveness in quiescent memory CD4+
Our microarray data, that was analyzed by the most stringent parameters set for gene clustering suggested that many genes from our final gene list appeared to have immediate interactions with each other. Quite remarkably, however, a number of differentially expressed genes that were excluded from the list, because of unclear immediate relevance to our data set, merged with the interactions in the gene map as functionally related to the genes of known functional importance. Altogether, differential expression of a significant number of genes that were connected to each other directly or through controlling “nodes” in our map support our findings that long-lived memory CD4+ T cells are dormant. These points strongly argue for the non-random nature of our microarray data.
Microarray data suggest that quiescent memory CD4+
T cells significantly reduce the expression of genes that induce cell proliferation and immune activation, while increasing the expression of genes that can protect cells from apoptosis and promote survival. The changes in profiles of genes belonging to the cluster of cytoskeletal rearrangement suggest that actin polymerization is prevented in rested memory T cells, confirming that the cells are not in activated but in quiescent state. The switch in gene expression corresponding to a state of quiescence may coincide with the disappearance of inflammation and depletion of circulating antigen, which appears to begin about 8 weeks from the onset of the viral entry in the infection models presented here. Recent reports indicate that memory T cells downregulate their activation genes and lower their metabolic activities59, 61, 62
. Interestingly, our characterization of long-lived memory T cells after over a year post immunization, pointed to a remarkable observation. In Vaccinia-OVA injected mice, over half of CD4 DO11.10 cells showed high expression of memory markers, such as CD44, CD127, and CD62L from which more than half did not undergo homeostatic proliferation. In contrast, only about 10% of CD4 DO11.10 T cells from OVA emulsified in CFA injected mice showed high expression of CD44, CD62L and CD127. Majority of CD4 DO11.10 cells in the latter group expressed intermediate levels of CD127 and low levels of CD44 and proliferated well. If CD44 is to be considered a more representative memory marker, one can distinguish Vaccinia-OVA immunization as a better inducer of long-lived memory CD4 T cells as compared to OVA/CFA. Because of the 9 months time past OVA/CFA immunization, yet persistence of a clear population of KJ1.26 positive cells, one might propose that a new undefined population of memory CD4 cells that divide rapidly, yet are CD44-
might have developed. Those findings are in agreement with the microarray data, indicating that injection of mice with Vaccinia-OVA led to fully developed CD4 memory T cells, whereas continuous release of antigen over a long period of time as expected in OVA-CFA63
did not efficiently induce long-lasting CD4 memory T cells. We like to suggest that OVA emulsified in CFA continues to be released and in a way mimics a chronic infection, whereas, Vaccinia-OVA infection clears in few days and leads to the generation of memory CD4 T cells that become fully rested and have their anti-stress genes turned on while being completely dormant. All these new findings document that perhaps dormant memory T cells survive longer and might be less harmful to self-tissues due to cross-reactivity.
Our findings highlight an important physiological process that takes place at the end of an infection, when, i)
the antigenic load is reduced, and ii)
memory T cells have developed and need to receive an inhibitory signal to cease proliferation and release of cytokines. Under such conditions, B cells bearing specific receptors for antigens are the natural choice for the immune system for capturing antigen at its lowest level and present it to the memory T cells for promoting a resting state. When the need arises to fight infection during challenge, the quiescent memory T cells get activated and exert their effector function. In all, our studies put forward a novel regulatory mechanism for CD4 memory T cells. Despite the general view that memory T cells are readily activated, our data reveal strict regulation on memory CD4+
T cells. First, lack of cell proliferation and reversibility by inflammatory cytokines fulfill the original definition of T cell clonal anergy64
. Anergized memory T cells maintain low metabolic activity and cell cycle progression, criteria that would preserve cellular energy and might be the key mechanism in long-term survival. However, the critical requirement of anergized memory T cells for inflammatory cytokines for reactivation is another control mechanism of responding to danger65
. Thus, memory T cells, while equipped to respond to antigens rapidly, also require second signals for the initiation of response, similar to naïve T cells. The need for an inflammation induced danger signal for the activation of memory T cells prevents them from self-reactivity.