Vav1 GEF activity is required for T cell mediated allograft rejection.

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression.

Differential promotion of hematopoietic chimerism and inhibition of alloreactive T cell proliferation by combinations of anti-CD40Ligand, anti-LFA-1, everolimus, and deoxyspergualin.

Allogeneic bone marrow (BM) engraftment for chimerism and transplantation tolerance may be promoted by combinations of costimulation blocking biologics and small molecular weight inhibitors. We showed previously in a mouse model that anti-CD40Ligand (anti-CD40L, CD154) combined with anti-LFA-1 or everolimus (40-O-(2-hydroxyethyl)-rapamycin) resulted in stable chimerism in almost all BM recipients, whereas anti-LFA-1 plus everolimus conferred approximately 50% chimerism stability. Here, we investigated whether this lower incidence could be increased with deoxyspergualin (DSG) in place of or in addition to everolimus. However, DSG and everolimus were similarly synergistic with costimulation blockade for stable hematopoietic chimerism. This correlated with allospecific T cell depletion and inhibition of acute but not chronic skin allograft rejection. Different treatments were also compared for their inhibition of alloreactive T cell proliferation in vivo. While anti-CD40L did not impair T cell proliferation, anti-LFA-1 reduced both CD4 and CD8 T cell proliferation, and combining anti-LFA-1 with everolimus or DSG had an additive inhibitory effect on CD4 T cell proliferation. Thus, despite their strong inhibition of alloreactive T cell proliferation, combinations of anti-LFA-1 with everolimus or DSG did not reach the unique potency of anti-CD40L-based combinations to support stable hematopoietic chimerism in this system.

Strongly reduced alloreactivity and long-term survival times of cardiac allografts in Vav1- and Vav1/Vav2-knockout mice.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection").