IL-10 promoter polymorphisms influence susceptibility to aGvHD and are associated with proportions of CD4+FoxP3+ lymphocytes in blood after hematopoietic stem cell transplantation.

Four hundred and ninety-five patients (390 and 105 grafted from unrelated and sibling (SIB) donors, respectively) and their donors were analyzed for the impact of interleukin-10 (IL-10) promoter genotype [rs18000896 (-1082 G/A), rs18000871 (-819 C/T) and rs18000872 (-592 C/A)] on the outcome of hematopoietic stem cell transplantation (HSCT). Patients having ACC haplotype were at a lower risk of acute graft versus host disease (aGvHD, grade > I) if transplanted from human leukocyte antigen (HLA) well-matched (10/10) unrelated donors (20/135 vs 39/117, P < 0.001, Pcorr = 0.002), which was not seen if patients were transplanted from either sibling (SIB) or poorly matched (<10/10) unrelated donors (MUD). In addition, GCC haplotype positive recipients of unrelated donor transplants tended to be more susceptible to aGvHD (68/199 vs 39/169, P = 0.019, Pcorr = 0.057). Multivariate logistic regression analysis in the MUD transplanted group showed that donor-recipient human leukocyte antigen (HLA) mismatch [odds ratio (OR) = 3.937, P = 0.001] and a lack of ACC haplotype in recipients (OR = 0.417, P = 0.013) played a significant role as independent risk factors of aGvHD grade > I. ACC carriers had higher proportions of FoxP3+ lymphocytes gated in CD4+ lymphocytes as compared with patients with other IL-10 haplotypes. It was seen at the time of hematological recovery (mean ± SEM: 3.80 ± 0.91% vs 2.06 ± 0.98%, P = 0.012) and 2 weeks later (5.32 ± 0.87% vs 2.50 ± 0.83%, P = 0.013); -592 C/A polymorphism was separately analyzed and it was found that AA homozygotes tended to have a higher incidence of aGvHD (8/15 vs 116/456, P = 0.034) and low proportions of FoxP3 CD4+ lymphocytes in blood (0.43 ± 0.22% vs 4.32 ± 0.71%, P = 0.051) measured 2 weeks after hematological recovery. Functional IL-10 polymorphism associated features influenced the risk of aGvHD with a positive effect of ACC on the pool of Treg in blood.

Association with the presence of naive T cells in chronic myeloid leukemia patients after allogeneic human stem cell transplantation and the lower incidence of chronic graft-versus host disease and relapse.

INTRODUCTION: Allotransplantation in chronic myeloid leukemia (CML) patients offers long-lasting remissions, which largely depend on immunologic surveillance of alloreactivity. Alloreactivity in CML patients has a durable potential. However a large proportion of relapsing patients, who have to undergo donor lymphocyte treatment is still abundant. METHODS: We studied a group of 31 CML patients post allogeneic transplantation for their level of T-cell receptor excision circles (TREC) and proportion of naive and memory/effector T cells in the peripheral blood (PB). TREC numbers were determined by quantitative PCR (qPCR) and T-cell subsets CD4(+)CD27(+)CD45RO(-), CD4(+)CD27(-)CD24RO(+), CD4(+)CCR7(+), and CD4(+)CCR7(-) by flow cytometry. Patients were analyzed for posttransplant chimerism, type of bcr-abl transcripts, and number of TREC in association with the presence of chronic graft-versus-host disease (cGVHD) and relapse. CML patients with TREC+ in PB had a higher proportion of CD4(+)CD27(+)CD45RO(-) cells (3.54 vs 2.45%; P = .105) and CD4(+)CCR7(+) cells (4.85 vs 2.67%; P = .007), and a lower proportion of CD4(+)CD27(-)CD45RO(+) cells (5.55 vs 9.09%; P = .037). The incidence of cGvHD was reduced among TREC+ CML patients (3/14 vs 11/17; P = .006). RESULTS: The 5 out of 31 CML patients who relapsed were characterized by the presence of b2a2, b3/a2 or both type of transcripts, a lack of TREC in the blood, and a lower proportion of naïve and effector/memory T cells. No association was observed between any of HLA specificities, type of bcr-abl transcripts and incidence of relapse. CONCLUSION: The presence of TREC is affected by chronic GvHD; TREC negativity may constitute a risk of mixed chimerism and relapse.

Low CXCR4 membrane expression on CD34(+) cells characterizes cells mobilized to blood.

SUMMARY: In this work, association between the presence and membrane density of CXCR4 and the effectiveness of mobilization was studied. Ninety G-CSF mobilized PBPC and 28 native BM (nBM) preparations obtained from healthy individuals for transplantation and BM obtained after G-CSF mobilized PBPC collection in 10 donors were investigated. Positivity for CD34, HLA-DR and CXCR4 were analysed in the three colour fluorescence. The cellular profile of PBPC differed from nBM preparations with respect to lower: (i) proportion of CD34(+) cells (0.64%+/-0.04 vs 0.92+/-0.07) and CD34(+)CXCR4(+) cells (0.30%+/-0.02 vs 0.61+/-0.07); (ii) contribution of CXCR4(+) cells to CD34(+) cells (52.2%+/-2.5 vs 62.2%+/-4.2); (iii) CXCR4 epitope density in CD34(+) cells (48.9+/-5.5 vs 94.7+/-10.4). PBPC yield for CD34(+) cells was correlated with the content of CD34(+) cells lacking CXCR4 in the leukapheresis product (R=0.38). In contrast, nBM harvested for transplantation was poor in CD34(+) cells if these cells were frequently CXCR4- (R=-0.49). The present study shows that CD34(+) cells mobilized to blood were characterized with a low proportion of CXCR4 and this associated with CD34(+) cell content in PBPC.

B-cell lymphoproliferative syndrome and peripheral blood CD20+ cells expansion after hematopoietic stem cell transplantation: association with fludarabine and anti-thymocyte globulin containing conditioning regimen.

Fifty-eight patients who received hematopoietic stem cell transplants during a 3-year period in our unit were followed for the symptoms of posttransplant B-cell lymphoproliferative syndrome (B-cell PTLD). Three cases showed lymph node enlargement; in 14, there was an excess of B cells in the blood. Histochemical staining of lymph nodes revealed CD20+ cell expansion in two cases, and in one, CD38+ and CD138+ cells. Kappa and lambda staining revealed poly- or oligoclonal expansion, which was characterized by the presence of Ki67+ cells in 10% to 50% of cells. In 14 cases, an excess of CD20+ cells were observed in blood. Clinical analysis revealed that patients with B-cell expansion in blood and/or in lymph nodes frequently showed fever and that some subjects displayed arthralgia, hemolytic anemia, and hepatitis. LMP-1-positive cells were observed in lymph nodes as well as EBV copies, whereas only a proportion of patients with the excessive CD20+ cells in blood were EBV positive. Notably, lymph node enlargement and CD20+ blood excess occurred significantly more frequently among patients receiving a Fludarabine (Flu) and anti-thymocyte globulin (ATG) conditioning regimen than those whose treatment lacked Flu independent of whether they received ATG (0.80 vs 0.44; P =.036).

Reactivations of cytomegalovirus, human herpes virus 6, and Epstein-Barr virus differ with respect to risk factors and clinical outcome after hematopoietic stem cell transplantation.

One hundred two recipients of hematopoietic stem cell transplants (HSCTs) 45, from siblings and 57 from matched unrelated donors, were followed for cytomegalovirus (CMV), human herpes virus (HHV) 6, and Epstein-Barr Virus (EBV) reactivation by quantitative polymerase chain reaction in the context of immunologic reconstitution and posttransplantation complications. CMV, EBV, and HHV6 DNA copies (>100 copies/10(5) cells) were detected in 34%, 27%, and 26% of patients, respectively. The presence of 100 copies of EBV or CMV was associated with posttransplant complications: 29/66 versus 6/36 (P<.01) or 24/66 versus 4/36 (P=.01). CMV reactivation was more frequent among patients with acute graft-versus-host disease grade≥I: 17/35 versus 18/67 (P<.05). Older patient age of adults>16 year (2/16 versus 33/86; P<.05) and, to a lesser extent, CMV IgG positivity before HSCT (34/84 versus 1/10; P=.08) or an HLA-mismatched graft (9/16 versus 26/86; P=.08) constituted risk factors for CMV reactivation, which resulted in a higher rate of bacterial pneumonia (7/11 versus 28/91; P=.04). EBV reactivation risk was associated with donor EBV IgG seropositivity (28/84 versus 0/10; P=.03) and donor female gender (18/47 versus 10/55; P=.03). In contrast to EBV and CMV, EBV reactivation itself was associated with encephalitis (5/8 versus 23/94; P=.013), which was also seen as a trend among HHV6 reactivations (8/8 versus 46/94; P=.08). Multivariate analysis demonstrated that these factors play independent roles in the reactivation of the investigated herpes viruses.

Interleukin-17-producing cells increase among CD4+ lymphocytes before overt manifestation of acute graft-versus-host disease.

Interleukin-17A is a hallmark of a subset of CD4+ lymphocytes called T(H)17. Allogeneic hematopoietic stem cell transplantation (HSCT) induces an immune response that facilitates graft acceptance, but if clinically apparent as acute graft-versus-host disease (aGvHD), it may adversely affect transplantation outcomes. TH17 cells are involved in the inflammatory processes associated with several diseases, including inflammatory bowel disease (IBD) as a prototype. In this study we investigated the presence of IL-17-producing cells among peripheral blood mononuclear cells (PBMC) of patients after HSCT. The 48 patients of median age 45 years (range, 1.0-64 years), experienced hematologic malignancies (n=45) or nonmalignant disorders (n=3), treated with matched unrelated (n=24) or sibling (n=24) transplants. We examined IL-17-producing cells in alloreactive reactions after HSCT. PBMC were stimulated with BD Leukocyte Activation Cocktail (Ionomycin, Brefeldin A, and phorbol myristic acetate (PMA)) in the presence of BD GolgiStop. After stimulation the cells were labeled with anti-CD4 and intracellular anti-IL-17A monoclonal antibodies. IL-17+ cell proportions were analyzed in the CD4+ lymphocyte gate. We observed that patients at the time of hematologic reconstitution had higher proportions of IL-17-producing cells than healthy control subjects (0.73±0.13 vs 0.19±0.06%; P=.019). Fourteen patients displayed the first symptoms of aGvHD at the time of hematologic reconstitution, when they showed lower proportions of IL-17+ cells among CD4+ lymphocytes than their counterparts lacking aGvHD at a similar time after transplantation (0.29±0.09 vs 0.73±0.13%; P=.024). Eight patients developed aGvHD after hematologic reconstitution (median, 34 days). All of these patients displayed lower proportions of IL-17-producing CD4+ cells on the day of aGvHD compared with their initial measurements preceding this complication (0.34±0.14 vs 1.07±0.37%; P=.01).

The CXCL12-3'A allele is associated with a higher mobilization yield of CD34 progenitors to the peripheral blood of healthy donors for allogeneic transplantation.

The interaction between CXCL12 and its receptor CXCR4 plays a crucial role in the homing and mobilization of haematopoietic progenitors. We investigated the putative association between a CXCL12 gene polymorphism, the G --> A transition at position 801 in the 3'-untranslated region (3'UTR), and the yield of CD34(+) progenitors in 65 healthy allogeneic transplant donors who received G-CSF. Importantly, in this setting, the analysis was not biased by background disease or chemotherapy. The 3'UTR CXCL12 G801A polymorphism was detected using a PCR-RFLP technique with the MspI restriction enzyme and the frequency of CD34(+) progenitors was assessed by flow cytometry. The frequency as well as the number of CD34(+) progenitor cells in the first leukapheresis product was significantly higher from donors with the CXCL12-3'A allele compared to GG homozygotes (P<0.05 in both cases), especially for subjects with the CXCL12-3'AA homozygous genotype (P<0.01 in both cases). Moreover, more leukaphereses were needed to obtain the required number of CD34(+) progenitors for transplantation from CXCL12-3'GG homozygous donors compared to the CXCL12-3'A carriers (P=0.003). In conclusion, the CXCL12-3'A allele was associated with a higher yield of CD34(+) cells from healthy donors of PBPC for allogeneic haematopoietic SCT.

G-CSF-mobilized leukapheresis products: cellular characteristics and clinical performance in allografting.

Twenty-five G-CSF-mobilized leukapheresis products (mLP) were screened for cellular composition, including CD34+DR-, CD34+DR+ and leukocyte profile, to compare with 5 native (unstimulated) LP (nLP) and 16 BM inoculi. G-CSF stimulation led to an increase in CD34+ cells and CD15+ cells but did not influence the lymphocyte content of mLP. Two groups of 14 and 16 patients were allografted with phenotypically defined mLP (1-4 mLP were used for each patient) and BM, respectively. mLP used for allografting had significantly more CD34+ cells, including CD34+DR- cells, monocytes, T cells, and B cells as compared with BM inoculi. Patients were followed for median observation time of 289 days and 409 days for the mLP (PBPC) and BM groups, respectively. The two groups were well matched in regard to age, sex, and stage of disease, with a slight prevalence of major blood group incompatibility (7 of 14 versus 3 of 16) and a lower donor/recipient weight ratio (0.8+/-0.2 vs 1.5+/-0.6, p = 0.002) in the PBPC group. Granulocyte and platelet recovery was faster in the PBPC group than in the BM group. The time of reaching 20,000/microl platelets but not 500/microl granulocytes correlated with the number of CD34+ cells in each inoculum. The survival curves of the PBPC and BM groups were similar, as was the incidence of acute GvHD (aGvHD). This was also valid for aplastic anemia cases (7 and 5 patients in the PBPC and BM group, respectively), who benefited from a high number of CD34+ grafted cells but did not experience aGvHD. Thus, mLP do not appear to elicit aGvHD with higher frequency than BM and may be preferable for hematotherapy.

Adjuvant spleen ultrafiltrate immunotherapy in unresectable advanced non small cell lung cancer.

The purpose of this study was to assess the possible benefit of adjuvant spleen ultrafiltrate immunotherapy in advanced lung cancer patients on chemotherapy. Twenty-six patients with inoperable non-small cell lung carcinoma were eligible to this study and randomised divided into two groups. The two groups received additionally to standard chemotherapy--three times once a month--placebo or spleen ultrafiltrate (Prosplen), respectively, given to the patients for 14 days beginning from the 7th day after each chemotherapy. To evaluate the effects of spleen ultrafiltrate, the time of haematological recovery and profile of peripheral blood lymphocytes and clinically number of days with fever and oncological response were documented. During the observation time patients receiving spleen ultrafiltrate had a higher number of leukocytes (p = 0.01) and higher counts (p = 0.03) and percentage of granulocytes (p < 0.001) including nadir values (p = 0.05) in blood. The positive effect was also seen in natural killer cells (p = 0.005) but not in T cells compartment. This could be of clinical significance because patients receiving spleen ultrafiltrate presented less frequently febrile episodes than patients in the placebo group (75 vs. 127 days with body temperature > 38 degrees C, p = 0.007) and had less frequently highly elevated serum C-reactive-protein levels (p = 0.02). Notably, 2 out of 12 patients receiving in addition to chemotherapy spleen ultrafiltrate and 7 out of 14 lacking this adjuvant treatment showed tumor progression during the treatment. Serum C3 levels were associated with progression of disease in both groups (p = 0.03). Overall, spleen ultrafiltrate receiving patients had lower serum C3 values than placebo receiving patients. Serum IgM levels were rather high in the placebo group (p = 0.033). Spleen ultrafiltrate is thought to benefit patients on palliative chemotherapy in advanced metastatic cancers.