We have shown that the homeostasis of antigen-specific CD4+
T cells, in the steady state as well as during a strong antigen-driven pathogenic response, is regulated by the same non-clonal neighboring T cells, that out-compete the targeted T cell for specific recognition of a sub-threshold peptide-MHC trophic ligand. These data help resolve several outstanding questions on the control of CD4+
T cell dynamics, discussed below, and suggest a conceptual framework for envisioning the functional architecture of the peripheral T cell repertoire (see Figure S7
The first of these is regarding the role of public vs private factors in peripheral T cell homeostasis (Figure S7B
). The extensive dataset in this report argues that competition between CD4+
T cells for generic trophic resources is unlikely to be the major regulator of helper T cell frequency in vivo
. Clearly, a variety of such factors are critical for T cell survival in a global sense (Marrack and Kappler, 2004
). In the absence of signals from the γc
cytokines, for example, a profound lymphopenia is observed – although CD4+
T cells (of an activated phenotype) are less affected (DiSanto et al., 1996
; Lantz et al., 2000
). But, because T cells widely express receptors for these factors, it is not easy to explain how such a public competition can ensure the stable maintenance of individual specificities within a diverse repertoire. We resolved this question using a cellular strategy - packing mice with defined populations of T cells that can compete with the target T cell for some or all of the public factors. This strategy avoids the complexities of using genetic knockouts or blocking antibodies for individual factors and replaces it with cells that can compete physiologically for these resources. An argument can be made that it is still a low density sampling of the T cell universe, since the few TCR transgenics used in our packing experiments may not represent the full range of possibilities in a diverse repertoire. In this context, the negative results within our in vivo
screen provide a more striking illustration. Here the bulk of the pools, effectively representing >99.9% of the peripheral T cell population from a polyclonal repertoire, fail to modulate the density of 5C.C7 T cells. While these data clearly eliminate public factors as the sole determinants of peripheral homeostasis, it is still possible that they play a critical but secondary role in the process.
Of the cognate factors (sensed via the TCR) that remain, the restricting MHC element itself (IEk
for 5C.C7) was also not sufficient, because the similarly restricted A1(M) T cell did not affect the frequency of 5C.C7. A competition for the cognate antigen itself is certainly widely reported in models that use acute immunization (Smith et al., 2000
). However, in the chronic antigen model, two T cells specific for the same antigen with TCRs that are known to have different affinities for IEk
-PCC (AND & 5C.C7) did not affect each other’s numbers. Although the lack of an effect on AND numbers in particular could be attributed to the known cross-reactivities of AND to other ligands on IEk
, 5C.C7 (not known for such cross-reactivities) also remains unaffected when competing with AND for PCC.
Finally, several elegant studies have shown that competition between large numbers of identical clones of T cells interfere with the survival and response of each other (Hataye et al., 2006
; Moses et al., 2003
; Troy and Shen, 2003
). Indeed, clonal competition does work in our model as well, when measured by the steady state survival () or acute activation of 5C.C7 T cells (Sojka et al., 2004
). However, in the absence of a clear mechanism for this phenomenon, it was difficult to envision how it would operate within a polyclonal repertoire. In our data, the lifespan of 5C.C7 T cells was similar in hosts with a normal polyclonal population and in TCR transgenic mice with 10–15 million 5C.C7 T cells. This would imply that there are as many relevant clones in the polyclonal repertoire to mimic the clonal abundance of a monoclonal TCR transgenic animal. But most strikingly, the 5C.C7 T cells in a PCC+
but lymphopenic host routinely expand to 10–15 million. If clonal competition was a dominant determinant of the frequency of 5C.C7 T cells, these abundant clones should have efficiently interfered with one another - or for that matter a 2nd
cohort of 5C.C7 T cells (Singh et al., 2006
). The isolation of the Vα2+
deletor T cell finally allowed us to resolve these paradoxical observations and propose a unifying model. This deletor was not a clonal competitor of 5C.C7 but shares a sub-threshold ligand with it (Figure S7C
). Most importantly, it did not share any cognate antigen specificity with its target that we could detect.
This last property is crucial, since one of the consequences of chronic antigen stimulation in vivo
is the induction of T cell tolerance – in this case, by a process involving the tuning of TCR proximal signaling molecules (Choi and Schwartz, 2007
; Singh and Schwartz, 2003
). As a result, a T cell that is constantly stimulated by agonistic self (or in some cases chronic-pathogen derived) antigens would get blunted in its ability to transduce signals via the TCR. This is likely to render that T cell a poor competitor for antigens. In essence, the tuning process eliminates the distraction afforded by clonal competition and allows one to hone in on the relevant activity within the truly polyclonal repertoire. Since the deletor also inhibits the proliferation of 5C.C7 T cells in PCC-negative lymphopenic hosts (in a manner similar to clonal competition), it is likely that the unifying feature underlying both phenomena is indeed the recognition of the same universe of self-ligands.
Other than the shared recognition of a sub-threshold ligand, the deletor T cell does not seem to possess a unique property that defines it as a separate lineage. But further studies are required to clearly establish if the deletor capable receptor does also trigger a new gene expression signature in the T cells. Furthermore, while the deletor activity did not enrich with regulatory T cells (Tregs), it must be pointed out that it was not depleted in the Foxp3+ve population either. In addition, although the early expansion of the 5C.C7 T cells was also blunted slightly by the Vα2+
deletor T cells, this effect was not as pronounced as the effect of total polyclonal T cells. It is likely that other mechanisms such as Tregs cells may therefore be involved during this phase (Vanasek et al., 2006
Finally, the nature of the sub-threshold self-ligand itself raises interesting questions. At least one peptide that anchors the interaction between the deletor and 5C.C7 is one recently identified as a positively selecting ligand for the 5C.C7 TCR (Ebert et al., 2009
). While it is tempting to speculate that a direct correlation might exist between thymic selection and peripheral regulation, previous studies have failed to validate a linear relationship (Bender et al., 1999
; Clarke and Rudensky, 2000
; Ebert et al., 2009
; Ernst et al., 1999
; Goldrath and Bevan, 1999
; Kieper et al., 2004
). In fact in our experiments, polyclonal T cells that are selected in a thymus that did not afford an IEk
positively selecting signal to 5C.C7 T cells still possessed deletors capable of regulating them. Therefore, a more critical requirement might simply be for the ligand to be non-agonistic but consistently available on self MHC molecules in quantities that are limiting enough for a competitive process to operate. In many cases, positively selecting ligands are likely to satisfy these criteria and this might in fact be a strong teleological reason for retaining peptide-specific positive selection in the thymus. But certainly other sources of these ligands can arise in the periphery (from tissue-specific antigens, products of commensal organisms etc.) which might even qualitatively modulate the complexity of T cell repertoires in different tissue locations even before an antigen-specific response.
In this context, the ability of the deletor to regulate 5C.C7 numbers even in the presence of chronic PCC presentation is quite intriguing. This implies that interactions with the “non-stimulatory” weak ligands (that allow the deletor to dominate) are still relevant even though a stimulatory agonist is available. One potential explanation could be that the sub-threshold ligands and the agonists elicit qualitatively different signals downstream of the TCR. The former may in fact be more potent at triggering some pro-survival signals within the T cell than even the agonist. Alternately, the sub-threshold ligands may be segregating to specialized subsets of APCs which might allow the interacting T cells to obtain secondary survival signals. The segregation of the peripheral ligand on special APCs could also explain the ability of numerically fewer deletors to control more abundant target T cells after a clonal expansion. Since the relevant niches are limited, a few potent deletors could effectively block access to these, at least temporally. These mutually non-exclusive models will require further experimental validation, but have important consequences for our understanding of peripheral T cell responses.
These data are the strongest experimental evidence yet, in support of an emerging model envisioning the peripheral T cell repertoire as subdivided into small homeostatic units or colonies (Figure S7D
) (Hao et al., 2006
; Hataye et al., 2006
; Leitao et al., 2009
; Min et al., 2004
; Takada and Jameson, 2009
). Each colony can be defined as a group of TCRs that share the recognition of a specific set of endogenous sub-threshold ligands - although their cognate agonists differ greatly. In such a model, the size of each colony would likely depend on the amounts of the sub-threshold ligand presented in vivo
and be strictly regulated by intra-colony competition for these ligands. Within each colony, individual clones would be able to respond to their disparate cognate antigens during an infection or injury (Figure S7E
). But most importantly, after the clearance of antigen (or the induction of tolerance), the number of antigen-specific T cells would be controlled primarily on the basis of competition between members of its own colony (Figure S7F
). Since this process avoids rampant bystander losses that might be a side effect of competition for public resources, the overall diversity of the repertoire would be minimally affected. The teleological advantage of such a mechanism would be to maintain the broad diversity of the naive and memory T cell repertoire, especially in the context of multiple recurrent infections from diverse pathogens. Clearly such a model would have profound implications on our attempts to manipulate the immune response in various clinical contexts. Learning to identify and manipulate the dynamics of individual micro-colonies could be key to developing vaccines capable of generating long lasting antigen-specific T cells or conversely, ameliorating autoimmunity by reducing the life-span of auto-antigen-specific effectors.