Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process.

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha- l-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4- d + 20.5 g/l KNO3 ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

Hairy Root Cultures-A Versatile Tool With Multiple Applications.

Hairy roots derived from the infection of a plant by Rhizobium rhizogenes (previously referred to as Agrobacterium rhizogenes) bacteria, can be obtained from a wide variety of plants and allow the production of highly diverse molecules. Hairy roots are able to produce and secrete complex active glycoproteins from a large spectrum of organisms. They are also adequate to express plant natural biosynthesis pathways required to produce specialized metabolites and can benefit from the new genetic tools available to facilitate an optimized production of tailor-made molecules. This adaptability has positioned hairy root platforms as major biotechnological tools. Researchers and industries have contributed to their advancement, which represents new alternatives from classical systems to produce complex molecules. Now these expression systems are ready to be used by different industries like pharmaceutical, cosmetics, and food sectors due to the development of fully controlled large-scale bioreactors. This review aims to describe the evolution of hairy root generation and culture methods and to highlight the possibilities offered by hairy roots in terms of feasibility and perspectives.

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-L-iduronidase.

The Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha-L-iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N- and O-glycosylation that characterize the alpha-L-iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N-glycosylation sites of the IDUA, a single N-glycan composed of a core Man3 GlcNAc2 carrying one beta(1,2)-xylose and one alpha(1,3)-fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O-glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins.

Perturbations of the CD8(+) T-cell repertoire in CVID patients with complications.

A higher chronic expansion of effector cytotoxic CD8(+)DR(+) T-lymphocytes has been reported in common variable immunodeficiency (CVID) patients with complications such as splenomegaly, autoimmune disease and/or granulomatous disease. In order to document the features associated with this T cell activation involving the CD8(+) T-compartment, we examined the diversity of the alpha/beta TCR repertoire of the patient's CD8(+) T-lymphocytes using the qualitative analysis of the CDR3 lengths (Immunoscope). Ten CIVD patients were enrolled in this study, four without complications (Group 1), six with complications (Group 2). All patients exhibited non-gaussian altered CDR3 length distributions, albeit to different extent within the different Vß families. CVID patients with activated CD8(+) T-cells show a reduction of their TCR repertoire diversity which is more severe in patients with complications. Viral reactivations such as CMV are suspected to be part of the mechanisms underlying immunosenescence.

Identification of a gene expression profile associated with operational tolerance among a selected group of stable kidney transplant patients.

Despite their utility, immunosuppressive treatments have numerous side effects, including infectious complications, malignancies and metabolic disorders, all of which contribute to long-term graft loss. In addition to the development of new pharmaceutical products with reduced toxicity and more comfortable modes of administration, tailoring immunosuppression according to the immune status of each patient would represent a significant breakthrough. Gene expression profiling has been shown to be a clinically relevant monitoring tool. In this paper, we have assessed the overall long-term kidney transplant outcome and attempted to identify operationally tolerant-like patients among recipients with stable clinical status at least 5 years post-transplantation. We thus measured a combination of noninvasive blood biomarkers of operational tolerance in a cohort of 144 stable patients and showed that only 3.5% exhibited a gene expression profile of operational tolerance, suggesting that such a profile can be detected under immunosuppressive therapy but that its frequency is low in kidney transplant recipients when compared with liver transplant recipients. We suggest that a rational approach to patient selection, based on a combination of clinical and biological characteristics, may help to provide a safer method for identification of patients potentially suitable for immunosuppressive drug weaning procedures.

Analysis of the peripheral T-cell repertoire in kidney transplant patients.

The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+) /CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation.

Failure of glatiramer acetate to modify the peripheral T cell repertoire of relapsing-remitting multiple sclerosis patients.

Glatiramer acetate (GA) is a random copolymer used as an immunomodulatory treatment in relapsing-remitting multiple sclerosis (RR-MS). Its mechanisms of action are poorly understood, and several hypotheses have been put forward, the majority of which rely on in vitro studies. It has been hypothesised that further to processing by APC, GA could provide a large number of different epitopes with a possible sequence similarity to auto-antigens, which are able to stimulate a large proportion of T cells. Given that in a previous study we showed that the circulating T cells of MS patients present more alterations of the Vbeta T cell receptor (TCR) usage than normal individuals, we explored the possible effect of GA on the ex vivo T cell repertoire of MS patients. Here we used quantitative PCR and electrophoresis to longitudinally analyse (and without any ex vivo stimulation), the CDR3 length distribution (LD) and the amount of Vbeta TCR, as well as various cytokines, in the blood T cells of 10 RR-MS patients before and after 3 months and 2 years of GA treatment. In addition, we also determined the status of responder and non-responder patients after 24 months of GA treatment based on clinical and radiological criteria. We found no significant modification of cytokine production, Vbeta TCR mRNA accumulation or CDR3-LD in the patients after short-term and long-term treatment. In addition, we did not observe any difference in CDR3-LD in the GA responder patients (n=6) compared to non-responder patients (n=4). Focusing our study on responder patients, we performed TCR repertoire analysis in the CD4+ and CD8+ compartment. Alterations of CDR3-LD were predominantly found in the CD8+ compartment, without any significant influence of GA treatment. Finally, the T cell repertoire variations in MS patients treated with GA and healthy controls were equivalent. Collectively, our data suggest that GA therapy does not induce significant variations in cytokine production or TCR usage in MS patients.

Immunoproteasome beta subunit 10 is increased in chronic antibody-mediated rejection.

Chronic active antibody-mediated rejection is a form of late rejection with a poor prognosis. To identify specific markers of this, we analyzed several microarray studies in the literature and performed mRNA profiling of 65 biopsies and 165 blood samples of a large cohort of renal transplant patients with precisely characterized pathologies. Immunoproteasome beta subunit 10 was found to be specifically increased in the graft and blood samples during chronic active antibody-mediated rejection and was also significantly increased in rat cardiac allografts undergoing acute rejection as well as chronic active antibody-mediated rejection. This syndrome is characterized by chronic transplant vasculopathy associated with diffuse C4d staining and circulating donor-specific antibodies. Using this animal model, we found that administration of the proteasome inhibitor, Bortezomib, delayed acute rejection and attenuated the humoral response in both the acute phase and established state of this syndrome in a dose-dependent manner. Following treatment with this reagent, donor-specific antibodies and C4d deposition were reduced. These studies highlight the role of the proteasome in chronic rejection and identify this molecule as a marker of this syndrome.

Company Profile: TcLand Expression.

TcLand Expression is a fully integrated molecular diagnostics company that holds a pioneering position in personalized medicine focusing on immunology (transplantation and autoimmune disorders). The company has a well-balanced pipeline of blood-based gene-expression biomarkers and companion diagnostics in development. The company's state-of-the-art central laboratories are International Organization for Standardization (ISO) 17025-accredited for quantitative PCR. TcLand Expression has expertise in all the steps of biomarker development, from research and development, bioinformatics and biostatistics to clinical and regulatory affairs, market access and production. Through gene-set discovery, external and clinical validation in large multinational studies, TcLand Expression is developing diagnostic and prognostic blood tests to help make personalized medicine a reality for transplant recipients and patients with autoimmune diseases.

Statistical analysis of CDR3 length distributions for the assessment of T and B cell repertoire biases.

Complementarity-determining region 3 (CDR3) length distribution analysis explores the diversity of the T cell receptor (TCR) and immunoglobulin (Ig) repertoire at the transcriptome level. Studies of the CDR3, the most hypervariable part of these molecules, have been frequently used to identify recruitment of T and B cell clones involved in immunological responses. CDR3 length distribution analysis gives a clear perception of repertoire variations between individuals and over time. However, the complexity of CDR3 length distribution patterns and the high number of possible repertoire alterations per individual called for the development of robust data analysis methods. The goal of these methods is to identify, quantify and statistically assess differences between repertoires so as to offer a better diagnostic or predictive tool for pathologies involving the immune system. In this review we will explain the benefit of analyzing CDR3 length distribution for the study of immune cell diversity. We will start by describing this technology and its associated data processing, and will subsequently focus on the statistical methods used to compare CDR3 length distribution patterns. Finally, we will address the various methods for assessing CDR3 length distribution gene signatures in pathological states.

Serial blood T cell repertoire alterations in multiple sclerosis patients; correlation with clinical and MRI parameters.

A significant skewing of the peripheral T cell repertoire has been shown in relapsing-remitting multiple sclerosis (MS). Most of the studies already performed in this field are cross-sectional and therefore, little is known of the T cell repertoire evolution over time in MS and the correlation of T cell repertoire variation with clinical and MRI parameters. This study was performed on serially harvested frozen PBMC from nine untreated MS patients (27 samples) and 14 healthy individuals. The blood T cell repertoire of each patient was analysed at the complementarity determining region 3 (CDR3) level and compared with a monthly MRI scan performed over a six month period with assessment of T2 lesion load and gadolinium enhancing lesions. A highly significant blood T cell repertoire skewing was observed in MS patients as compared with healthy controls (p<0.01). In addition, the number of altered Vbeta families correlated significantly with both the T2 lesion volume and the number of gadolinium enhancing lesions as assessed by MRI (Spearman correlation tests, r=0.51 and r=0.44, p<0.01 and p<0.05 respectively). Furthermore, the variation of the number of altered Vbeta families over time also correlated with the appearance of new gadolinium enhancing lesions (r=0.36, p=0.05). These findings which need confirmation on larger serial cohorts, suggest an association between the magnitude of TCRBV CDR3 length distribution alterations in the peripheral blood of MS patients and the disease process.

Exhaustive depletion of graft resident dendritic cells: marginally delayed rejection but strong alteration of graft infiltration.

BACKGROUND: Donor dendritic cells (DDC) are believed to sustain direct recognition leading to acute allograft rejection. However, DDC are also required for tolerance induction in various models. METHODS: We studied the effect of DDC depletion on major histocompatibility complex (MHC) mismatched rat heart allografts in a strain combination characterized by a DDC-dependant tolerance induction. Grafts were depleted of DDC either by pretreating donors with cyclophosphamide (CyP) or by being parked in an intermediate recipient treated with cyclosporine A (CsA). RESULTS: CyP depleted 95% of resident DC and no specific donor MHC class II staining was observed in parked grafts. Parked grafts survived significantly but only moderately longer than untreated grafts (10.8+/-1.9 days vs. 6.5+/-0.5 days; P<0.05). Compared to unmodified grafts, on day 5 after transplantation, the magnitude of the graft infiltrate was dramatically decreased in DDC-depleted grafts, with IgG deposition within the grafts at the time of rejection. In parallel, the cytokine transcript levels were also lower in these grafts on day 5, but reached levels similar to those of unmodified grafts by day 7, indicating a delayed pattern of rejection. CONCLUSIONS: Taken collectively, these data suggest that DDC depletion has a greater effect on the capacity of tolerance induction than the rejection process.

Compartmentalization of TCR repertoire alteration during rejection of an intrabrain xenograft.

Xenograft rejections of embryonic pig neural cells implanted into the adult rat striatum occurs within 3-4 weeks, following a dramatic T cell infiltration. Little is known about the cross-talk between the brain and peripheral lymphoid tissues which results in this recruitment and lymphocyte homing. To better characterize the dynamics of the T cell response against xenogeneic neural cells implanted into the brain parenchyma, we used both qualitative and quantitative methods to follow the alterations of the CDR3 length distribution (CDR3-LD) of the TCR (T cell receptor) beta chain in the transplanted striatum and compared this response to that observed in the deep cervical lymph nodes, spleen, and blood. Data showed that the T cell repertoire diversity was highly altered in the recipient brain during xenograft rejection. Comparison of the alterations of the CDR3-LD between several animals revealed a single public alteration in the Vbeta20 family, and many private alterations of the CDR3-LD which differed from one infiltrated brain to another. Alterations of the T cell repertoire were also observed in lymphocytes homed into the deep cervical lymph nodes. However, they differed from the alterations detected in the infiltrated brains. Conversely, no significant alteration of the CDR3-LD was detected in the spleen or in the blood. These data suggest that the deep cervical lymph nodes play an active role in the process of xenograft recognition or/and rejection. However, they also indicate that the fate of T cells homed in the brain and deep cervical lymph nodes differs.

Operationally tolerant and minimally immunosuppressed kidney recipients display strongly altered blood T-cell clonal regulation.

Most kidney transplant recipients who discontinue immunosuppression reject their graft. Nevertheless, a small number do not, suggesting that allogeneic tolerance state (referred to operational tolerance) is achievable in humans. So far, however, the rarity of such patients has limited their study. Because operational tolerance could be linked to anergy, ignorance or to an active regulatory mechanism, we analyzed the blood T-cell repertoire usage of these patients. We report on comparison of T-cell selection in drug-free operationally tolerant kidney recipients (or with minimal immunosuppression), recipients with stable graft function, chronic rejection and healthy individuals. The blood T cells of operationally tolerant patients display two major characteristics: an unexpected strongly altered T-cell receptor (TCR) Vbeta usage and high TCR transcript accumulation in selected T cells. The cytokine transcriptional patterns of sorted T cells with altered TCR usage show no accumulation of cytokine transcripts (IL10, IL2, IL13, IFN-gamma), suggesting a state of hyporesponsiveness in these patients. Identification of such a potential surrogate pattern of operational tolerance in transplant recipients under life-long immunosuppression may provide a new basis and rationale for exploration of tolerance state. However, these data obtained in a limited number of patients require further confirmation on larger series.

Different patterns of TCR beta chain regulation following allo- and xeno-transplantation.

BACKGROUND: In the concordant hamster-to-rat cardiac xenograft model, recipients treated with cobra venom factor for the first 10 days following transplantation and daily with Cyclosporine A (CsA) do not reject their grafts. However, when CsA is withdrawn on day 40, an acute cellular rejection occurs within 4 +/- 1 days. Allografts performed in the same conditions are rejected within 18 +/- 4 days. METHODS: In this model, we have compared graft infiltrating T cells through both a quantitative (number of Vbeta transcripts) and qualitative (CDR3 length distribution) assessment of the T cell receptor (TCR) beta chain transcriptome in allo- and xeno-transplantations. RESULTS: We report striking differences in TCR usage at day 15 following allo- and xeno-transplantation as well as during rejection following CsA withdrawal. The number of Vbeta transcripts was high in both rejected allo- and xenografts. However, whereas in xenografts acute rejection occurred without skewing of Vbeta CDR3 length distribution, T cells infiltrating allografts during rejection after CsA interruption had a highly altered CDR3 length distribution pattern. In addition, using a correspondence factor analysis of the beta chain transcriptome, we show that some families can clusterize and can discriminate allo- or xeno-patterns at the level of both the number of Vbeta transcripts and the CDR3 length distribution. CONCLUSIONS: Our data show that, in vivo, even in the hamster-to-rat concordant combination, the anti-xenograft T cell response is strong and will likely represent another challenge for xenotransplantation.

Blood T-cell Vbeta transcriptome in melanoma patients.

Tumor-cells have been shown to elicit MHC-restricted and antigen-specific T-cell responses. In this article, we used a new approach to study T-cell responses in tumor-bearing patients based on a global representation of the Vbeta-transcriptome, making it possible to grade CDR3-length distribution (CDR3-LD) alterations. Six patients with advanced melanoma disease, from whom blood samples were taken before and serially after tyrosinase-A peptide vaccination, were studied. The PBMC from patients displayed highly significant Vbeta transcriptome alterations as compared to healthy individuals. Similar Vbeta alterations could be detected both in PBMCs and at the tumor site. After vaccination, Vbeta alterations could also be observed by gauging individually their transcript level but not their cell-surface expression. Some Vbeta families exhibited high Vbeta/HPRT transcript ratios (e.g., Vbeta1), which represented up to 44% of the whole transcriptome, a situation that was not reflected by an increase in the percentage of T cells that expressed the corresponding protein and was not observed in normal individuals. In several instances, CDR3-LD altered T cells exhibited MHC-restricted and tumor-specific IFNgamma or GM-CSF production. Finally, we show that the presence of a tumor and probably vaccination can affect Vbeta transcriptome patterns and induce specific clones reactive to autologous tumor or vaccinating peptides. In combination with other methods, such an approach should help in identifying the clones actually involved in the response against the tumor.

Blood T-cell receptor beta chain transcriptome in multiple sclerosis. Characterization of the T cells with altered CDR3 length distribution.

Multiple sclerosis is an inflammatory demyelinating disease of the CNS associated with T cells autoreactive for myelin components. In this study, we analysed the T-cell receptor (TCR) usage of the variable beta (Vbeta) chain transcriptome in the blood of multiple sclerosis patients at various stages of the disease using a global and quantitative comparison of the complementarity-determining region 3 length distribution (CDR3-LD) of transcripts of the 26 Vbeta genes. We investigated 35 patients: 12 with a high risk of multiple sclerosis, 10 with clinically definite multiple sclerosis, 13 with a relapsing-remitting worsening and active multiple sclerosis and 13 healthy individuals. Cells bearing the TCR transcripts with altered CDR3-LD were sorted and studied for CD4 or CD8 phenotype, cytokine transcript accumulation and response to human myelin basic protein (MBP). We show that patients from all the groups have a significantly skewed blood T-cell repertoire. Vbeta transcriptome patterns were more altered in patients from the clinically definite multiple sclerosis group and the worsening and active multiple sclerosis group than in the high risk group. The T cells sorted from Vbeta families with altered CDR3-LD concerned both CD4 and CD8 T cells, with a more pronounced skewing in the CD8 compartment. These cells displayed a significantly increased level of interferon-gamma, interleukin-2 and tumour necrosis factor-alpha transcripts compared with their counterparts from the healthy individual group. Furthermore, using interferon-gamma enzyme-linked immunospot (ELISPOT) assays, T cells from four out of seven altered Vbeta families tested from multiple sclerosis patients responded to human MBP, whereas no response was observed with human albumin or with altered Vbeta families from healthy individuals. Our data support the concept of an early autoimmune component in the disease and emphasize the possible involvement of CD8-positive T cells in multiple sclerosis.

Tolerance induction in rats, using a combination of anti-CD154 and donor splenocytes, given once on the day of transplantation.

BACKGROUND: Donor-specific tolerance induction remains an attractive objective that generates much research in the field of transplantation. Unfortunately, most of the protocols available involve pregraft conditioning, making these treatments incompatible with clinical applications. METHODS: LEW.1A rats were grafted with histoincompatible LEW.1W hearts. On the day of transplantation, recipients were treated with anti-CD40L combined with donor splenocytes. The hearts were evaluated for graft survival; cellular infiltrate and intragraft cytokines were determined using real-time reverse transcriptase-polymerase chain reaction. Tolerance induction was assessed by skin grafting and adoptive transfers. RESULTS: The combination of a single injection of anti-CD40L and donor splenocytes, given on the day of surgery, allowed 40% of cardiac allografts to survive long-term (mean survival time=66.3 day). The cellular composition or the extent of graft infiltrate was not modified but was associated with a massive decrease of proinflammatory cytokines expression within the graft. Long-term survivors accepted donor-matched skin grafts, and leukocytes harvested from these animals transferred tolerance into irradiated freshly grafted recipients. CONCLUSION: A combination of costimulation blockade and donor cells, given once at the time of transplantation, is sufficient to induce allograft tolerance in rats.

Different qualitative and quantitative regulation of V beta TCR transcripts during early acute allograft rejection and tolerance induction.

Recently, using a global method of T cell repertoire analysis, we showed that purified naive T cells confronted in vitro with allogeneic APCs in a direct pathway-restricted MLR up-regulate their Vbeta mRNAs without exhibiting skewing of complementarity-determining region 3 (CDR3) length distribution. In this report, using this approach, we show in vivo that Vbeta transcript regulation and CDR3 length distribution follow the same pattern during acute rejection of MHC-incompatible heart allografts. In contrast, in tolerance induction by priming of recipients with donor cells, the vigorous Vbeta mRNA accumulation with Gaussian CDR3 length distribution is abolished, providing a possible explanation for the down-regulation of activated T cells in tolerant animals. In addition, tolerated grafts harbor T cells with a highly altered repertoire, suggestive of self-restricted presentation with some patterns corresponding to previously identified regulatory cells.