Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection

Abstract

Background: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection.

Methods: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts.

Results: The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal.

Conclusions: Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.

Figure 1

Differential effects of AZD2014 and RAPA on mTOR signaling and inhibition of DC generation and T cell proliferation in vitro. (A) CD11c⁺DC generated from B6 mouse bone marrow (BM) cells, as described in the Materials and Methods, were incubated for 1 hour with either RAPA or AZD2014, then for an additional hour in the absence or presence of LPS (100ng/ml). The cells were washed, lysed and proteins resolved by SDS-PAGE. The influence of RAPA or AZD2014 on signaling downstream of mTORC1 (4EBP1 threonine 37/46) and mTORC2 (Akt S473) was analyzed by Western blot. Relative expression is plotted for n=3 independent experiments per condition. (B) DC were generated in the presence of GM-CSF and IL-4 and exposed to either RAPA or AZD2014 (AZD), starting on day 2 of culture. On day 7 of culture, the DC were enumerated (total number of cells per culture dish is shown). (C) On Day 7 of culture, control (durg vehicle-treated) CD11c⁺ DC and CD11c⁺ DC exposed to either RAPA or AZD2014 were treated with or without LPS (100ng/ml) for 16–18 hours, washed with PBS, stained and analyzed by flow cytometry. Quantitation of CD86, B7-H1 (= programed death ligand-1) and MHC class II (I–A^b) expression (relative MFI) across multiple experiments is shown. (D) Bulk T cells were isolated by negative selection from B6 mouse spleen and labeled with VPD450, as described in the Materials and Methods. They were stimulated with αCD3/CD28 Dynabeads (1:2) in 96-well, round-bottom plates for 72 hours in the presence of RAPA or AZD2014 added at the start of culture. CD4 and CD8 T cell proliferation was assessed by flow cytometry. Proliferating cells were identified as CD4⁺VPD450^lo or CD8⁺VPD450^lo and % proliferation relative to the total parent population determined. Data are from n=3 independent experiments *p<0.05; **p<0.01; ***p<0.001.

Figure 2

AZD2014 administration induces thymocyte depletion and enriches for thymic Treg. AZD2014 (AZD; 10mg/kg bid i.p.), RAPA (1mg/kg i.p.) or drug vehicle was administered daily for 9 days to naïve B6 mice. Thereafter, the mice were euthanized, thymi excised, weighed and processed for histology, or total thymocytes isolated, counted and analyzed by mAb staining and flow cytometry. (A) and (B) Both AZD2014 and RAPA induced marked thymic involution with profound cortical and medullary atrophy. (C) The total number of thymocytes as well as (D) double-positive (CD4⁺CD8⁺) T cells and (E) and (F), single positive CD4⁺ and CD8⁺ T cells were markedly depleted by both AZD2014 or RAPA. (G) Although the incidence of Treg (CD4⁺CD25⁺Foxp3⁺) increased significantly in thymi of both AZD2014- and RAPA-treated mice (H) the absolute number of Treg diminished in these groups. Data are from 3 animals per condition. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 3

AZD2014 administration suppresses immune cell populations in secondary lymphoid tissue but does not enrich for Treg. AZD2014 (AZD), RAPA or drug vehicle was administered daily to naïve male B6 mice for 9 days. Thereafter, mice were euthanized, spleens excised, weighed and either processed for histology or total splenocytes isolated, counted and analyzed by mAb staining and flow cytometry. (A) and (B) Representative spleen histology and spleen weights from drug vehicle-, AZD2014- or RAPA-treated mice. The cellularity of both the red and white pulp was depleted markedly in drug-treated compared with vehicle-treated mice. (C) The mean total number of splenocytes, as well as (D–H) the absolute numbers of CD4, CD8, Treg (CD4⁺CD25⁺Foxp3⁺) and Tfh cells (CD4⁺PD1^hiCXCR5^hi) were reduced by both AZD2014 and RAPA. F, Although the incidence of Treg in the CD4 T cell compartment was either unaffected (AZD2014) or reduced (RAPA) (G) both drugs reduced the absolute number of Treg. (I–L) AZD2014 and RAPA suppressed the mean absolute numbers of other immune cells in the spleen, including CD11c⁺DC, B cells and NK cells. NKT cells were not affected significantly. Data are from 3 animals per condition. *p<0.05; **p<0.01; ***p<0.001.

Figure 4

AZD2014 inhibits alloreactive T cell proliferation in vivo. (A) The protocol for BALB/c I-Eα-derived allopeptide, drug vehicle, RAPA or AZD2014 (AZD) administration following adoptive transfer of VPD450-labeled 1H3.1 TCR-transgenic (tg) CD4⁺ T cells (CD90.1⁺Vβ6⁺) is shown. All mice, other than the “no peptide” group, received allopeptide 48 hours after adoptive transfer of cells. (B) Gating strategy for determining percent T cell proliferation. Adoptively transferred tg T cells were identified in the spleen by flow cytometry as CD3⁺CD4⁺CD90.1⁺Vβ6⁺ cells. Then, VPD450^−lo cells were gated and percent proliferation determined. (C) Percent proliferation 6 days after adoptive transfer of alloreactive tg T cells. Data are from 3–5 animals per condition and n=2 experiments were performed. **p<0.01, ****p<0.0001.

Figure 5

AZD2014 prolongs heart allograft survival. (A) The protocol used for AZD2014 (AZD) or RAPA administration following heart transplantation. Starting on day 3 posttransplant, a 9-day course of either AZD2014 (10mg/kg i.p. twice daily), RAPA (1 mg/kg i.p. daily), or drug vehicle was administered. (B) Actuarial graft survival curves; numbers of transplanted mice in each group are shown in parentheses; (C) Histological appearance of heart allografts (H & E stain; horizontal bar indicates 200 μm); (D-E) T cell populations (Tfh and Treg) in the spleen and Treg:Tem ratios 7 days and 21 days posttransplant. n=3 graft recipients per group *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 6

AZD2014 inhibits alloreactive T cell proliferation and IFNγ production by host T cells. On day 7 posttransplant, splenocytes from graft recipients were stimulated with PMA and ionomycin and T cell intracellular cytokine expression (IFNγ; IL-2) assessed by flow cytometry. (A) Cytokine production by T cells from representative mice in each group; (B) Means + SD from n=2–5 animals per group. (C) Antidonor and antithird party CD4⁺ T cell proliferative responses in 5 day ex vivo MLR cultures. *p<0.05; **p<0.01.

Figure 7

Influence of AZD2014 on B cell responses and DSA production. (A) Total as well as IL-10⁺ B cells were determined on day 7 and day 21 posttransplant in host spleens by mAb staining and flow cytometry. (B) Antibody deposition in the heart allografts was examined by immunofluorescence at 7 and 21 days posttransplant. Total IgG is shown in green, CD31 (a marker of endothelial cells) is shown in white, actin is shown in red and nuclei are stained with hoescht (blue). A naïve B6 heart is shown for comparison. Horizontal bars indicate 207μm. (C) Donor-specific alloAb (DSA) (IgG1, IgG2c and IgG2b) in serum 21 and 100 days posttransplant was quantified by flow cytometry. n=3–5 animals per group *p<0.05; ***p<0.001.