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Background & Aim Mesenchymal Stromal Cells (MSCs) are involved in several clinical trials in the field of Advanced Therapeutic Medicinal Products (ATMPs) for their properties and therapeutic potential. However, the production of MSCs is currently done in multi-plates culture chambers (CellSTACK, Corning) that only allow the production of a limited number of cells and it's time consuming. This therefore represents a constraint, especially for allogeneic uses. Now, the major challenge is to increase this production capacity. The use of a bioreactor would overcome this problem. The Xpansion® bioreactor (Pall) is composed of a controller and single-use bioreactors. Similar in concept to CellSTACK, the Xpansion bioreactors are composed of stacked cell culture plates that are sold in four variations: 10, 50, 100, and 200. Although the plastic of the bioreactor plates is identical to those of the CellSTACK, the feasibility and equivalence of quality and expansion of the MSCs produced using the Xpansion bioreactor had to be demonstrated. Methods, Results & Conclusion Preliminary tests were carried out on the smallest model of the Xpansion bioreactor product line: The XP10 (10 culture plates). All experiments were performed in parallel with a CellSTACK manipulation. The tests were performed using MSCs at passage 0 (from bone marrow or adipose tissue) or raw materials. Quantitative and qualitative tests were carried out at the end of the culture. From raw material or MSCs, cell densities obtained after 6 days of culture are equivalent in both the Xpansion bioreactor and CellSTACK, demonstrating similar cell growth. The average cell expansion rates in the Xpansion (XP10) bioreactor and CellSTACK are 24.39 times and 21.69 times (n=3), respectively. The flow cytometry analysis showed that the phenotype and viability of the MSCs from the bioreactor were not altered by this culture method. Preliminary tests on the Xpansion bioreactor have demonstrated the feasibility of MSCs cell expansion without affecting the quality of the finished product compared to the current method in CellSTACK. In addition, using a bioreactor allows the production of cells in large quantities by managing only one culture chamber while reducing the risk of contamination and operation time. Additionally, using a bioreactor improves monitoring of culture parameters and scalability. Manufacturing MSCs in this bioreactor would make the graft of a larger number of patients from the same initial collection possible.

Evidence from animal models replicating postradical prostatectomy erectile dysfunction (pRP-ED) suggests intracavernous injection of bone marrow-mononuclear cells (BM-MNCs) as a promising treatment approach for pRP-ED. We conducted a phase 1/2 pilot clinical trial of intracavernous autologous BM-MNC injection to treat pRP-ED (NCT01089387). Twelve patients with localized prostate cancer and vasculogenic pRP-ED refractory to maximal medical treatment were divided into four equal groups treated with escalating BM-MNC doses (2×10 7 , 2×10 8 , 1×10 9 , 2×10 9 ). Tolerance was the primary endpoint. Secondary endpoints were the effects on erectile function and penile vascularization at 6 mo, as assessed using the International Index of Erectile Function-15 and Erection Hardness Scale questionnaires, and color duplex Doppler ultrasound. We measured the peak systolic velocity in cavernous arteries and assessed endothelial function using the penile nitric oxide release test. No serious side effects occurred. At 6 mo versus baseline, significant improvements of intercourse satisfaction (6.8±3.6, 3.9±2.5, p =0.044) and erectile function (17.4±8.9, 7.3±4.5, p =0.006) domains of the International Index of Erectile Function-15 and Erection Hardness Scale (2.6±1.1, 1.3±0.8, p =0.008) were observed in the total population. Spontaneous erections showed significantly greater improvement with the higher doses. Clinical benefits were associated with improvement of peak systolic velocity and of % penile nitric oxide release test and sustained after 1 yr. Our results need to be confirmed by phase 2 clinical trials. Patient summary We report a phase 1/2 pilot clinical trial investigating cell therapy with injection of bone marrow mononucleated cells to treat postradical prostatectomy erectile dysfunction. No serious side effects occurred. Improvements of erectile function and penile vascularization were noted. Further studies are required to confirm these preliminary results.

We recently reported stage I of a phase 1/2 clinical trial of cell therapy to treat postradical prostatectomy erectile dysfunction (INSTIN, INtra-cavernous STem-cell INjection clinical trial, NCT01089387). In this first stage, four doses of intracavernous autologous bone marrow mononuclear cells (BM-MNCs) were tested in 12 patients. Here, we report the results of stage II, in which six additional patients received the optimal dose identified in stage I (10 9 BM-MNCs), and the long-term results in the 12 patients included in stage I. The objectives were to assess the safety and efficacy of this new treatment. In stage II, no patients had side effects, and the erectile function improvements were similar to those seen in stage I: after 6 months, significant improvements versus baseline were noted in International Index of Erectile Function-15 intercourse satisfaction (7.8±3.1 vs 2.2±3.4, p =0.033) and erectile function (18±8.3 vs 3.7±4.1, p =0.035) domains. In stage I patients, after a mean follow-up of 62.1±11.7 mo, there were no prostate cancer recurrences, and erectile function scores were somewhat lower compared with the 1-yr time point. These findings suggest that intracavernous BM-MNC injections are safe and improve erectile function. The decline in erectile function over time suggests a need for assessing repeated injections. Patient summary We report a phase 1/2 pilot clinical trial of cell therapy consisting in intracavernous injection of bone marrow mononuclear cells to treat postradical prostatectomy erectile dysfunction. Erectile function was improved after 6 mo in the patients given 1×10 9 cells. No serious side effects (life threatening or requiring hospitalisation) occurred after a mean follow-up of 62.1 mo in the first 12 patients.

We previously demonstrated that intracardiac delivery of autologous peripheral blood‐derived CD34+ stem cells (SCs), mobilized by granulocyte‐colony stimulating factor (G‐CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+‐SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G‐CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+‐cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. stem cells translational medicine 2019;8:822&832

Introductory paragraphThe successful production of Hematopoietic Stem and Progenitor Cells (HSPCs) from human pluripotent sources is conditioned by transgene delivery1-5. We describe here a dedicated and tractable one step, GMP-grade, transgene-free and stroma-free protocol to produce HSPCs from human induced pluripotent stem cells (hiPSCs). This procedure, applied to several sources of hiPSCs with equal efficiency, is based on a directed differentiation with morphogens and cytokines to generate a cell population close to nascent HSPCs or their immediate forerunners i.e., hemogenic endothelial cells6-9. Following engraftment into immunocompromised recipients, this cell population was proved capable of a robust myeloid, lymphoid and definitive red blood cell production in sequential recipients for at least 40 weeks. Further identification of the repopulating cells show that they express the G protein–coupled receptor APELIN (APLNR) and the homing receptor CXCR4. This demonstrates that the generation of bona fide HSPCs from hiPSCs without transgenes is possible and passes through an early endo-hematopoietic intermediate. This work opens the way to the generation of clinical grade HSPCs for the treatment of hematological diseases and holds promise for the analysis of HSPC development in the human species.

Background: For the last 40 years, the technique of extracorporeal photopheresis has constantly developed. Among irradiation systems, those called 'off-line' allow the validation of the quality of the cell therapy product. The inhibition of the proliferation of lymphocytes after UVA irradiation is usually verified by the tritiated thymidine assay as in vitro proliferation assay. The document presented here describes the results obtained while performing the setting up of an alternative proliferation assay using flow cytometry according to ISO 15189:2007 Standard. Methods: Cells samples taken before and after UVA irradiation were labeled with CFSE and then cultured with phytohemagglutinin. After 3 days, an analysis of the CFSE staining was realized by flow cytometry. In order to validate the shift in the method used according to Standard, the following tests were performed: 1) comparison with the reference method, 2) robustness test, 3) reagents stability. Results: Comparison method demonstrated that the sensitivity of the CFSE test is 100%, the specificity is 89% and the concordance is almost complete. The CFSE test is robust regarding parameters like cell concentration or PHA concentration. PHA and CFSE are stable for 6 months and one year, respectively. Conclusion: Validation of this alternative test, according to the ISO 15189:2007 Standard, has demonstrated good concordance with reference method. The results of the robustness and stability of reagents are appropriate for its routine use. Thus, the benefits of alternative technique make it a wise choice for the quality control of ECP in a cell therapy laboratory. © 2014 Clinical Cytometry Society.

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.

It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4(+)CD25(+) T cell compartment. Here, we describe an original method to purify human CD4(+)CD25(+)CD152(+) T lymphocytes as living cells by forcing the exportation of CTLA-4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4(+)CD25(+) T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4(+)CD25(+)CD152(+) T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen-specific T cells than CD4(+)CD25(+)CD152(-) T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA-4, we demonstrate that indeed CTLA-4 is involved in CD4(+)CD25(+)CD152(+) T cell regulatory activity, while suppressive cytokines are not.

The frequent alteration of human leucocyte antigen (HLA) class I molecule expression observed in non-Hodgkin's lymphomas (NHL), similarly to solid tumours, has been reported to favour tumoral escape from the immune system. In order to identify the underlying mechanisms, we analysed 15 HLA defective NHL including partial (n = 10) and total class I (n = 5) loss, as well as HLA class II defects (n = 5). The HLA defect concerned HLA-A and -B antigens in 14 of 15 cases. In the cases with partial defect, the use of specific allelic monoclonal antibodies detected a defect of both alleles of A or B loci in six of seven tested cases. Allelic reverse transcription polymerase chain reaction (RT-PCR) demonstrated defects in six of nine cases, including four alterations of both A and B mRNA alleles. Real-time quantitative RT-PCR (RQ-PCR) did not detect the HLA-DR transcript in the two negative HLA-DR lymphomas, contrasting with the presence of CMH II transactivator (CIITA) transcript. Loss of heterozygosity (LOH) was detected in nine of 14 cases through variable pattern of nine microsatellites markers of the HLA locus. Taken together, these findings demonstrate the complexity and the variability of the mechanisms underlying HLA protein deficiencies with a high frequency of LOH. The diversity of these mechanisms indicates the importance of positive selection of HLA altered clones in the development of these NHL cases.

OBJECTIVES Three recently identified NOD2/CARD15 mutations have been described associated with an increased susceptibility Crohn's disease (CD). Our aim was to examine the potential association of these NOD2 mutations with CD and different subsets of CD phenotypes in our population. METHODS Two hundred and five well-defined CD patients from north-western France and 95 ethnically matched healthy controls were genotyped for mutations R702W, G908R and Leu1007insC by DNA sequencing. Allele and genotype frequencies of NOD2 variants were examined in the whole series of CD and in different subgroups of CD phenotypes defined by the clinical characteristics of the Vienna classification (age at diagnosis, location and behaviour) or by histological features (granuloma). RESULTS Carriers of at least one NOD2/CARD15 variant were significantly more frequent in CD than in controls (38.0% versus 20.0%, P < 0.002), and the R702W allele was the most significant contributor to this NOD2 association with CD. Homozygotes and compound heterozygotes combined had a higher risk of CD (odds ratio = 12.0, P < 0.0026) than simple heterozygotes for any variant (odds ratio = 2.2, P < 0.013) compared with subjects with no variant. Univariate analysis revealed that carriage of at least one NOD2 mutation was significantly associated with ileal involvement (P < 0.03), and stricturing evolution (P < 0.0015). Granuloma was associated with an excess of the R702W allele (16.1% versus 8.0%, Pc < 0.035), and was correlated with a young age at diagnosis, whatever the NOD2/CARD15 genotype. Multivariate analysis demonstrated that carriage of NOD2/CARD15 mutants, especially R702W, was primarily and independently associated both with stricturing evolution of CD and the presence of granuloma. CONCLUSIONS In our population, all NOD2/CARD15 mutant genotypes, especially compound heterozygosity, were found to increase the risk of CD, but R702W was the sole allele showing a significant association with CD. In addition, we confirm the positive and independent association of the R702W mutation with stricturing behaviour and describe a second one with the presence of granuloma.

Monoclonal rat anti-idiotypic antibody (Ab2) BR3E4 mimicking the colorectal carcinoma (CRC)-associated epitope CO17-1A induced antigen-specific humoral and cellular immune responses in mice and rabbits. Ab2 BR3E4 was administered in a phase I trial to CRC patients either as intact IgG or as F(ab′) 2 coupled to keyhole limpet hemocyanin (KLH). There was a trend for the F(ab′) 2 -KLH-immunized patients to show higher immune response rates (18/21 and 5/15 patients with anti-anti-idiotypic antibodies and T cells, respectively) than the IgG-immunized patients (15/23 and 3/15 patients positive). Clinical responses were rare in these patients with liver metastases.

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with rhigher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful in guiding early implementation of additional treatments in hematopoietic stem cell transplantation.

Il existe 18 unites de therapie cellulaire (UTC) a l’EFS. Les centres de soins peuvent etre eloignes de ces UTC et des delais variables entre la preparation des PTC et leurs injections aux patients sont ainsi observes. La stabilite des PTC a fait l’objet d’une validation pour obtenir les autorisations procedes pour chaque PTC. Nous avons realise une etude nationale sur la stabilite des greffons decongeles de CSH autologues de sang peripherique afin d’avoir des donnees sur un nombre plus eleve de produits. Il s’agissait pour chacune des 18 UTC de decongeler 5 PTC en attente de destruction. Les greffons decongeles etaient conserves en poche entre 4 oC et 10 oC et des echantillons etaient preleves a T0, T0 + 2 heures (h), T0 + 4 h et T0 + 6 h. Pour chaque echantillon ont ete determines: la viabilite des CD34+ et des CD45+ et la concentration des CD34+ viables pour calculer le rendement de recuperation par rapport a la congelation. Ces donnees ont ete obtenues par cytometrie en flux en utilisant une technique de quantification en simple plateforme. Le travail presente correspond a un point d’etape. Sept sur 18 UTC ont fourni a ce jour des resultats pour 25 PTC (28 % des PTC prevus). Cette premiere cohorte suggere que la qualite des PTC serait encore acceptable apres 6 heures de conservation entre 4 °C et 10 °C puisque la viabilite des CD34+ passe de 87,9 % en moyenne a T0 a 84,7 % a T0 + 6 h. Nous n’avons pas observe de correlation entre la contamination en cellules granuleuses des PTC ou la concentration de CNT a la congelation et le rendement de recuperation des CD34+. Plus de donnees sont necessaires pour consolider ces premieres conclusions et effectuer une etude multivariee.

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.

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.

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.

For the last 40 years, the technique of extracorporeal photopheresis (ECP) has constantly developed. Among irradiation systems, those called "off-line" allow the validation of the quality of the cell therapy product. The inhibition of the proliferation of lymphocytes after ultraviolet irradiation (UVA) is usually verified by the tritiated thymidine assay as in vitro proliferation assay. The document presented here describes the results obtained while performing the setting up of an alternative proliferation assay using flow cytometry according to ISO 15189:2007 Standard.Cells samples taken before and after UVA irradiation were labeled with CarboxyFluorescein Succinimidyl Ester (CFSE) and then cultured with phytohemagglutinin-A (PHA). After 3 days, an analysis of the CFSE staining was realized by flow cytometry. In order to validate the shift in the method used according to Standard, the following tests were performed: 1) comparison with the reference method, 2) robustness test, 3) reagents stability.Comparison method demonstrated that the sensitivity of the CFSE test is 100%, the specificity is 89%, and the concordance is almost complete. The CFSE test is robust regarding parameters like cell concentration or PHA concentration. PHA and CFSE are stable for 6 months and one year, respectively.Validation of this alternative test, according to the ISO 15189:2007 Standard, has demonstrated good concordance with reference method. The results of the robustness and stability of reagents are appropriate for its routine use. Thus, the benefits of alternative technique make it a wise choice for the quality control of ECP in a cell therapy laboratory.

International audience; Acute graft-versus-host disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell transplantation. CD4(+)CD25(hi) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine models. Based on these observations, we examined their role in the prevention of aGVHD in patients who underwent transplantation with peripheral blood-mobilized hematopoietic stem cells after administration of granulocyte colony-stimulating factor. The effects of the G-CSF on the phenotype, frequency, and function of CD4(+)CD25(hi) T cells were analyzed in grafts and after transplantation to determine whether these cells were regulatory T cells. CD4(+)CD25(hi) T cells could be detected at the same frequency before and after granulocyte colony-stimulating factor administration in the donors' peripheral blood. The isolation of these cells from the grafts or from the recipients' peripheral blood after transplantation revealed that they were suppressive to the same extent as T(reg) isolated from healthy volunteers. Their number and frequency were estimated in the grafts and the results indicated that protection against aGVHD was not dependent on the T(reg) amount transferred to the recipients. Similarly there was no correlation between the number of circulating CD4(+)CD25(hi) T cells in the recipients' peripheral blood during the early period after transplantation and the outcome of aGVHD.

SummaryHaematopoietic 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 (Treg) frequency in the blood of transplanted patients; and (ii) the presence of CD4+ CD25+ CD152+ natural Treg 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 Treg to foster the induction of a tolerogenic environment and improve PSC transplantation favourable outcome.

Purpose: A phase I/II trial was conducted to evaluate clinical and immunologic responses after intralymphatic and intranodal injections of mature dendritic cells.Experimental Design: Fourteen patients with a metastatic melanoma received matured dendritic cells, loaded with Melan-A/MART-1 and/or NA17-A peptides and keyhole limpet hemocyanin. The cells were matured overnight with Ribomunyl, a toll-like receptor ligand, and IFN-γ, which ensured the production of high levels of interleukin-12p70. Dendritic cells were injected at monthly intervals, first into an afferent lymphatic and then twice intranodally. Immunologic responses were monitored by tetramer staining of circulating CD8+ lymphocytes and delayed-type hypersensitivity tests.Results: Dendritic cell vaccination induced delayed-type hypersensitivity reactivity toward NA17-A-pulsed, keyhole limpet hemocyanin–pulsed, and Melan-A-pulsed dendritic cells in 6 of 10, 4 of 11, and 3 of 9 patients, respectively. Four of the 12 patients analyzed by tetramer staining showed a significantly increased frequency of Melan-A-specific T cells, including one patient vaccinated only with NA17-A-pulsed dendritic cells. Furthermore, 2 of the 12 analyzed patients had a significant increase of NA17-A-specific T cells, including one immunized after an optional additional treatment course. No objective clinical response was observed. Two patients were stabilized at 4 and 10 months and three patients are still alive at 30, 39, and 48 months.Conclusions: Injections into the lymphatic system of mature peptide-loaded dendritic cells with potential TH1 polarization capacities did not result in marked clinical results, despite immunologic responses in some patients. This highlights the need to improve our understanding of dendritic cell physiology.

Mutated epidermal growth factor receptor (EGF-RvIII, ΔEGF-R, and de2–7 EGF-R) is the result of an 801-bp deletion within the extracellular domain of wild-type EGF-R and is expressed by breast carcinomas, but not by normal breast tissues. EGF-RvIII is expressed both on the surface and in the cytoplasm of tumor cells. Thus, EGF-RvIII is a potential tumor-specific target for both Abs and T cells. However, it is not known whether breast cancer patients can raise immune responses to EGF-RvIII expressed by their tumors. The demonstration of EGF-RvIII-specific immune responses in patients would suggest that immunization of patients with EGF-RvIII vaccines is feasible, because these vaccines may boost a pre-existing immune response. We have evaluated humoral and cellular immune responses to EGF-RvIII in 16 breast cancer patients and three healthy donors. Seven of 16 patients developed EGF-RvIII-specific Abs that bound to isolated EGF-RvIII protein or the protein expressed by EGF-RvIII-transfected mouse fibroblasts. The Abs that bound to EGF-RvIII did not bind to wild-type EGF-R, and anti-EGF-RvIII Abs were not found in the sera of healthy donors. Three patients had EGF-RvIII peptide-specific lymphoproliferative responses, and two of these patients also had humoral immune responses. Humoral and cellular immune responses correlated with EGF-RvIII expression by patients’ tumors in most cases. These studies demonstrate that breast cancer patients specifically recognize EGF-RvIII with an overall immune response rate of 50%, suggesting that patients may benefit from vaccination against EGF-RvIII, boosting pre-existing immune responses.

The frequent alteration of human leucocyte antigen (HLA) class I molecule expression observed in non-Hodgkin's lymphomas (NHL), similarly to solid tumours, has been reported to favour tumoral escape from the immune system. In order to identify the underlying mechanisms, we analysed 15 HLA defective NHL including partial (n = 10) and total class I (n = 5) loss, as well as HLA class II defects (n = 5). The HLA defect concerned HLA-A and -B antigens in 14 of 15 cases. In the cases with partial defect, the use of specific allelic monoclonal antibodies detected a defect of both alleles of A or B loci in six of seven tested cases. Allelic reverse transcription polymerase chain reaction (RT-PCR) demonstrated defects in six of nine cases, including four alterations of both A and B mRNA alleles. Real-time quantitative RT-PCR (RQ-PCR) did not detect the HLA-DR transcript in the two negative HLA-DR lymphomas, contrasting with the presence of CMH II transactivator (CIITA) transcript. Loss of heterozygosity (LOH) was detected in nine of 14 cases through variable pattern of nine microsatellites markers of the HLA locus. Taken together, these findings demonstrate the complexity and the variability of the mechanisms underlying HLA protein deficiencies with a high frequency of LOH. The diversity of these mechanisms indicates the importance of positive selection of HLA altered clones in the development of these NHL cases.

We report the results of a genome-wide screen for linkage disequilibrium (LD) in multiple sclerosis (MS) performed on 200 cases, 200 controls and 200 case–parent trios from France employing pooled DNA methodology. A total of 3510 microsatellite markers supplied through the GAMES collaborative were analysed and ranked according to their evidence for association. The most promising 117 markers were then followed up in a two-step validation process. In the first step, additional PCR of the DNA pools was performed in order to refine the ranking order. In the second step, markers were genotyped in individual cases and parents from the trio families. Seven markers showing nominally significant allele frequency differences between affected and unaffected emerged—D6S265, D12S1064, TNFa, D7S1824, D14S1426, D14S605 and D21S2051. These potential associations will require confirmation in further studies.

The breakdown of tolerance to autologous bacterial flora has been implicated as a major factor contributing to the initiation and perpetuation of chronic inflammation in inflammatory bowel diseases (IBD). To test whether bacterial DNA is at the origin of inflammation in IBD, we have examined the response of lamina propria (LPMC) or peripheral mononuclear cells (PBMC) and purified T cells from IBD patients and control patients to stimulations with a set of oligodeoxynucleotides (ODNs) characterized by the presence or absence of cytosine-guanosine dinucleotides (CpG) and/or 3' poly-guanosine (poly-G) extension. Furthermore we have evaluated the costimulatory activities of these ODNs on T cells activated via CD2 or CD3 pathway. We demonstrated that CpG ODNs induce higher proliferation of LPMC from inflammatory intestinal mucosa compared to healthy mucosa. We confirmed that CpG ODNs do not directly costimulate peripheral blood T cells activated by CD3 pathway. Finally, we revealed that CpG or non-CpG ODNs with 3' poly-G extension inhibit completely CD2 activation of purified PB or LP T-cells whereas only CpG ODNs without poly-G extension enhance proliferation and IFN-gamma production of PB T cells stimulated by CD2 pathway only in presence of NK and NK T cells. Our data suggest that NK T cells may be the primary target of ODNs and play a crucial role in indirect T-cell activation by ODN.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system with a genetic component. Until now, the more consistent association with the disease is found with the major histocompatibility complex, especially HLA-DRB1*1501-DQB1*0602 haplotype. In this report, we demonstrate the interaction of Cytotoxic T Lymphocyte-associated antigen 4 (CTLA-4 [CD152]) gene with DRB1*15 haplotype in multiple sclerosis genetic susceptibility. Our data were obtained from two European independent family-based studies including 610 multiple sclerosis family trios. Ann Neurol 2003;54:119-122, Fondation pour la Recherche Médicale (ARS 2.09 Bertrand Fontaine), by Association pour la Recherche sur la Sclérose en Plaques, by Italian Foundation for Multiple Sclerosis, by INSERM, and by the French Ministry of Research (Centre d'Investigations Cliniques Pitié‐Salpétrière and Centre de Resources Biologiques)

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.

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.

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.

Mit dem monoklonalen Antikorper Mab C017-1A (Panorex ®) konnte erstmals in einer randomisierten Studie ein Therapievorteil von 30% in der adjuvanten Behandlung von kolorektalen Karzinomen gezeigt werden [1]. Das dazugehorige tumorassoziierte Antigen CO17-1A/GA733, ein 40 kD Glykoprotein, wird zu uber 80% auf kolorektalen und Pankreaskarzinomen exprimiert und findet sich auf > 80% der Zellen innerhalb eines Tumors [2, 3]. An Nacktmausen konnte gezeigt werden, das GA733-positive Tumoren durch Behandlung mit den monoklonalen Antikorpern GA733 und CO17-1A lysiert werden konnten [4, 5, 6]. Die extrazellulare Domane des Antigens konnte jetzt in einem Baculovirus-System synthetisch hergestellt und mit Aluminiumhydroxid (ALUM) als Adjuvans prazipitiert erstmals in einer Phase I/II Studie klinisch eingesetzt werden; die erzeugte Immunantwort und die Toxizitat wurden untersucht.

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.