HLA obstacle in Haploidentical bone marrow transplant (BMT)

冰绿茶

The mechanism study of across HLA obstacle in Haploidentical bone marrow transplant (BMT)  
4 u* v/ X: X, `; E    Haploidentical HSCT can be available immediately to almost all patients, who need allogeneic HSCT without the wait and expense of donor searching. However, the resultant high incidences of lethal GVHD and host versus graft failure/rejection (HVGR) make the procedure almost prohibitive.
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  h7 J4 ?+ O: \6 L* X: K5 l    Intensified chemo-radiation conditioning in attempting to prevent graft rejection and eradicate leukemia has been associated with significantly increased regimen related toxicity and incidence of acute GVHD.  It is known that the number of donor T cells in the grafts is associated with the development of GVHD and vigorous ex-vivo donor T-cell depletion (TCD) decreases GVHD. Unfortunately, the benefit of ex-vivo TCD is often offset by increased graft rejection and delayed immune reconstitution, which may increase infections with mortality as high as 40% and leukemia relapse.We designed a haploidentical HSCT protocol without ex-vivo T cell depletion based on the knowledge that marrow preparation contains 10 times fewer mature T cells than peripheral blood stem cell (PBSC) preparation. Granulocyte colony stimulating factor (G-CSF) donor priming enhances engraftment and reduces the incidence of acute GVHD. We then extended our transplant study from match to mis-match related and developed current halpo-BMT protocol. In our initial phase during February 1999 and November 2000, 15 patients with high-risk hematological malignancy underwent haploidentical G-CSF primed marrow transplants.  Nine patients survived more than one year. Three patients died of acute GVHD in the initial study. 0 k6 B! H3 y9 O( h( ~

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$ @& s" F- L0 K- d- ^- j1 q    In order to decrease  incidences of lethal GVHD , we then improved the protocol with the addition of anti-CD25 mAb to the protocol and focused on the engraftment and sequential immunosuppressants during transplant to prevent both HVGR and GVHD with the eventual goal to achieve a stable engraftment without clinical GVHD. We report here the results in the last 104 patients (acute myeloid leukemia,n=25;acute lymphoblastic leukemia,n=45;chronic myeloid leukemia ,n=32;myelodysplastic syndromes,n=2) with leukemia all of whom were transplanted under our modified protocol. Age ranged from 2 to 50 years with a median of 24 years. All patients were at high risk because 49 were actually in relapse at transplant, 23 were in second or later CR and even the 32 patients in CR1 or CP were at high risk because of unfavourable prognostic features. All donors were HLA-haploidentical relatives sharing at least two major histocompatibility complex (MHC) antigen mismatches with the recipients by polymerase chain reaction sequence-specific primers (PCR-SSP) and high-resolution molecular genetic typing. Overall 103/104 patients established trilineage engraftments confirmed by bone marrow exams one-month post-transplant. Median days to achieve an ANC > 0.5×109/L and platelets > 20×109/L were 19 (range 13-27 days) and 25days (range16-51 days). Eleven patients died before day 100. Ninty-three patients survived over 100 days and 66 of the 93 patients had platelet counts >50×109/L by day +100 (71%). Chimerism analysis demonstrated full donor chimerism (>95% of donor cells) at 1 month post transplant among all patients. 17 of 104 patients developed grade II-IV acute GVHD.  The incidences of acute II-IV GVHD were 16.7%. Seven of them died of severe acute GVHD. 17 of 61 patients surviving at present progressed to chronic extensive GVHD. One patient died from chronic extensive GVHD. The numbers of CD3+CD8+ T cells, CD19+ B cells and CD16+/CD56+ NK cells recovered rather rapidly to normal levels within 6-12 months post transplant.  Absolute CD3+CD4+ cell counts recovered above 200/ul within six months and returned to normal in 18 months. Follow up lasted more than 3 months in all patients with a median of 23 months (range 3- 56 months).  The actual survival rate at 1 years was 61.6%.17 patients died with transplant related complications: GVHD - 6, infections - 6， viral hepatitis -1，heart failure – 2,other-2. The disease recurrence were 28 patients. 26 of them died from disease recurrence. Relapse occurred mainly in patients who were already  in relapse at transplant. Only 6 of 45 patients who were in any CR or CP at transplant have so far relapsed. 3 significant adverse factors emerged in cox analysis: active disease,donor age >25 years and patients age >18 years. Patients who were transplanted at the relapse state had significantly lower survival rates (30.6%) compared to the patients who were transplanted in non- relapse (70.5%) (p 0.043). Patients whose donor age were >25 years had significantly lower survival rates (42.5%) compared to the patients whose donor age were <25 years (70.8%) (p 0.04). Donor-vs-recipient NK cell alloreactivity exerts a specific graft-vs-AML effect in the absence of GVHD. In fact, leukemia relapse was largely controlled in myeloid leukemia recipients(AML or CML),with only 11 out of 59 relapsing. The probability of event-free survival is 65.6% in myeloid leukemia (AML and CML) and 37.8% in ALL patients. The probability of EFS was better in the AML relapse whose transplant included donor vs recipient NK cell alloreactivity than in those whose transplant did not.
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    G-CSF primed marrow rather than G-CSF-mobilized PBSCs was used to facilitate engraftment and to reduce mature donor T cells in the grafts. Further more, G-CSF reduced lymphocytes in the marrow and modulated subsets of T cells resulting in a significantly decreased CD4/CD8 ratio. It is known that cytokines produced by CD4+ and CD8+ T cells can be characterized into two patterns, Th1 (IL-2 and interferon-gamma) and Th2 responses (IL-4 and IL-10). Th1 type responses are critical for acute GVHD and treatment that induces the Th2 response reduces GVHD. G-CSF polarizes T cell differentiation from Th1 to Th2 type cells and induces Th2 responses with production of IL-4 and IL-10. Second, the TBI conditioning was well tolerated. TBI is administered to condition regimen demonstrating faster engraftment kinetics, especially speed platelet recovery. Third, GVHD prophylaxis with sequential immunosuppressants is crucial in haploidentical BMT, especially without ex-vivo T cell depletion. We chose CsA and MTX as a baseline and added different immunosuppressants at different stages of the transplant course with an emphasis on up front prevention.  ATG (Fresenius S,half life 2 weeks) was given on days –4 to –1 to suppress and/or deplete host lymphocytes to prevent HVGR as well as to suppress/deplete infused donor lymphocytes in vivo to prevent GVHD.  MMF, as a purine analog is a powerful inhibitor of lymphocyte proliferation.  We added MMF on day +7 to prevent rapid proliferation of donor lymphocytes at this stage. In current study we added a new immunosuppressant, anti-CD25 mAb Basiliximab, which binds specifically to IL-2 receptor-a (IL-2Ra) of activated T-cells with high affinity and is a potent inhibitor of IL-2 mediated T-cell activation and proliferation. Therefore, proper immune reconstitution after allogeneic HSCT becomes another crucial factor in determining risk of infection and disease relapse.  Our protocol did not use any ex-vivo donor T cell depletion. Immune reconstitutions were all 6-18 months faster than that reported by many other studies. Here, we present the 104 leukemia patients who received G-CSF primed HLA-haploidentical marrow transplants with improved GVHD prophylactic regiment.   The clinical results with high engraftment rate, low incidences of GVHD, and proper immune reconstitution suggest that this difficult goal is attainable. Given our current results, the full haplotype mismatched transplant should be in early stage disease, selection of an NK alloreactive and more young donors .

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