ALBERT

Description: CD4+CD25+ regulatory T cells (Treg) have been hypothesized to control the development and progression of autoimmunity by suppressing autoreactive T cells. Treg are characterized by constitutive expression of membrane CD25 and intracellular expression of the transcription factor forkhead box (FOX) P3. FOXP3 and NF-AT1 have key roles in regulatory T-cell development and function: induction of FOXP3 in naïve T cells by gene transfer results in gain of a regulatory phenotype, and NF-AT1 modulates transcription of FOXP3. Treg display their suppressive properties on CD4+ CD25- T cells when activated via the T cell receptor or Toll-like receptor-2 (TLR2). Decreased numbers of FOXP3-positive regulatory Tregs have been associated with impaired immune homeostasis. Treg numbers are deficient in patients with active systemic lupus erythematosus, GVHD, and autoimmune hepatitis; in multiple sclerosis, decreased FOXP3 impairs Treg function. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by immune-mediated destruction of hematopoietic stem cells. To examine expression of CD4+CD25+ T-cells in this disease, peripheral blood mononuclear cells from AA patients, sampled at diagnosis and prior to immunosuppressive therapy (n=18), were examined by flow cytometry: CD4+CD25hi+ T-cells were markedly reduced or absent in patients compared to healthy controls (n = 12, 0.08± 0.01 vs 0.4± 0.1%, p=0.001). CD4+CD25hi+FOXP3+ T-cells were also significantly lower in patients (0.05± 0.01 vs 0.32± 0.1, p=0.001). By immunoblot, we observed significantly decreased and often undetectable FOXP3 protein levels in CD4+CD25+ T cells from patients (n=8) compared to controls (n=6). Low FOXP3 protein levels correlated with decreased FOXP3 mRNA levels, as measured by RT-PCR and quantitative real-time PCR experiments (n=5, p=0.03). There were no differences in TLR2 in immunoblots between patients and healthy controls despite differences in FOXP3 expression. Patients (n=8) with low FOXP3 levels also showed decreased or absent NF-AT1 protein levels. These data collectively implicate a transcriptional mechanism for FOXP3 down-regulation. In confocal microscopy, purified CD4+CD25+ T cells from patients (n=5) showed undetectable NF-AT1 and FOXP3 levels as compared to controls (n=3). Four patients studied 3–6 months after first sampling and post-immunosuppressive treatment showed increased Tregs and FOXP3 expression; four further patients in complete remission after immunosuppressive treatment had increased CD4+CD25hi+ T-cells as compared to treatment-naïve patients. CD4+CD25+FOXP3+ regulatory T-cells are decreased in most patients with AA, probably through transcriptional regulation; decreased NFAT1 could explain low FOXP3 expression and Treg frequency. Treg employed as cellular therapy in a murine model of immune-mediated AA can prevent T cell-mediated marrow destruction (Chen J and Young NS, unpublished data). Treg defects are now implicated in autoimmune marrow failure in aplastic anemia.

Description: The primary granule proteins (PGP) neutrophil elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1 which can induce cytotoxic T lymphocyte (CTL) responses in chronic myeloid leukemia (CML). The relative contribution of PR3 and ELA2 to PR1 expression is not known. We previously found that higher levels of PR3 and ELA2 gene expression in CD34+ progenitor cells were associated with longer survival in CML patients. Eradication of leukemia depends on the elimination of leukemic stem cells which are thought to reside within the CD34+ progenitor cell pool. We therefore studied PGP expression and T cell response to PR1 in 23 CML patients and their HLA-identical family donors prior to T-cell depleted allogeneic stem cell transplantation (SCT) with T-cell add-back on day 45–100 post-SCT. CD8+ T cells and CD34+ progenitor cells were purified from mononuclear cells (MNC) of cryopreserved leukapheresis products from HLA-A*0201+ CML patients. Following reverse transcription of RNA from MNC and CD34+ cells, ELA2 and PR3 gene expression was measured using real-time quantitative polymerase chain reaction (RQ-PCR). To assess PR1-CTL responses, T2 cells were loaded with PR1 peptide (VLQELNVTV) at 0.1, 1, and 10μM for 2h, irradiated and subsequently co-cultured with CD8+ T cells for 3h. PR1-CTL response was measured as interferon-γ mRNA expression (relative to CD8) obtained by RQ-PCR. PR1-specific CTL responses were detected in 5/23 CML patients and 6/16 HLA-identical donors. In CML patients, pre-SCT expression of both PR3 and ELA2 in MNC was strongly correlated with the expression in CD34+ cells (p=0.009 and p=0.0006 respectively). There was an inverse relationship between PR1-CTL response in CML patients pre-SCT and PR3 or ELA2 expression (p=0.02 and p=0.01 respectively). This data suggest that both PR3 and ELA2 expression in CD34+ cells and their progeny are a potential source of PR1. However, high expression of these proteins may result in selective deletion of PR1 T cell clones. The presence of PR1-responses in HLA-identical donors was associated with an improved overall survival post-SCT. However, there was no impact of PR1-response in either CML patients pre-SCT or donors, on time to achieve molecular remission post-SCT but a greater proportion of patients whose donors did not have a PR1-response succumbed to fatal graft-versus-host disease. These findings support post-transplant vaccination strategies with PR1 peptide to eradicate minimal residual disease but in patients who are not eligible for SCT, the lower PR1 response in the presence of high PGP expression suggests that there is a risk that vaccines given to patients with minimal residual disease could induce tolerance. Figure Figure

Description: Allogeneic HSCT is a curative therapy for leukemia and other hematologic malignancies and disorders. Acute Graft-versus-Host Disease (aGvHD) is a significant cause of morbidity and mortality that limits the success of HSCT. No large analysis of this complication has been recently performed. Risk factors for aGvHD after HLA-matched sibling myeloablative unmanipulated HSCT were analyzed in 1960 adult (≥18 yrs) patients treated for AML (n=761), ALL (n=303), or CML (n=896) and reported to the CIBMTR registry from 1995–2002 by 226 centers worldwide. All patients received cyclosporine+methotrexate (CSA+MTX) alone (85%) or CSA+MTX+other agents (15%) for aGvHD prophylaxis. 635 (32%) patients developed grade II-IV aGvHD before day +100 post–HSCT. Outcome was measured as time from HSCT to onset of aGvHD Grade II-IV with death as a competing risk. Statistically significant risk factors for aGvHD in the univariate analysis were: Age ≥ 40 vs 〈 40 at HSCT, RR (95% CI, P) =1.35 (1.16–1.58, P=0.0001); Race, White/Black vs. Asian/Hispanic, RR=1.65 (1.31–2.08, P 0.05 to remove each clinical factor from the model. Significant independent predictors of aGvHD Grade II-IV risk were: Conditioning Regimen, CyTBI vs BuCy, RR (95% CI, P) RR=1.4, (1.2–1.7, P 80, RR=1.3 (1.05–1.5, P=0.014) and recipient/donor CMV status, at least one + vs −/ −, RR=0.8 (0.7–0.99, P=0.04). For pts ≥ 40 yrs at BMT, PB was not an independent risk factor for aGvHD Grade II-IV. There are modifiable risk factors for aGvHD Grade II-IV which include conditioning regimen, stem cell source (in younger patients) and CMV negative donors for CMV negative recipients. There are non-modifiable risk factors such as recipient age, race and gender and underlying diagnosis. Identifying patients at high risk for aGvHD Grade II-IV may allow for individualized risk modification and result in altering treatment strategies.

Description: NK cell alloreactivity in HLA-identical sibling stem cell transplantation (SCT) is not well understood. We previously showed that higher (greater than the median of 150/ ul) NK30 (absolute NK count on day 30 post transplant in recipient) correlated with a particular “favorable” KIR combination (2DL5A, 2DS1, 3DS1) in the donor. Both high NK30 and “favorable” KIR correlated with improved transplant outcome after T-depleted myeloablative sibling donor SCT for myeloid malignancies. We therefore studied the readout of NK cell subset ratio, CD107a degranulation level and different KIR receptor expression level within each NK subset among these patients. Cryopreserved mononuclear cell from 16 pairs of donor pre-transplant sample with his/her corresponding recipient day 30 post-transplant sample were thawed, recovered overnight and incubated with K562 cells at an E:T ratio of 5:1 togather with CD107a-FITC. The incubation product was stained with multicolor fluorochrome antibody cocktails. Events were acquired on an LSR-II flow cytometer. Data was analysed by Flowjo software and Student t-test was performed on those readouts previously mentioned within donor and recipient samples, each is further grouped by high versus low NK30, favorable vs unfavorable KIR and mild (grade 0, 1) versus severe (grade 2 and above) acute GVHD (aGVHD). In recipient day 30 post-transplant samples, the high NK30 group had significantly higher CD56+CD16+ NK subset ratio (p

Description: The advent of imatinib, and subsequently, other tyrosine kinase inhibitors (TKIs) has relegated allogeneic stem cell transplantation (SCT) to second-or third-line therapy for chronic phase-chronic myeloid leukemia (CML). A significant shortcoming of TKIs in the large majority of good-responder patients is the persistent detection of BCR-ABL transcripts despite achievement of complete cytogenetic response (CCyR) or major molecular response (greater than 3-log reduction of BCR-ABL levels below a standardized baseline in minimal residual disease (MRD) measurements from total mononuclear cells). The presence of this MRD, and the demonstration that primitive CD34+ cells from patients in CCyR still harbor BCR-ABL (Bhatia, et al, Blood 2003) strengthens the opinion that TKIs do not eradicate all CML cells. Conversely, SCT has apparently cured some CML patients with more than 20 years follow-up. Of note, MRD is also quite frequently found in CML patients more than 5 years post-SCT, and donor lymphocyte infusions (DLI) are usually given to patients who satisfy criteria for molecular relapse. We compared the level of BCR-ABL transcripts in primitive hematopoietic cells from patients who were treated with T-depleted SCT with scheduled T-cell addback from D+45 to D+100 post-SCT (n=34) with levels in those taking TKIs (n=4). CD34+ progenitor cells were isolated from leukapheresis collections at D+120 post-SCT (n=13), peripheral blood from D+60 – 66 months post-SCT (n=19), bone marrow aspirates in patients on long-term follow-up (4–10 years) post-SCT (n=8) or 15 – 18 months post-TKI treatment alone (n=4); serial post-SCT samples were available in 6 patients. Using fluorescence activated cell sorting, hematopoietic stem cells (HSC, CD34+CD38-Lin-CD90+), common myeloid progenitors (CMP, CD34+CD38+Lin-IL3Rα+CD45RA-) and granulocyte-macrophage progenitors (GMP, CD34+CD38+Lin-IL3Rα+CD45RA+) were collected. BCR-ABL expression in primitive CD34+ subpopulations and total leukocytes from peripheral blood (PB) was measured using real-time quantitative polymerase chain reaction, with the sensitivity to detect one BCR-ABL-positive cell in 106 normal cells. A median of 112 x 106 mononuclear cells (range 16 – 582 x 106) were available per patient sample, with a median of 3 x 106 CD34+ cells (range 1 – 90 x 106) sorted. There was no difference between the number of HSC, CMP or GMP CD34+ cells collected between MRD negative or MRD positive-post-SCT patients, or TKI-treated patients. All patients with negative MRD in PB (n=12 post-SCT, n=1 post-TKI) did not have detectable BCR-ABL transcripts in primitive CD34+ subpopulations. Furthermore, in PB MRD-positive patients, 3/21 (14%) post-SCT and 2/2 (100%) post-TKI did not have detectable BCR-ABL transcripts in HSC, CMP or GMP populations. 18/21 (86%) PB MRD-positive patients who were post-SCT had detectable BCR-ABL transcripts in at least one primitive CD34+ subpopulation. A rise in BCR-ABL levels in GMP populations tended to herald impending relapse. In post-SCT patients on long-term follow-up with persistent PB MRD positivity not fulfilling criteria for molecular relapse, the highest BCR-ABL levels were in HSC populations. In comparable patients with a major molecular response, post-SCT patients appeared to harbor a greater amount of residual leukemia cells in CD34+ subpopulations than TKI-treated patients. Our observations suggest that although SCT is a curative treatment for CML, the graft-versus-leukemia effect may eliminate only more mature leukemic CD34+ subpopulations in some patients who have enduring positive MRD post-SCT, with persistence of the most primitive leukemic HSCs, which are presumably constrained in patients who do not fulfill criteria for relapse. Conversely, TKI appears to reduce the number of BCR-ABL-positive primitive CD34+ subpopulations, especially GMPs and CMPs, more efficiently. Our data support TKI-treatment as an adjunct to DLI to treat CML relapse post-SCT, and concurrent vaccination strategies which are able to target surface proteins on HSC to eradicate disease.

Description: CMV reactivation after allogeneic stem cell transplantation remains a major cause of post-transplant morbidity but may be mitigated by strong memory T cell responses in the transplanted donor T cell repertoire. To induce CMV specific T cell responses in CMV seronegative donors and to boost CMV reactivity in seropositive stem cell donors we vaccinated donors and healthy volunteers with the ALVAC-pp65 live attenuated canarypox vaccine (sanofi pasteur, Lyon, France). A safety study in 4 normal donors confirmed that apart from variable local reactions, an accelerated vaccine schedule was well tolerated. We then studied a similar accelerated regimen giving 1.0 ml intramuscular ALVAC on days 0, 5, and 10 and measured CD4+ and CD8+ T-cell responses to a CMV pp65 peptide library using flow cytometry to measure the frequency of interferon-gamma producing cells. Blood was drawn for testing on days 0, 5, 10, 30, 60, and 90. Positive responses were defined as a two fold increase of interferon-gamma production compared to unstimulated lymphocytes. Four seropositive and eight seronegative individuals were studied. Of the 8 seronegative individuals, 4 were found to have pre-vaccine CD4+ and CD8+ responses to pp65 and were considered to be CMV-experienced individuals. Three of these 4 individuals had a CD4+ and CD8+ response exceeding baseline at some time between days 30 and 90. All 4 CMV-naïve individuals had responses to ALVAC-pp65 by day 30. Of the four seropositive individuals, 3 lacked detectable CD4+ and 2 lacked CD8+ T cell responses at baseline. The 2 subjects that lacked both CD4+ and CD8+ responses at baseline had positive responses with vaccination. To explore the possiblility that immediate early responses might be rapid and transient in CMV-exposed individuals, three donors (one seronegative) were monitored daily during the first 10 days of the vaccine series. All showed significant responses 1–4 days after second vaccination (see figure). Ten patients (8 with hematological malignancies) were recipients of HLA-matched stem cell transplants from vaccinated donors. Four reactivated CMV, two were recipients of T cell depleted transplants and no patient had CMV disease. These results show that ALVAC-pp65 is a well-tolerated vaccine with the potential to induce pp65 cellular immunity in CMV-naïve individuals and boost responses in CMV-experience individuals. However, the kinetics of the CMV response differs according to whether the T cell response exists at baseline but interestingly does not correlate with serostatus. To deliver a high frequency of pp65 specific T cells to transplant recipients, the optimum schedule for CMV exposed donors appears to be after second vaccination. In contrast, longer periods are required in CMV-naïve donors. Based on these findings, we will use appropriately-timed cell collections from ALVAC-pp65 vaccinated donors to explore whether CMV-exposed transplant recipients can be protected from CMV reactivation. Figure Figure