T cells express two distinct integrin heterodimers that contain the α4 integrin subunit,α4β7 and α4β1. It is critical to define the mechanisms that control the expression of α4β7 and α4β1 on T cells, as these integrins promote homing to the gut (α4β7) and to extra-intestinal sites, such as the BM and brain (α4β1) (3
). In this study, we demonstrate that changes in the expression of β1 integrin reciprocally alter α4β7 expression on CD4 T cells. We show that this regulation occurs at the protein level, where the α4 integrin subunit preferentially pairs with the β1 integrin subunit when both β1 and β7 integrin are present. We identify the abundance of α4 integrin as the major driver of α4β7 expression on CD4 T cells and demonstrate its importance in RA-induced α4β7 upregulation. Finally, by tracking an endogenous antigen-specific population of CD4 T cells following infection, we demonstrate that the absence of β1 integrin results in enhanced α4β7 expression and altered localization of early memory CD4 T cells.
Naïve CD4 T cells express low levels of both the α4β1 and α4β7 integrins. When we crossed mice with “floxed” alleles of the β1 integrin gene with CD4-Cre transgenic mice, the loss of β1 integrin on naïve CD4 T cells was associated with increased expression of α4β7. These results are similar to recent findings from an independently derived line of conditional β1-deficient mice (10
). We show that this change in α4β7 expression has functional significance, as β1−/− T cells exhibit enhanced adhesion to MAdCAM-1 in vitro and increased localization to PP in vivo. Since successful cell surface integrin expression requires intracellular α/β heterodimer formation, we suggest that the availability of the α4 integrin subunit for pairing regulates α4β7 integrin expression on T cells. Several lines of evidence suggest that the α4 integrin preferentially pairs with β1 integrin. First, we do not observe increased β1 integrin expression or function on β7-deficient CD4 T cells. Instead, loss of β7 integrin expression results in decreased cell surface expression of α4 integrin. This suggests that all available β1 integrin associates with α4 integrin, even when β7 integrin is present. Staining of permeabilized cells further supports this hypothesis, as there are abundant levels of intracellular β7 integrin, but not β1 integrin, in naïve CD4 T cells. The inability of β7 integrin over-expression to reduce β1 integrin expression in another system is also consistent with this model (4
). Second, over-expression of β1 integrin on naïve CD4 T cells results in a dose-dependent decrease in α4β7 integrin expression without altering α4 integrin expression. Thus, increased levels of β1 integrin can effectively out-compete β7 integrin for association with the limiting amount of the α4 subunit expressed in CD4 T cells. Thus, there is a hierarchy of β subunit pairing to the α4 integrin, with a “dominant” β1 integrin subunit that modulates the expression of the other heterodimer, α4β7.
The functional relevance of this integrin subunit pairing hierarchy is revealed by our analysis of β1 and α4β7 integrin expression following CD4 T cell activation. Activation of control T cells with anti-CD3/CD28 antibodies for 3 days resulted in increased β1 integrin expression and loss of α4β7 integrin expression even before the first cell division. These results are consistent with our over-expression data demonstrating β1 integrin as the dominant α4 integrin pairing partner. In contrast, β1−/− T cells exhibit dramatically elevated levels of α4β7 expression. Expression of α4 integrin on T cells is also increased following activation, although the level of surface α4 integrin was slightly lower on β1−/− T cells. This is likely due to the overall lower levels of β subunits available for pairing with α4 integrin in β1−/− T cells. These findings suggest that the preferential pairing of β1 integrin with α4 integrin is critical for the suppression of α4β7 expression following T cell activation.
Our work supports a model where β1 integrin expression modulates α4β7 expression by controlling the abundance of α4 integrin available to pair with β7 integrin. In a naïve T cell, all available β1 integrin pairs with α4 integrin and is expressed on the cell surface as α4β1. Any remaining free α4 integrin is then available for pairing with β7 integrin, resulting in a low level of α4β7 integrin expression on the cell surface. The identification of an intracellular pool of β7 integrin in naïve CD4 T cells suggests that β7 integrin is expressed in excess of the available α4 integrin. We also demonstrate that α4β7 cell surface expression is enhanced when the α4 integrin subunit is over-expressed in naïve T cells, even though mRNA levels for β7 integrin are not altered. This suggests that intracellular β7 integrin serves as a reservoir of β7 integrin available for pairing with free α4 integrin. Our model is consistent with microarray data that shows human, β7-high memory CD4 T cells have an increase in mRNA transcript for α4 integrin but not β7 integrin (40
). The relative abundance of β1 integrin to α4 integrin is critical, as over-expression of both α4 and β1 integrin results in suppression, rather than induction, of α4β7 expression. In this situation, the excess β1 integrin subunits likely associate with the free α4 integrin subunits, thereby suppressing α4β7 heterodimer formation. Overall, our model predicts that the precise ratio of β1 to α4 subunit is critical for controlling the expression of α4β7. A ratio favoring β1 integrin results in suppression of α4β7, while a ratio favoring α4 integrin results in increased α4β7 expression.
Previous work has shown that RA produced by intestinal DCs induces α4β7 expression on T cells (21
), but the exact cellular mechanism for how this occurs is unknown. CD8 T cells activated by intestinal DCs are reported to have increased mRNA transcript for α4 integrin but not β1 or β7 integrin (41
). We find that RA-treated, activated CD4 T cells have a similar pattern of enhanced α4 integrin protein and mRNA without alterations in β1 or β7 integrin mRNA. Thus, RA-induced α4β7 expression is driven by increased abundance of α4 integrin. As a result, the β1 to α4 subunit ratio favors the α4 integrin. Under these conditions, α4 integrin is no longer a limiting pairing partner. Thus, β7 integrin can pair with excess α4 integrin not bound to β1, resulting in increased α4β7 cell surface expression. Our finding that over-expression of α4 integrin mimics the effects of RA treatment on α4β7 expression supports this model. The precise balance between the β1 and α4 subunits remains critical, as the over-expression of β1 integrin in RA-treated CD4 T cells suppresses the induction of α4β7. Here, the addition of excess β1 integrin elevates the β1 to α4 subunit ratio, resulting in suppression of α4β7 expression. These results highlight the importance of the stoichiometry of the α4, β1 and β7 integrin subunits in determining the relative levels of α4β1 and α4β7 that are expressed on the surface of a T cell. This mechanism of regulation of integrin expression may also be applicable to other T cell subsets that express integrin subunits that share a common integrin subunit binding partner (42
To examine changes in integrin expression following T cell activation in vivo, we utilized peptide:MHCII tetramer-based enrichment approaches to monitor changes in integrin expression on a polyclonal population of antigen-specific CD4 T cells following Listeria monocytogenes
). This approach avoids possible alterations in activation kinetics, homing molecule expression, and memory generation and maintenance that have been reported when using high cell number adoptive transfer (46
). In the spleen, we unexpectedly detected high levels of α4β7 on ~50% of activated control CD4 T cells at the peak of the response (day 5). Although splenic DCs have been reported to induce α4β7 on CD8 T cells in vitro, this has not been reported for CD4 T cells in vivo (50
). A transient increase in the availability of the α4 subunit following T cell activation in the spleen may explain the high percentage of β1 integrin expressing α4β7-high CD4 T cells recovered 5 days following infection. By day 18, the majority of activated control CD4 T cells expressed high levels of β1 integrin and low levels of α4β7, while a smaller subpopulation had a β1-low α4β7-high “gut-homing” phenotype (~8%). Our results suggests that CD4 T cells with this “gut-homing” phenotype have a mechanism to suppress β1 integrin abundance at the protein and/or mRNA level. In contrast to the results obtained with control CD4 T cells, activation of antigen-specific β1−/− CD4 T cells resulted in uniformly elevated expression of α4β7 at all time points examined. This is consistent with our model that β1 integrin expression is critical for suppressing α4β7 expression and modulating cell surface α4β7 following CD4 T cell activation.
Control and β1−/− T cells exhibited comparable kinetics of CD4 T cell expansion, contraction, and maintenance in the spleen following Listeria monocytogenes
infection. Although the number of β1−/− 2W1S-specific CD4 T cells was lower at all time points examined than controls, this difference was not statistically significant. Overall, this indicates that β1 integrin expression is not essential for the expansion or maintenance of a polyclonal population of CD4 T cells in the spleen following antigen challenge. However, there were clear differences in the localization of activated CD4 T cells, with lower numbers of β1−/− T cells in the BM and higher numbers in the PP. As a source of IL-7 and IL-15, the BM appears to function as a reservoir for the long-term maintenance of CD8 T cells (14
). IL-7 and IL-15 are also important for memory CD4 T cell survival (53
) and recent work suggests that CD4 memory T cells reside in the BM and associate with stromal cells that produce IL-7 (55
). Although less memory β1−/− CD4 T cells are maintained in the BM, we do not detect a significant loss of peptide-specific CD4 T cells in the spleen out to day 120 post-infection. It is possible that enhanced α4β7 expression on β1−/− T cells promotes aberrant entry into other sites where similar survival signals could be relayed.
In summary, we have identified a novel mechanism of integrin regulation based on pairing hierarchy, where β1 integrin dominates β7 integrin pairing with their common α4 subunit. This mechanism of regulation is critical for controlling the level of α4β7 integrin on naïve CD4 T cells, the changes in α4β7 expression that occur during the course of T cell activation, and the subsequent localization of early memory CD4 T cells. Our findings present a means for the intentional modulation of α4β7 integrin expression on CD4 T cells. The targeted modulation of α4β7 integrin is of clinical interest with its known involvement in the progression of IBD(19
) and, more recently, as a co-receptor for HIV (56