Engraftment of primates with G-CSF mobilized peripheral blood CD34+ progenitor cells expanded in G-CSF, SCF and MGDF decreases the duration and severity of neutropenia.

We used a primate model of autologous peripheral blood progenitor cell (PBPC) transplantation to study the effect of in vitro expansion on committed progenitor cell engraftment and marrow recovery after transplantation. Four groups of baboons were transplanted with enriched autologous CD34+ PBPC collected by apheresis after five days of G-CSF administration (100 microg/kg/day). Groups I and III were transplanted with cryopreserved CD34+ PBPC and Groups II and IV were transplanted with CD34+ PBPC that had been cultured for 10 days in Amgen-defined (serum free) medium and stimulated with G-CSF, megakaryocyte growth and development factor (MGDF), and stem cell factor each at 100 etag/ml. Group III and IV animals were administered G-CSF (100 microg/kg/day) and MGDF (25 microg/kg/day) after transplant, while animals in Groups I and II were not. For the cultured CD34+ PBPC from groups II and IV, the total cell numbers expanded 14.4 +/- 8.3 and 4.0 +/- 0.7-fold, respectively, and CFU-GM expanded 7.2 +/- 0.3 and 8.0 +/- 0.4-fold, respectively. All animals engrafted. If no growth factor support was given after transplant (Groups II and I), the recovery of WBC and platelet production after transplant was prolonged if cells had been cultured prior to transplant (Group II). Administration of post-transplant G-CSF and MGDF shortened the period of neutropenia (ANC < 500/microL) from 13 +/- 4 (Group I) to 10 +/- 4 (Group III) days for animals transplanted with non-expanded CD34+ PBPC. For animals transplanted with ex vivo-expanded CD34+ PBPC, post-transplant administration of G-CSF and MGDF shortened the duration of neutropenia from 14 +/- 2 (Group II) to 3 +/- 4 (Group IV) days. Recovery of platelet production was slower in all animals transplanted with expanded CD34+ PBPC regardless of post-transplant growth factor administration. Progenitor cells generated in vitro can contribute to early engraftment and mitigate neutropenia when growth factor support is administered post-transplant. Thrombocytopenia was not decreased despite evidence of expansion of megakaryocytes in cultured CD34+ populations.     

Recombinant human thrombopoietin augments mobilization of peripheral blood progenitor cells for autologous transplantation.

This study assessed the ability of various schedules of recombinant human thrombopoietin (rhTPO) to enhance mobilization of peripheral blood progenitor cells (PBPCs) in 134 patients with cancer undergoing high-dose chemotherapy and autologous PBPC transplantation. Patients received the study drug on days 1, 3, and 5 before initiation of granulocyte colony-stimulating factor (G-CSF) 10 microg/kg/day on day 5 and pheresis starting on day 9. Randomly assigned treatments on days 1, 3, and 5 were: group 1 (n=27) placebo, placebo, rhTPO 1.5 microg/kg; group 2 (n=27) rhTPO 1.5 microg/kg, placebo, placebo; groups 3 (n=28) and 4 (n=22) rhTPO 0.5 microg/kg on all 3 treatment days; and group 5 (n=30) placebo on all 3 treatment days. After high-dose chemotherapy and PBPC transplantation, groups 1 through 4 received rhTPO 1.5 microg/kg days 0, +2, +4, and +6 with either G-CSF 5 microg/kg/day (groups 1-3) or granulocyte-macrophage colony-stimulating factor 250 microg/m(2)/day (group 4). Group 5 received placebo plus G-CSF 5 microg/kg/day. The addition of rhTPO to G-CSF increased median CD34+ cell yield/pheresis in cohorts in which rhTPO was started before day 5, with higher yields in groups 2 (2.67 x 10(6)/kg) and groups 3 and 4 (3.10 x 10(6)/kg) than in group 1 (1.86 x 10(6)/kg) or group 5 (1.65 x 10(6)/kg) (P=.006 across groups). Comparing rhTPO to placebo, higher percentages of patients achieved the minimum yield of CD34+ > or =2 x 10(6)/kg (92% v 75%; P=.050) as well as the target yield of CD34+ > or =5 x 10(6)/kg (73% v 46%; P= .041). rhTPO-treated patients required fewer phereses to achieve minimum (P= .011) and target (P= .015) CD34+ cell values. rhTPO given after transplantation did not speed platelet recovery. No neutralizing antibodies were observed. We conclude that rhTPO can safely enhance mobilization of PBPC, reduce the number of leukapheresis, and allow more patients to meet minimal cell yield requirements to receive high-dose chemotherapy with PBPC transplantation.     

CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.     

Clinical applications of CD34(+) peripheral blood progenitor cells (PBPC)

Recently, a number of devices have been developed for the positive selection of CD34(+) peripheral blood progenitor cells (PBPC) for clinical use in autologous or allogeneic transplantation. The rationale for CD34(+) selection is based on clinical studies showing a two- to five-log reduction of contaminating tumor cells in patients with breast cancer, multiple myeloma and low-grade lymphoma. In addition, a three- to five-log reduction of T cells can be obtained by CD34(+) selection in both autologous grafts for patients with autoimmune disease resistant to conventional therapy and allogeneic grafts to reduce the incidence and severity of acute graft-versus-host disease. Transplantation of positively selected autologous CD34(+) PBPC results in a rapid and stable neutrophil and platelet engraftment in patients who received an infused dose of at least 2.0 x 10(6) CD34(+) cells/kg. Results from randomized trials suggest that time to engraftment is not different compared to unmanipulated PBPC autografts. However, close monitoring for infectious complications (e.g., cytomegalovirus disease) is required. Allogeneic CD34(+) PBPC have also been successfully transplanted and, using novel technologies, megadoses of purified CD34(+) PBPC can be obtained and used to overcome histocompatibility differences betweeen allogeneic donor and patient resulting in stable engraftment, even in a haploidentical setting. Additional randomized phase III trials are required to determine whether tumor cell purging or lymphocyte depletion by CD34(+) cell selection will have a significant impact on progression-free and overall survival in both autologous and allogeneic transplantation.     

Natural killer cells prime the responsiveness of autologous CD4+ T cells to CTLA4-Ig and interleukin-10 mediated inhibition in an allogeneic dendritic cell-mixed lymphocyte reaction

Cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig) and interleukin (IL)-10 are immunomodulatory molecules which target CD28 costimulation by acting either directly or indirectly on the CD80/86 receptors on dendritic cells (DCs). This study examined the effect of combined treatment with CTLA4-Ig and IL-10 on T-cell responsiveness in a dendritic cell-mixed lymphocyte reaction (DC-MLR). T cells derived from nylon wool enrichment (NWT cells) demonstrated 15% (P = 0.006) and 10% (P = 0.0015) inhibition of proliferation with suboptimal doses of IL-10 (5 ng/ml) and CTLA4-Ig (20 ng/ml), respectively. Combined treatment with both agents resulted in 38% inhibition (P = 0.004) of the MLR response compared with untreated controls. In contrast to NWT cells, which consisted of CD4+, CD8+ and CD56+ (NK) cells, purified CD4+ T cells were less responsive to immunomodulation by CTLA4-Ig and IL-10. Repletion of the CD4+ T cells with NK cells restored IL-10 and CTLA4-Ig mediated immunomodulation, suggesting a role for NK cells in the regulation of DC-T-cell interactions. The specific effect of NK cells on DC activation was demonstrated by CD80 up-regulation on DCs in the absence of T cells. However, in the absence of DCs, NK cells augmented the proliferation of autologous CD4+ T cells stimulated by anti-CD3 monoclonal antibody (mAb), which was blocked by CTLA4-Ig. It is proposed that, in the MLR, immunomodulation by suboptimal CTLA4-Ig and IL-10 is influenced by cellular interactions of NK cells with DCs and T cells involving DC lysis and costimulation. Thus, NK cells prime both DCs and T cells to low doses of CTLA4-Ig and IL-10 during alloimmune responses, providing evidence for the potential interaction between innate and adaptive immunity.     

G-CSF-mobilized CD34+ cells cultured in interleukin-2 and stem cell factor generate a phenotypically novel monocyte

To study the early stages of development from stem cells of the CD56+ cell population [which includes natural killer (NK) cells], granulocyte-colony stimulating factor-mobilized peripheral blood CD34+ cells from healthy donors were sorted to >99% purity and cultured in the presence of stem cell factor and interleukin (IL)-2. After 3 weeks in culture, the majority of cells acquired CD33, with or without human leukocyte antigen-DR and CD14. In 20 stem cell donors tested, 8.7 +/- 8.8% of cells were CD56+. Two major CD56+ subsets were identified: CD56(bright), mainly CD33- cells (7+/-10%, n=11) with large, granular lymphocyte morphology, and CD56dim, mainly CD33+ (2.5+/-2, n=11) cells with macrophage morphology. The CD56bright population had cytoplasmic granzyme A but lacked killer inhibitory receptor, suggesting they were immature NK cells. The CD56dim, CD33+, population lacked NK markers. They may represent a minor subset of normal monocytes at a developmental stage comparable with the rare CD56+ CD33+ hybrid myeloid/NK cell leukemia. Consistent with a monocyte nature, CD56dimCD33+ proliferated and produced a variety of cytokines upon lipopolysaccharide stimulation, including IL-8, IL-6, monocyte chemoattractant protein-1, and macrophage-derived chemokine but not interferon-gamma. In a short-term cytotoxicity assay, they failed to kill but powerfully inhibited the proliferation of the NK-resistant cell line P815. The generation of CD56+ cells was negatively regulated by hyaluronic acid and IL-4, indicating that extracellular matrix may play an important role in the commitment of CD34+ cells into CD56 myeloid and lymphoid lineages.     

Reconstitution of T-cell repertoire after autologous stem cell transplantation: influence of CD34 selection and cytomegalovirus infection.

The period of immunodeficiency following autologous hematopoietic stem cell transplantation is characterized by transient expansions of CD8+CD45RO+CD57+ T lymphocytes, displaying markers of an activated phenotype. Most evidence suggests that this early reconstitution results from proliferation of mature T cells that have survived conditioning or were transferred with the graft. Although homeostatic mechanisms are thought to act in maintaining total T-cell numbers, the degree to which antigen-driven expansions contribute and the nature of the stimulating antigens remain unclear. CD34 selection of stem cell grafts reduces the available T-cell pool, potentially delaying immune reconstitution and resulting in increased infective complications. In the allogeneic transplantation setting, lymphopenia has been associated with cytomegalovirus (CMV) infection risk and, if persistent, with adverse outcome. We prospectively studied patients undergoing CD34-selected (n = 13) or unselected (n = 13) autologous hematopoeitic stem cell transplantation for immune reconstitution and CMV infection. No significant differences were demonstrated between graft types with respect to lymphocyte subset recovery, T-cell receptor beta-chain variable region spectratype diversity, or CMV DNA detection rates (45% versus 40%). CMV infection was associated with a trend toward higher rather than lower CD8+ counts at 6 weeks posttransplantation (P =.08) that became significant by 3 months (P=.007), and that was associated with decreased T-cell receptor beta-chain variable region spectratype diversity (P =.01). CMV-specific HLA-tetramer analysis demonstrated transient expansions with CDR3 lengths corresponding to those of some of the major posttransplantation T-cell expansions demonstrated by spectratype analysis suggesting that CMV-specific T cells contribute to the pattern of immune reconstitution.     

Generation of antigen-presenting cells for soluble protein antigens ex vivo from peripheral blood CD34+ hematopoietic progenitor cells in cancer patients

Peripheral blood CD34⁺ hematopoietic progenitor cells (PBPC) mediate hematopoietic reconstitution in cancer patients after autologous transplantation and can be expanded ex vivo in the presence of colony-stimulating factors. This study shows that functionally active antigen-presenting cells (APC) for soluble proteins are generated and expanded in these PBPC cultures. CD34⁺ cells were cultured ex vivo in medium containing stem cell factor, interleukin-1β (IL-1β), IL-3, IL-6, and erythropoietin (EPO). The cells from these cultures developed into very potent APC of tetanus toxid and purified derivative of tuberculin for autologous T cells in vitro. The antigen-presenting capacity of these cells was maintained for at least 38 days of culture. These APC resembled immature cells of the myelomonocytic cell lineage by surface marker, immunocytochemistry and ultrastructural analysis. Such APC might be able to present antigens from certain tumors to the immune system.     

Phenotype analysis of tumour-infiltrating lymphocytes and lymphocytes in peripheral blood in patients with renal carcinoma

INTRODUCTION: When checking tumour growth, a number of observations indicate that the immune system plays a significant role in patients with renal cell carcinoma (RCC). Infiltration by lymphocytes (tumour infiltrating lymphocytes, TILs) is more prevalent in RCC than any other tumours. T lymphocytes are the dominant population of TIL cells. Views concerning the role of T lymphocytic subpopulations, B lymphocytes and NK cells in an anti-tumour response are not established. AIM: The aim is to determine the phenotype and activation of T and B lymphocytic subpopulations and NK cells and to compare their representation in tumour stroma and peripheral blood lymphocytes (PBL) in patients with RCC. MATERIAL AND METHODS: Samples of peripheral blood taken from the cubital and renal veins and tumour stroma cells were obtained from 44 patients in the course of their surgeries carried out due to primary RCC. TILs were isolated from mechanically disintegrated tumour tissue. Immunophenotype multiparametric analysis of PBL and TILs was carried out. Their surface and activation characteristics were determined by means of flow cytometer. RESULTS: CD3+ T lymphocytes (69.7%) were the main population of TILs. The number of CD3+/CD8+ T lymphocytes was significantly higher in TILs, 42.6% (p < 0.01), while CD4+ T lymphocytes were the majority population in peripheral blood, 41.35% (p < 0.001). The representation of CD3+/69+ T lymphocytes was significantly higher in TILs, 32.9%, compared to PBL (p < 0.001). On the contrary, the numbers of CD3+/CD25+, CD8+/57+ and CD4+/RA+ (naive CD4+ T lymphocytes) were higher in PBL (p < 0.001). The differences in representation of (CD3-/16+56+) NK cells and CD3+/DR+ T cells in TILs and PBL were not significant. CONCLUSION: The above-mentioned results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (tumour/PBL). CD3+/CD8+ T lymphocytes are the dominant lymphocytic population of TILs. The knowledge of the phenotype and functions of effector cells, which are responsible for anti-tumour response, are the basic precondition for understanding the anti-tumour immune response and the cause of its failure.     

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells.

Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to introduce foreign genes into human cord blood CD34+ cells and evaluate gene expression in CD34+/CD38(dim) and committed myeloid progenitors (CD33+, CD11b+). CD34+ cells were cultured in X-VIVO 10 supplemented with thrombopoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency and cell viability measured by flow cytometry using enhanced green fluorescent protein (EGFP) as a reporter indicated 31% +/- 2% EGFP+ /CD34+ efficiency and 77% +/- 3% viability as determined 48 hours post-electroporation. The addition of allogeneic cord blood plasma increased the efficiency to 44% +/- 5% with no effect on viability. Of the total CD34+ cells 48 hours post-electroporation, 20% were CD38(dim)/EGFP+. CD34+ cells exposed to interleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into CD33+ and CD11b+ cells, and 9% +/- 3% and 8% +/- 7% were expressing the reporter gene, respectively. We show that electroporation can be used to introduce foreign genes into early hematopoietic stem cells (CD34+/CD38(dim)), and that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33+, CD11b+) in response to corresponding cytokines. Further investigation is needed to determine the transgene expression in functional terminal cells derived from the genetically modified CD34+ cells, such as T cells and dendritic cells.     

Natural killer (NK) T cells are significantly decreased in the peripheral blood of patients with rheumatoid arthritis (RA).

The number of NK T cells was measured in relation to the Th1/Th2 imbalance observed in RA. Peripheral blood samples of patients with RA (n = 60) and healthy controls (n = 36) were stained with anti-NK receptor 1A (anti-NKR-P1A), anti-CD56, and anti-CD3 MoAbs, and examined by three-colour flow cytometry. NK T (NKR-P1A+CD3+) cells in the peripheral blood were decreased in RA compared with the controls: 25 +/- 20/microl versus 143 +/- 53/microl (P < 0.0001). CD56+CD3+ cells were also decreased in RA: 60 +/- 46/microl versus 116 +/- 54/microl (P < 0.0001). The decrease was significant when adjusted to the number of total lymphocytes (P < 0.0001) or NK (CD56+CD3-) cells (P < 0.0001), and showed no correlation with age, sex, disease duration, disease activity, functional class, x-ray stage, drug treatment, joint score, grip strength, C-reactive protein, rheumatoid factor or erythrocyte sedimentation rate of the patients. The results show that the levels of NK T cells are depressed in the peripheral blood of patients with RA, suggesting that the measurement of NK T cells in peripheral blood may have clinical importance for a Th1-type autoimmune disease like RA.     

Favorable treatment outcome in non-Hodgkin's lymphoma patients with "poor" mobilization of peripheral blood progenitor cells.

Our purpose was to evaluate the outcome and costs of high-dose chemotherapy and autologous peripheral blood progenitor cell (PBPC) transplantation in patients with the inability to mobilize sufficient numbers of PBPCs to allow rapid engraftment after PBPC transplantation. We treated 172 consecutive non-Hodgkin's lymphoma (NHL) patients with cyclophosphamide and granulocyte colony-stimulating factor followed by apheresis to collect PBPCs. The cells were separated on a Percoll gradient and purged with monoclonal antibodies and complement. The patients were categorized as "good" mobilizers if a collection of > or =2 x 10(6) CD34+ cells/kg was obtained (n = 138, 80%) or "poor" mobilizers if <2 x 10(6) CD34+ cells/kg were obtained (n = 34, 20%). With a median follow-up of 3.5 years, there is no statistically significant difference in actuarial event-free survival, overall survival, or relapse for good mobilizers compared with poor mobilizers. However, there was a trend toward increasing nonrelapse, transplantation-related mortality of 11.8% for poor mobilizers versus 3.6% for good mobilizers (P = .08) and early death from all causes including relapse within 120 days (poor 20.6% versus good 8.7%, P = .06). The total cost for bone marrow transplantation-related care was significantly higher, at $140,264 for poor mobilizers versus $80,833 for good mobilizers (P = .0001). The population of patients with NHL who mobilize PBPCs poorly into the circulation have a higher cost for posttransplant support. However, there is no significant difference in relapse, event-free survival, or overall survival for such patients compared with those who mobilize PBPCs easily.     

Mobilization kinetics of CD34(+) cells in association with modulation of CD44 and CD31 expression during continuous intravenous administration of G-CSF in normal donors

The aim of the present study is to evaluate the kinetics of CD34(+) cells and investigate the potential modulation of CD44 and CD31 expression on CD34(+) cells during continuous i.v. administration of G-CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G-CSF (10 microg/kg/day) was administered for four consecutive days through continuous 24-h i.v. infusion. For measurement of complete blood counts, CD34(+) cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G-CSF (steady-state) and after 4, 8, 24, 48, 72, 96, and 120 h of G-CSF administration. The percentage and absolute number of CD34(+) cells significantly increased at day 3 (0. 55 +/- 0.09%, 51.12 +/- 24.83 x 10(3)/ml) and day 4 (0.47 +/- 0.09%, 46.66 +/- 24.93 x 10(3)/ml), compared to the steady-state level (0. 06 +/- 0.09%, 2.03 +/- 5.69 x 10(3)/ml). At day 3 to day 5 following the onset of G-CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34(+) cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%-70% and 40%-90% lower than that of controls. We conclude that continuous i.v. administration of G-CSF apparently results in more rapid mobilization of CD34(+) cells, and downregulation of CD44 and CD31 on CD34(+) cells is likely to be involved in the mobilization of PBPC after treatment with G-CSF.     

Purification of human hemopoietic stem cells for transplantation in Thailand

Stem cell transplantation (SCT) has become the therapy of choice for many hematologic and immunologic disorders. At present, only 25% of patients have suitable HLA-identical donors. In an attempt to increase the donor pool for SCT in Thailand and Southeast Asia, we developed a program whereby parents and mismatched siblings can be used as donors. In this preliminary study, after granulocyte-colony-stimulating factor (G-CSF) was given to adult donors, peripheral blood stem cells (PBSC) were collected and CD34+ cells purified using a CliniMACS immunomagnetic device (Miltenyi Biotec, Germany). In seven experiments, purified CD34+ cells could be obtained from G-CSF-stimulated PBSC in large numbers (1.71 +/- 0.19 x 10(8)), with high purity (93 +/- 2.4%) and excellent recovery (64.28% - 85.62%). Immune reactive T and NK cells were adequately depleted to less than 0.2%. The purification procedure can be completed within 3 hours. In conclusion, a clinical stem cell purification program using this novel device is now established in Thailand and for the first time in Southeast Asia. This should allow further development of advanced SCT therapy including haploidentical and mismatched CD34+ SCT for patients' lacking HLA-identical donors in this region.     

Possible role of natural killer cells in pemphigus vulgaris - preliminary observations

Pemphigus vulgaris (PV) is an autoimmune blistering disease that affects the skin and multiple mucous membranes, and is caused by antibodies to desmoglein (Dsg) 1 and 3. Natural killer (NK) cells have a role in autoimmunity, but their role in PV is not known. NK cells in the peripheral blood leucocytes (PBL) of 15 untreated Caucasian patients with active PV were studied and compared with healthy controls for the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules. CD56+ CD16- CD3- NK or CD56+ CD16+ CD3- NK cells from the PBL of PV patients co-express MHC class II and co-stimulatory molecule B7-H3 without exogenous stimulation. CD4+ T cells from the PBL and perilesional skin of PV patients were co-cultured with CD56+ CD3- NK cells from the PBL of the same patients; in the presence of Dsg3 peptides underwent statistically significant proliferation, indicating that NK cells functioned as antigen-presenting cells. Supernatants from these co-cultures and serum of the same patients with active PV had statistically significantly elevated levels of interleukin (IL)-6, IL-8 and interferon-gamma, compared with controls indicating that the NK cells stimulated CD4+ T cells to produce proinflammatory cytokines. In these experiments, we present preliminary evidence that NK cells may play a role in the pathobiology of PV.     

NK cells become Ki-67+ in MLC and expand depending on the lack of ligand for KIR on stimulator cells in IL-2 supplemented MLC

Mixed lymphocyte culture (MLC) response was measured with the use of Ki-67 monoclonal antibody and responding cells were verified by CD3 and CD56 surface markers staining. Stimulator cells were discriminated from responder cells on the basis of forward and side scatter. Allogeneic, but not autologous MLC had Ki-67+ responder cells in lymphocyte gate at the end of the culture. In allogeneic MLC T cells and natural killer (NK) cells were in a similar proportion Ki-67+ (mean +/- SD: 59.25% +/- 9.72% versus 61.75% +/- 13.2%). Ki-67+ NK cells had higher CD56 mean fluorescence intensity than those lacking Ki-67 (745+/-357 versus 196+/-56 p < 0.0001). NK cells contribution to responding lymphocytes was positively correlated with the percentage of Ki67+ cells in NK population by the end of the culture (r = 0.74, p = 0.002). NK cells response in MLC increased upon supplementation of the culture medium with human recombintant interleukin-2 (IL-2). Responder cells from single individual were tested with 8 Bw4+ and 8 Bw4- as well as with 9 CNK1+ and 9 CNK1- stimulators. In IL-2 supplemented MLC killer inhibitory receptor expressing cells expanded when ligands for this receptor were absent in stimulating population. Consequently, stimulator cells lacking Bw4 promoted NKB1+ cells expansion (7.2% +/- 3% versus 3.6% +/- 1%, p = 0.0031), whereas HLA-C NK1 negative stimulators promoted CD158a+ cells expansion (9.6% +/- 4.8% versus 6% +/- 2.6%, p = 0.0385).     

Graves' disease is associated with an altered CXCR3 and CCR5 expression in thyroid-derived compared to peripheral blood lymphocytes

The mechanisms by which T cells accumulate in the thyroid and support the autoimmune process in patients with Graves' disease (GD) are poorly understood. Chemokines and their receptors may be involved in this process. We have analysed the expression of CXCR3 and CCR5 as Th1-specific chemokine receptors, CCR3 as a marker for Th2 cells, CXCR4 (expressed on unprimed, naive T cells) and CCR2 (known to be involved in autoimmunity) on peripheral blood (PBL) and thyroid-derived lymphocytes (TL) using flow cytometry. Chemokine receptor expression on PBL of GD patients (n = 16) did not differ from that of normal controls (n = 10). In GD, CXCR3+ (67.3 +/- 4.0% versus 45.7 +/- 2.1%) and CCR5+ T cells (42.5 +/- 3.4% versus 18.8 +/- 2.1%) showed a significant enrichment in the TL compared to PBL. The positive cells were contributed mainly by the CD4+CD45R0+ subset. TL are mostly primed CD45R0+ T cells, but surprisingly, they had significantly higher levels of CXCR4+ cells among TL (96.2 +/- 1.0%) compared to PBL (66.8 +/- 4.2%). However, CXCR4 has been induced during in vitro isolation of TL. There was no correlation between chemokine receptors and the level of TSH-receptor and thyroid peroxidase autoantibodies. CCR3+ and CCR2+ cells remained unchanged in TL compared to PBL. We could confirm the results using RT PCR and immunohistology. In summary, TL showed a different chemokine receptor pattern compared to PBL from the same patient. This indicates a role for CXCR3 and CCR5 in the recruitment of T cells to the thyroid in GD.