Quality controls on cord blood unit contiguous segments: recommendation of the SFGM-TC.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) set up its fourth annual series of workshops which brought together practitioners from all of its member centers. These workshops took place in September 2013 in Lille. Literature and intra-laboratories studies suggest that attached segment is representative of cord blood unit (CBU). Nevertheless, some discrepancies have been observed when analyzing large data registries. To address these issues, we have listed recommendations to increase the standardization of segment processing and quality control (QC), information on units of measurement and specifications and action to be taken in case of out of specifications QC results on segment.

[Cord blood banking: from theory to an application]. / La collecte de sang placentaire: du concept à la réalité.

Cord blood units are now routinely used as an alternative source of haematopoietic stem cells from unrelated donors for allogeneic transplantation. In France, cord blood units are collected in a network of more than 70 maternity hospitals in relationship with 11 public cord blood banks part of the Réseau Français de Sang Placentaire. Unrelated cord blood unit donation is an altruistic act, anonymous and free. Donors are selected on medical criteria. Then, only cord blood unit containing more than 100 × 10(7) total nucleated cells and more than 1.8 × 10(6) CD34+ cells are cryopreserved according to Réseau Français de Sang Placentaire recommendations. Cord blood units qualification will be completed by viral and functional testings and the clinical outcome of the newborn child 6 weeks after the collection. Since the last 5 years, cord blood banking growing in France in order to enhance the French registry of volunteer donors by increasing both the number and diversity of the donors listed and make available cord blood banking for transplantation.

Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells.

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.

Soluble human leucocyte antigen-G molecules in peripheral blood haematopoietic stem cell transplantation: a specific role to prevent acute graft-versus-host disease and a link with regulatory T cells.

Haematopoietic stem cell transplantation is often complicated by the life-threatening graft-versus-host disease (GVHD) which consists of an allogeneic reaction of the graft cells against the host organs. The aim of this study was to investigate the putative involvement of soluble human leucocyte antigen (sHLA) class I molecules, and particularly sHLA-G molecules, in the occurrence and/or prevention of acute GVHD (aGVHD) in allogeneic peripheral blood stem cell (PSC) transplantation. Whole sHLA class I molecules seem to be involved in aGVHD pathogenesis because detection of a high concentration of these molecules in the first month post allograft is correlated with aGVHD occurrence. Conversely, a high level of sHLA-G molecules before and after allograft could indicate good prognosis in PSC allograft transplantation. sHLA-G molecules seem to be involved in aGVHD prevention, not only because they are enriched in plasma of patients without aGVHD, but also because: (i) a positive correlation has been found between sHLA-G level and CD4+ CD25+ CD152+ natural regulatory T cell (T(reg)) frequency in the blood of transplanted patients; and (ii) the presence of CD4+ CD25+ CD152+ natural T(reg) is correlated with increased sHLA-G expression in in vitro mixed leucocyte reaction cultures. Altogether, these results support the immunomodulatory function of sHLA-G molecules that might create a regulatory network together with the natural T(reg) to foster the induction of a tolerogenic environment and improve PSC transplantation favourable outcome.

Anti-idiotypic antibody and recombinant antigen vaccines in colorectal cancer patients.

The colorectal carcinoma (CRC)-associated GA733 antigen (also known as CO17-1A, KS1-4, KSA or EpCAM) has been the target of a phase II/III randomized trial of passive immunotherapy with monoclonal antibody CO17-1A and phase I active immunotherapy trials with polyclonal anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope on the antigen. The CO17-1A antigen was molecularly cloned and the extracellular domain expressed in baculovirus (BV) GA733-2E. Whereas, anti-idiotypic antibody mimics a single epitope on the antigen, BV GA733-2E expresses multiple potentially immunogenic epitopes. In animals, the immunogenicity of BV GA733-2E in aluminum hydroxide was superior to that of anti-idiotype in the same adjuvant. Here, we compared the immunogenicity of anti-idiotypic antibody and GA733-2E antigen in CRC patients. These studies indicate that the antigen is superior to the anti-idiotype antibody in inducing humoral and cellular immunity in CRC patients.

Immunogenicity of recombinant GA733-2E antigen (CO17-1A, EGP, KS1-4, KSA, Ep-CAM) in gastro-intestinal carcinoma patients.

Targeting the GA733 antigen (also known as CO17-1A, EGP, KS1-4, KSA, Ep-CAM) by monoclonal antibody CO17-1A or anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope has induced prolonged survival and specific immune responses to the antigen, respectively, in colorectal cancer (CRC) patients. In pre-clinical studies in mice and rabbits, recombinant baculovirus-derived GA733-2E antigen was superior to anti-idiotypic antibodies at modulating specific immune responses. Our aim was to evaluate the immunogenicity and potential toxicity of alum-precipitated GA733-2E in a phase I trial in patients with resected CRC or pancreatic cancer. Six patients with advanced pancreatic carcinoma and 6 with CRC Dukes' stage A, B or C received between 4 and 7 doses of alum-precipitated GA733-2E at 50, 200 or 800 microg/dose at monthly intervals. Antibody binding to GA733-2E or antigen-positive CRC cells was determined, as were antigen-specific proliferative, cytolytic T-lymphocyte and delayed-type hypersensitivity responses. Six of the 12 patients developed antigen-specific humoral immune responses after immunotherapy, and 8 developed cellular immune responses. The overall immune response rate, including patients with humoral and/or cellular immune responses, was 83%. Median overall survival of the CRC and pancreatic cancer patients was 39.8 and 11.2 months, respectively. Following 18 years of single-epitope targeting of the GA733 antigen, immunization of patients against multiple epitopes of the antigen frequently induces an immune response in the absence of significant toxicity, despite relatively widespread expression of this antigen on normal epithelial cells.

Anti-idiotypic antibody (ab2) vaccines: coupling of Ab2 BR3E4 to KLH increases humoral and/or cellular immune responses in animals and colorectal cancer patients.

The colorectal carcinoma (CRC)-associated CO17-1A/GA733 antigen (Ag) has been the target of a phase II/III randomized trial of passive immunotherapy with monoclonal antibody CO17-1A (Ab1), and phase I active immunotherapy trials with polyclonal anti-idiotypic antibodies (Ab2) mimicking the CO17-1A or GA733 epitope of the Ag. However, monoclonal rat Ab2 BR3E4 directed against Ab1 CO17-1A was superior to polyclonal Ab2 in inducing antigen-specific humoral and cellular immune responses in mice and rabbits. Various forms of Ab2 BR3E4, i.e., BR3E4-F(ab')2 precipitated with aluminum-hydroxide (alum), BR3E4-F(ab')2 coupled to KLH and precipitated or non-precipitated with alum, and BR3E4-IgG in alum or incomplete Freund's adjuvant were compared for their capacity to induce in rabbits anti-anti-idiotypic antibodies (Ab3) that specifically bind to the CO17-1A Ag. BR3E4-F(ab')2 coupled to KLH and precipitated with alum was shown to induce the highest Ab3 titers, followed by Ab2 BR3E4-IgG in alum. Therefore Ab2 BR3E4 as intact IgG (IgG group) or as F(ab')2 coupled to KLH (KLH group), was administered in a phase I trial to 45 patients with CRC, stage Dukes'D (UICC stage IV), with the goal to modulate patients' immune responses to their tumors. Fifteen of 23 patients in the IgG group developed Ab3 binding specifically to Ab2, and in four of these patients the Ab3 also specifically bound to Ag-positive CRC cells. Lymphoproliferative responses to Ab2 and/or GA733-2E Ag stimulation were observed in three of these patients. Eighteen of the 22 KLH group patients tested developed Ab3 and the Ab3 bound specifically to CRC cells in eight patients. Five of the 15 KLH group patients tested developed lymphoproliferative responses to Ab2 and/or GA733-2E Ag. Thus, there was a trend for the KLH group demonstrating higher immune response rates than the IgG group. Clinical responses were rare in these patients with liver metastases.

Colorectal cancer vaccines: antiidiotypic antibody, recombinant protein, and viral vector.

The colorectal cancer antigen GA733 (also termed CO17-1A, KSI-4, Ep-CAM, KSA) has proved to be a useful target in passive immunotherapy with monoclonal antibody and in active immunotherapy with antiidiotypic antibodies in cancer patients. The GA733 antigen was molecularly cloned and expressed in baculovirus (BV), adenovirus (AV), and vaccinia virus (VV). Recombinant BV-, VV-, and AV-GA733 induced antigen-specific cytotoxic antibodies and proliferative and delayed-type hypersensitive lymphocytes. However, only the AV recombinant induced antigen-specific cytolytic T lymphocytes and regression of established tumors. Cured mice were protected against challenge with antigen-negative tumors, indicating antigen spreading of immune responses. In a model of active immunotherapy against the murine homologue of the human GA733 antigen, murine epithelial glycoprotein (mEGP), BV-derived mEGP protein in various adjuvants did not protect mice against a challenge with mEGP-positive tumors. AV mEGP, only when combined with interleukin-2, significantly inhibited growth of established mEGP-positive tumors. This is in contrast to the same vaccine expressing the human antigen that was effective without interleukin-2. AV GA733, in combination with interleukin-2, is a candidate vaccine for colorectal cancer patients.

Advances in cancer vaccine development.

Traditionally, cancer vaccines have used whole tumour cells administered in adjuvant or infected with viruses to increase the immunogenicity of the cells. With the identification of tumour-associated and tumour-specific antigens (TAA, TSA), antigen and epitope-specific vaccines have been designed. Compared to tumour cell vaccines, antigen and epitope vaccines are more specific and easier to produce in large quantities but may display lower immunogenicity and lead to the in vivo selection of antigen or epitope-negative escape tumour variant cells. The optimal vaccine will elicit both humoral and cellular immunity in the patients as both parameters have been positively correlated with the induction of beneficial clinical responses. The choice of adjuvant, costimulation and delivery mode greatly determines the outcome of vaccinations and may favour the induction of T-cell responses of T helper (Th)1, Th2, or both Th1 and Th2 types. Animal models of TAA vaccines must take into account the normal tissue expression of TAA, which may induce immunological tolerance to TAA. With the identification of homologues of human TAA in animals, novel experimental models of cancer vaccines which mimic the condition in patients are now available. Several vaccines comprising tumour cells, TAA or anti-idiotypic antibodies mimicking TAA have recently entered phase III of clinical evaluation.

DRB1*15 and DRB1*03 extended haplotype interaction in primary Sjögren's syndrome genetic susceptibility.

OBJECTIVE: Sjögren's syndrome (SS) is a chronic autoimmune disease with a genetic component. Among the genetic factors, the role of HLA class II genes has been suggested and a positive association with DRB1*03 allele has been described. However, there is no consensus on a unique HLA locus for this disease nor on the role of the HLA gene product in the disease. The aim of this study was to analyse prospectively MHC region involvement in the genetic susceptibility to SS by studying DRB1, DQB1, DPB1, TAP1, TAP2 genes and TNF microsatellites in a population of 45 primary SS patients. METHODS: All the polymorphisms studied were analysed at the genomic level using PCR-based methodologies. RESULTS: Concerning HLA class II alleles, the highest relative risk to develop the disease was associated with the DRB1*15-DRB1*0301 heterozygous genotype (17.8% vs 3.5% in controls - pc < 0.005, OR = 5.96). Analysing other genes located on the same region allowed us to further determine the DRB1 haplotypes at risk. For instance, the DRB1*0301 haplotype involved in the genetic susceptibility to SS was more often associated with the DPB1* 0201 and TNF-a2 alleles in SS patients than in controls. Moreover, all the DRB1*15-DRB1*0301 SS patients were TAP1-0101, TAP2-0101 homozygous, allowing us to deduce the extended genotype at risk as DRB1*15, TAP1-0101, TAP2-0101/DRB1*0301, TAP1-0101, TAP2-0101 which was carried by only 3 controls out of the 130 tested (p < 0.01, OR = 6.68). CONCLUSION: This study confirmed the role of the MHC region in the susceptibility to Sjögren's disease, and for the first time suggests a synergistic interaction between two HLA-DRB1 extended haplotypes in the genetic mechanisms controlling the disease.

TCR repertoire and cytokine profiles of cerebrospinal fluid- and peripheral blood-derived T lymphocytes from patients with multiple sclerosis.

The cerebrospinal fluid (CSF) represents an important source of T lymphocytes that could be involved in the inflammatory response occurring in the central nervous system in multiple sclerosis (MS). In order to investigate whether the Vbeta gene usage of CSF T lymphocytes is restricted, we analyzed the TCR Vbeta expression in twelve CSF expanded by in vitro culture compared to the paired in vitro-stimulated peripheral blood T lymphocytes. The overexpression of one or two Vbeta genes was demonstrated in ten CSF, but the type of Vbeta over expressed varied from one patient to another. For one patient, the Vbeta repertoire was also investigated by single cell cloning. High frequency of BV6S7-expressing T cell clones was observed in the CSF while no BV6S7 clone was derived from the peripheral blood T lymphocytes suggesting that these cells could be involved in the immunopathological process in the central nervous system (CNS). The cytokine patterns of the T cell clones derived from the CSF- and peripheral blood-T lymphocytes of this patient were determined. The CSF T cell clones produced higher levels of cytokines than the peripheral blood T cell clones. The high frequency of IL-4-producing-T cell clones observed in CSF demonstrate that T cells which could downregulate the inflammatory process are present in the CNS.

[1st successful immunization of pancreas and colorectal carcinoma patients with CO17-1A-vaccine]. / Erste erfolgreiche Immunisierung von Pankreas- und kolorektalen Karzinompatienten mit CO17-1A-Vakzin.

The baculovirus-derived extracellular domain of the CO17-1A antigen induced cellular and/or humoral immune responses in curatively resected colorectal and pancreatic cancer patients after at least three applications of the vaccine. The vaccine application was safe and non-toxic. The clinical efficacy of the vaccine needs to be evaluated in larger, randomized clinical trials.

TAP2 gene polymorphism contributes to genetic susceptibility to multiple sclerosis.

MS is an autoimmune demyelinating disease that has been known to be associated with the HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype. TAP1 and TAP2, two genes encoded within the MHC class II region between HLA-DP and -DQ loci, display genetic variability and are involved in the transport of antigenic peptides from the cytoplasm to the endoplasmic reticulum. Comparison of 116 MS patients with Caucasoid controls did not reveal any significant correlation between the previously described alleles of the TAP1 and TAP2 genes and MS. We report here an additional TAP2 dimorphism at codon 386, called I and J, corresponding to a silent mutation. An increased frequency of the J variant was observed in the patient population. The J mutation was not found in linkage disequilibrium with the HLA-DRB1*1501 allele and can be considered an additional genetic susceptibility marker of the disease.

In vitro cytotoxic effect of guinea-pig natural killer cells (Kurloff cells) on homologous leukemic cells (L2C).

The Kurloff cell (KC) of the guinea-pig develops natural killer cytotoxic activity in heterologous systems. We report in this paper the effective in vitro cytotoxic activity of the KC in a homologous guinea-pig system, i.e. against the guinea-pig target leukemic L2C cells. A dual-color flow analysis of homologous effector-target conjugates, using calcein-labeled KC and hydroethidine-labeled L2C shows a 40% frequency KC-L2C conjugation. The specific cytotoxicity of KC against L2C (78%) was estimated as the target-loss of green fluorescence due to hydrolysed carboxy-fluorescein diacetate after 4 hours at 37 degrees C. We propose that the Kurloff cell could be involved in surveillance against spontaneously arising leukemic cells, and this could be an explanation for the high degree of resistance to spontaneous or experimentally-induced cancers, in the guinea-pig.