In this study, we identified a critical function of CD44 in the regulation of memory generation in Th1 CD4 cells. Despite potential roles in migration and interactions with DC, CD44 was not required for the initial induction of a primary immune responses in vivo, or for the localization of naive or effector cells in either lymphoid or non-lymphoid tissues. This is probably because of redundancies in adhesion receptor usage that enable T cells to bypass its contribution and/or the ability of other HA binding receptors to perform these functions. However, CD44 plays a non-redundant role in regulating the survival of CD4 effector cells in the influenza model, which is dominated by a Th1 cell response. Without engagement of CD44, effector cells that have progressed through several rounds of division die by apoptosis, whereas agonist signaling via CD44 during the expansion phase can lead to enhanced in vivo accumulation of effector cells. Thus, the generation of a memory population in Th1 cells most likely depends upon engagement of CD44 on responding effectors during the primary response. We show that CD44 ligation can activate the PI3K/Akt signaling pathway in Th1 cells. The mechanism by which CD44 activates PI3K remains to be explored, but could be due to constitutive association with the src family kinases, Lck and Fyn (Rozsnyay, 1999
), or to associations with β1 integrins that mediate the survival response (Lee et al., 2008; Marhaba et al., 2006
; Nandi et al., 2004
Th1 cells may uniquely require this survival signal through CD44 because of elevated Fas levels and an inherent ability to rapidly assemble the DISC in response to Fas trimerization (Varadhachary et al., 1999
). Thus, we suggest that without engagement of CD44, the response to Fas-ligation cannot be overcome. Such a mechanism may not be necessary in Th2 cells, and possibly other subsets of T cells, because of overall lower levels of Fas expression in addition to a greater capacity to engage PI3K/Akt in response to TCR signaling or co-stimulation (Varadhachary et al., 1999
). It is of significance that activation of PI3K/Akt can block DISC formation by preventing the association of FADD and recruitment of pro-caspase 8 in CD4 cells (Jones et al., 2002
) and our studies lead us to favor this mechanism for regulation of Th1 cell survival.
Although CD44 can mediate resistance of tumor cells to apoptosis by death receptor ligation via FasL/Fas, DR5/TRAIL, and TNFR1/TNF-α by interfering with DISC assembly through the physical association of Fas and CD44 (Hauptschein et al., 2005
), this interaction occurs through variant isoforms. Isoforms that include variants v6 and v9 are in close proximity with Fas in the membranes of transfected Jurkat cells and thereby prevent Fas trimerization (Mielgo et al., 2006
). However, the lack of CD44 isoforms on CD4 cells activated in vivo after influenza virus infection (data not shown) or on Th1, Th2, or Th17 cells generated in vitro, which differ in their susceptibility to Fas-mediated death in the absence of CD44, further argues against sequestration of Fas as the only mechanism that accounts for a selective function of CD44 in Th1 cells. The lack of CD44 isoforms on CD4 cell subsets also argues against a mechanism whereby osteopontin-binding to CD44 variants containing v7 leads to activation of NF-κB and prevents mitochondrial death controlled by the transcription factor Foxo3a, a regulator of Bim (Hur et al., 2007
). Indeed, we did not detect changes of either pro- or anti-apoptotic Bcl-2 family proteins in WT compared to CD44−/− CD4 cells. Although differences in glycosylation of Th2 cells has been reported to account for resistance to cell death compared to Th1 and Th17 cells (Toscano et al., 2007
), the mechanism involves protection from binding of galectin-1, and we did not observe differences in the molecular weight of CD44 from Th1 and Th2 cells that would suggest significant differences in glycosylation. Since CD44 is up-regulated on activated and memory CD8 cells, we did not anticipate differences in their regulation that would suggest independence from CD44-mediated survival signals. However, there are many differences in the regulation of CD4 and CD8 cells, including in the programming to develop into memory cells after the initiation of a response (Kaech and Ahmed, 2001
; van Stipdonk et al., 2003
). Our data support the concept that internal signaling differences rather than external molecular variation account for the differences in regulation by CD44 on T cells.
A role for CD44 in regulating survival of CD4 cells engaged in an immune response in vivo has not been previously examined directly. However, protection from TCR-mediated AICD by CD44 has been suggested by in vitro studies of in vivo primed cells (Marhaba et al., 2003
). The results described herein, which show normal priming of CD4 cells in vivo irrespective of the presence of CD44 was on T cells or DCs, support the concept that engagement of CD44 in vivo is required for Th1 cells only after activation. Our results suggest that survival signals are transmitted in Th1 cells during the expansion phase of the effector response to influenza virus, which is profoundly compromised in the absence of CD44 or when adhesion binding of CD44 is blocked
A CD44-dependent survival mechanism remained operative in activated Th1 cells that were withdrawn from overt Ag stimulation by transfer into naive recipients. This result suggests that TCR signaling in the context of an effector response and the production of pro-inflammatory cytokines both of which can augment CD44-binding of HA (Marhaba et al., 2003
), are not necessary for the function of CD44 in promoting apoptosis resistance. Indeed, in the absence of an immune response, agonist engagement of CD44 in vivo promoted enhanced accumulation of CD4 cells. Our previous studies indicate that maintaining effector survival through co-stimulation can be key to the generation of robust memory in CD4 cells (Linton et al., 2003
; Linton et al., 2000
). In this regard, we propose that CD44 can be viewed as an ECM-dependent, Th1 cell-specific ‘co-stimulatory’ molecule that sustains effector cell response through survival and thereby supports the development of memory.
The homeostasis of many cell types is regulated by contact dependence, and signals from the ECM can be crucial to prevent cells from undergoing anoikis, or programmed cell death which can be due to intrinsic death resulting in mitochondrial permeabilization, and extrinsic death that is initiated by death receptors (Gilmore, 2005
). While the molecular mechanisms that lead to PI3K activation and the downstream targets in Th1 cells remain to be defined, our results support the concept that physical contacts of Th1 cells with HA in the immediate environment regulate processes during which CD44-dependent survival signals are engaged. By promoting optimal survival of effector CD4 cells engaged in an immune response (clonal burst), CD44 provides a previously unknown contribution to the development T cell immunity in vivo.