Abstract
Rho GTPases are well known regulators of actin dynamics, gene transcription, kinase pathways and cell cycle progression. RhoH, a hematopoietic specific, GTPase-deficient Rho GTPase was first defined as a hypermutable gene in diffuse large B cell lymphoma (Pasqualucci et al., 2001), although its role in the pathobiology of B cell malignancies still remains unclear. Subsequently, RhoH has been implicated in TCR signaling and T cell development in humans and mice. As RhoH is required for activation and localization of ZAP70 and LCK to the immune synapse, knockout of RhoH results in T cell deficiency (Gu et al., 2006). However, despite a profound T cell defect in Rhoh-/- mice, we previously noted an apparent paradoxical delay in disease onset in the Em-TCL1Tg murine model of B cell chronic lymphocytic leukemia (CLL) after deletion of RhoH. We previously demonstrated that this is partly due to Rhoh-/- CLL cell-intrinsic changes resulting in impaired access to supportive niches and defective microenvironmental interactions (Troeger et al., 2012). However, there is accumulating evidence that progressive immune dysregulation also plays an important role in CLL progression. Specifically, an increase in circulating follicular helper T cells (Tfh), inverted CD4:CD8 ratios, a predominance of a memory T cell phenotype, defective T cell motility and an impaired immunological synapse formation have been reported in CLL patients and Em-TCL1Tg mice (Hofbauer et al., 2011). Interestingly, the immunomodulatory drug lenalidomide has proven effective in modulating CLL-associated changes in T cell function and altered Rho GTPase activity (Ramsay et al., 2013) and we have previously demonstrated that lenalidomide treatment of CLL cells in vivo and in vitro resulted in decreased RhoH expression, suggesting that Rho GTPases are involved in T cell- B cell/CLL interactions. Moreover, lenalidomide treatment has been shown to restore immune synapse formation and T cell function in CLL patients indicating that RhoH may similarly modulate T cell- B cell crosstalk including modified LFA1 signaling.
Here we aimed to assess the impact of RhoH on the germinal center reaction and changes in T cell populations over time in knock-out mice and a murine model of CLL. We demonstrate that RhoH is required for normal germinal center (GC) formation and induction of T cell dependent B cell responses in vivo. Thus, while IgM levels were only mildly reduced in Rhoh-/- mice, these animals exhibited significantly reduced IgG1 serum levels 21 days after TNP-KLH treatment (WT vs. Rhoh-/-mice: IgG1 3706951ng/ml +/-871537 vs 122176ng/ml +/-14006; mean+/- SEM; p=0.01), indicating a defect in immunoglobulin class switching.
In keeping with these observations, we detected a severe defect in CXCR5+ Tfh cells in the spleens (WT vs Rhoh-/- mice: 10.09%+/-1.23 vs. 1.71%+/-0.45; mean+/- SEM; p=0.01) and peripheral blood (WT vs Rhoh-/- mice: 4.69%+/-0.51 vs 0.36% +/-0.13) of young Rhoh-/- mice, and a profound defect in both naïve CD4+ and CD8+ T cells. However, while over time in mice with CLL disease CD8+ activated effector memory T cells expanded in Em-TCL1Tg;Rhoh-/- to levels comparable to WT mice, deficiency in CD4+ Th cells persisted in Rhoh-/- animals. As it has been shown that CLL cell proliferation depends on Th cell support (Plander et al., 2009), we next aimed to assess the impact of the T cell phenotype on disease progression by performing adoptive transfer experiments. After 6 weeks, recipients of WT CLL cells demonstrated a significantly accelerated disease progression compared to those injected with Rhoh-/- CLL cells (WT vs. Rhoh-/- recipients:238.1+/-65.8 K/µl vs 195.2+/-54.8 K/µl WT CLL cells; and 2.3+/-1.4 K/µl vs 5.9+/-2.5 K/µl Rhoh-/- CLL cells) and accordingly also demonstrated improved survival (survival probability: WT CLL: 0.4+/-0.15 vs. 0.3+/-0.14 and Rhoh-/- CLL: 0.75+/-0.13 vs. 0.82+/-0.12; p<0.05).
These data clarify that the lack of CD4+ Th cell support and impaired GC reaction due to RhoH-deficiency have little impact on disease progression in this model of CLL. The findings further confirm that the slower disease progression and improved survival observed in the murine model of Rhoh-/- CLL is mainly mediated by cell autonomous characteristics of the CLL cells and a sufficiently sustained immune surveillance by CD8+ T cells. Thus, inhibition of RhoH may represent an attractive tool for future targeted therapies in CLL.
Disclosures
No relevant conflicts of interest to declare.
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