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Acute myeloid leukemia (AML) is a heterogeneous leukemia characterized by the blockage of myeloid differentiation at different stages, which define distinct AML subtypes. We have recently reported that the ligation of CD44 with 2 activating monoclonal antibodies (mAbs), A3D8 and H90, triggers terminal differentiation of leukemic blasts in AML-M1/2 to AML-M5 subtypes, which are the most frequent ones. However, fresh AML blasts have short in vitro lifespans. Therefore, to find relevant in vitro cellular models for further studying the mechanisms involved in CD44-induced differentiation, we investigated whether CD44 ligation with A3D8 and H90 mAbs can induce terminal differentiation of THP-1, NB4, and HL60 cells, each interesting models of AML-M5 (monoblastic subtype), AML-M3 (promyelocytic subtype), and AML-M2 (myeloblastic subtype), respectively. We also study whether CD44 ligation induces a loss of proliferative capacity, an important feature of late-stage myeloid differentiation. In the second part of our study, we investigated whether A3D8 and H90 anti-CD44 mAbs can induce the differentiation and inhibit the proliferation of KG1a cells, which are very immature AML-M0 blasts. Using functional, antigenic, and cytologic criteria, we presently show that A3D8 and/or H90 induce terminal differentiation of THP-1, HL60, and NB4 cell lines and strongly inhibit their proliferation. Interestingly, cell-specific effects of H90 and A3D8 are observed. We also observe that incubation with A3D8 for 3 to 6 days induces an apoptotic cell death that is moderate in the case of THP-1 and HL60 cells and massive in the case of NB4 cells. Finally, our results demonstrate for the first time that it is possible to reverse the leukemic blockage of KG1a cells by using both an anti-CD44 mAb and retinoic acid. This result may provide a new experimental basis for a differentiative therapy in AML-M0 patients.

Blockage in myeloid differentiation characterizes acute myeloid leukemia (AML); the stage of the blockage defines distinct AML subtypes (AML1/2 to AML5). Differentiation therapy in AML has recently raised interest because the survival of AML3 patients has been greatly improved using the differentiating agent retinoic acid. However, this molecule is ineffective in other AML subtypes. The CD44 surface antigen, on leukemic blasts from most AML patients, is involved in myeloid differentiation. Here, we report that ligation of CD44 with specific anti-CD44 monoclonal antibodies or with hyaluronan, its natural ligand, can reverse myeloid differentiation blockage in AML1/2 to AML5 subtypes. The differentiation of AML blasts was evidenced by the ability to produce oxidative bursts, the expression of lineage antigens and cytological modifications, all specific to normal differentiated myeloid cells. These results indicate new possibilities for the development of CD44-targeted differentiation therapy in the AML1/2 to AML5 subtypes.

Our studies show that activated autoreactive inflammatory T cells specific and autoantibodies to collagen (V) and cardiac myosin are consistently detected after lung and heart transplantation, respectively. Clonal expansion of these T cells occurs only after an alloresponse, but once activated they can induce on their own rejection of allogeneic and even syngeneic transplants. Indirect rather than indirect alloresponse triggers autoimmunity after transplantation presumably via antigen mimicry between autoantigen peptides and donor MHC peptides. Also, it is plausible that inflammation and tissue damage associated with initial alloresponse to donor MHC antigens causes the release of formerly sequestered autoantigens. This may result in the presentation of some cryptic self-determinants thereby triggering an autoimmune process at the site of the graft. Finally, tolerance induction to cardiac myosin and collagen (V) results in long-term survival and reduced pathogenesis of heart and lung allografts, respectively. This suggests that, after transplantation, the inflammatory alloresponse to donor MHC triggers a cascade of events including autoimmunity to tissue antigens, a phenomenon that is essential to the actual rejection.

CD44-ligation with the anti-CD44 antibodies A3D8 and H90 triggers terminal differentiation in AML1/2 to AML5 blasts (Charrad et al, Nat Med, 5:669, 1999). Here we show that A3D8 and H90 inhibits the proliferation of myeloid leukemia cell lines HL60, NB4, THP1 and KG1a (by a factor 2 - 5 at day 2). Using three specific criteria: NBT assay, expression of differentiation antigens CD11b, CD14 and/or CD15, and cytological modifications we presently show that H90 is as efficient as retinoic acid to trigger terminal granulocytic differentiation of HL60 (myeloblastic) and NB4 (promyelocytic) cells. Moreover, like RA, H90 provokes the degradation of PML-RARα oncoprotein in NB4 cells. This differentiation is maximum at days 3 to 4. H90 is unable to induce any significant differentiation of either THP1 (monoblastic) or KG1a (most immature myeloblast) cells. The differentiation-inducing activity of A3D8 is similar to that of H90 in HL60 cells but not in the other cell lines. Indeed, unlike H90, A3D8 induces an important apoptotic cell death in NB4 cells (about 50% apoptotic cells at day 3, as shown by acridine orange assay) and it induces terminal differentiation of the monoblast-like THP1 leukemia cells. KG1a cells, which are most immature, cannot be significantly differentiated by either H90 or A3D8. However, simulataneous treatment with on one hand H90 or A3D8, on the other hand RA (which is inactive when used alone), induces full monocytic differentiation of KG1a cells. These data show for the first time that 1) CD44-ligation inhibits the proliferation of leukemia cells, 2) its differentiation and apoptosis-inducing ability depends on the antibody and the myeloid cell type, and 3) the differentiative effect is greatly improved by RA in most immature KG1a cells.