Tolerance Induction by Immature Dendritic Cells Is Mediated by Distinct MHC Dependent and Independent Mechanisms: A Novel Role for Perforin, Granzyme A and Toll Like Receptor 7.

Abstract 65

The mechanism underlining tolerance induction by immature myeloid dendritic cells (IMDC) is poorly understood. To address this issue, we generated mouse IMDC from hematopoietic stem cells (HSC) via differentiation to early myeloid cells (10 days with a cytokine cocktail containing: TPO, Flt-3L, SCF, IL-3 and IL-6) and further differentiation to IMDC (10% GM-CSF for an additional 10 days). The phenotype of the cells obtained at the end of this procedure was CD11c⁺, CD11b⁺, B220⁻, CD80⁺, MHC class II ^{+ Low} and CD86⁻.

Addition of the IMDC to long term ( 3 days) mixed lymphocyte reaction (MLR) in which TCR transgenic (Tg) CD4⁺ or CD8⁺ alloreactive T cells are reacted against their cognate MHC antigens, led to a marked inhibition of proliferation (reaction ratio 1 IMDC: 2 T cells). This suppression was found to be mediated In short term MLR ( 5 hours) through killing of the effector cells.

Killing of CD4⁺ alloreactive T cells was found to be MHC independent; namely, IMDC expressing MHC not recognized by the TCR transgene exhibited similar inhibition as that exhibited by IMDC expressing the cognate MHC (55±3% versus 59±4% killing upon addition of cognate MHC or non-cognate MHC IMDC, respectively).

As previously shown, this non-specific killing is likely mediated by the NO system, as it could be completely abrogated by pre-treatment with N (G)-nitro-L- arginine methyl ester (L-NAME ) (5±4% and 4±3% killing upon addition of pre- treated IMDC bearing cognate MHC or non-cognate MHC, respectively).

In contrast, killing of CD8⁺ alloreactive T cells by IMDC was only slightly reversed by L-NAME (74±3% killing with non-treated IMDC bearing cognate MHC, compared to 68±4% after pre treatment with L-NAME), and the remaining killing activity was found to be MHC dependent. Thus, in contrast to cognate IMDC, very low killing of alloreactive TCR transgenic CD8 T cells was exhibited by non-cognate IMDC in the presence of L-NAME (5±2% killing). When tested in a mouse model of T cell mediated bone marrow (BM) allograft rejection, only IMDC of donor, but not of third party origin, could prolong graft survival upon infusion of 3×10⁶ IMDC (median survival of 24 versus 10 days respectively, p<0.05) suggesting that only the MHC dependent tolerance mechanism is operative in this graft rejection model.

Surprisingly, the MHC dependent killing exhibited by IMDC in short term MLR was completely lost when using IMDC generated from perforin KO mice or when IMDC were pre-treated with Bapta-AM or Concanamycin A (6±4%, 5±3% and 3±3% killing, respectively). Thus, killing of the responding alloreactive CD8⁺ T cells by their cognate IMDC involves a perforin/granzyme mediated mechanism. Indeed, Electron Microscopy (EM) analysis revealed that IMDC were loaded with granules containing perforin and Granzyme A, mostly localized in proximity to the cell membrane. While these granules could barely be detected in early myeloid cells, formation of granules was markedly enhanced upon differentiating into IMDC.

Further EM analysis revealed that recognition of the IMDC by alloreactive CD8⁺ T cells induced granules polarization towards the area of contact. Live video imaging (using perforin and granzyme A immunostaining) further supported the suggestion that this granule polarization leads to specific killing of the responding CD8⁺ T cell. Furthermore, pre-treatment of IMDC with a blocking peptide for Toll like receptor 7 (TLR7) but not for TLR1, TLR5, TLR6, TLR9, TLR10, or TLR11, completely abolished this granule degranulation, and the eventual killing of alloreactive CD8⁺ T cells (blocking TLR7 reduced the MLR inhibition from 68±4% to 8±3%, p<0.01). Thus, MHC dependent killing of alloreactive CD8⁺ T cells by IMDC is likely triggered by TCR recognition of the cognate MHC on the IMDC which, in turn, activate Toll receptor signaling leading to degranulation and granule mediated killing.

In conclusion, IMDC can induce immune tolerance through two distinct mechanisms. The first, mediated by the NO system, is MHC independent and is limited to ablation of CD4⁺ T cell responses. The second, mainly targeting CD8⁺ T cells, is MHC dependent and is mediated by cytotoxic granules in the IMDC which polarize towards the contact area upon recognition by the T cells. This MHC- specific mechanism is clearly dependent on perforin and is specifically triggered by TLR7 ligation. The latter mechanism was predominant when testing IMDC in a mouse model for T cell mediated BM allograft rejection.