Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial.

OBJECTIVE: Regulatory T cells (Tregs) prevent autoimmunity and control inflammation. Consequently, any autoimmune or inflammatory disease reveals a Treg insufficiency. As low-dose interleukin-2 (ld-IL2) expands and activates Tregs, it has a broad therapeutic potential. AIM: We aimed to assess this potential and select diseases for further clinical development by cross-investigating the effects of ld-IL2 in a single clinical trial treating patients with 1 of 11 autoimmune diseases. METHODS: We performed a prospective, open-label, phase I-IIa study in 46 patients with a mild to moderate form of either rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, psoriasis, Behcet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis and sclerosing cholangitis. They all received ld-IL2 (1 million IU/day) for 5 days, followed by fortnightly injections for 6 months. Patients were evaluated by deep immunomonitoring and clinical evaluation. RESULTS: ld-IL2 was well tolerated whatever the disease and the concomitant treatments. Thorough supervised and unsupervised immunomonitoring demonstrated specific Treg expansion and activation in all patients, without effector T cell activation. Indication of potential clinical efficacy was observed. CONCLUSION: The dose of IL-2 and treatment scheme used selectively activate and expand Tregs and are safe across different diseases and concomitant treatments. This and preliminary indications of clinical efficacy should licence the launch of phase II efficacy trial of ld-IL2 in various autoimmune and inflammatory diseases. TRIAL REGISTRATION NUMBER: NCT01988506.

Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses.

Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I3 ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients. Here, we describe these optimized flow cytometry panels, showing that they provide the advanced analysis of T cells (including regulatory T cells), B cells, NK cells, MAIT cells, myeloid cells, monocytes, and dendritic cells. Most of the panels have been dried to improve standardization of the labeling and the entire procedure can be performed on less than 2 ml of whole blood. These deep immunophenotyping flow cytometry panels constitute a powerful tool for the monitoring of immune blood cells and will hopefully lead to the discovery of new biomarkers and potential therapeutic targets in autoimmune and cancer clinical trials. © 2018 International Society for Advancement of Cytometry.

High-resolution repertoire analysis reveals a major bystander activation of Tfh and Tfr cells.

T follicular helper (Tfh) and regulatory (Tfr) cells are terminally differentiated cells found in germinal centers (GCs), specialized secondary lymphoid organ structures dedicated to antibody production. As such, follicular T (Tfol) cells are supposed to be specific for immunizing antigens, which has been reported for Tfh cells but is debated for Tfr cells. Here, we used high-throughput T cell receptor (TCR) sequencing to analyze the repertoires of Tfh and Tfr cells, at homeostasis and after immunization with self- or foreign antigens. We observed that, whatever the conditions, Tfh and Tfr cell repertoires are less diverse than those of effector T cells and Treg cells of the same tissues; surprisingly, these repertoires still represent thousands of different sequences, even after immunization with a single antigen that induces a 10-fold increase in Tfol cell numbers. Thorough analysis of the sharing and network of TCR sequences revealed that a specific response to the immunizing antigen can only, but hardly, be detected in Tfh cells immunized with a foreign antigen and Tfr cells immunized with a self-antigen. These antigen-specific responses are obscured by a global stimulation of Tfh and Tfr cells that appears to be antigen-independent. Altogether, our results suggest a major bystander Tfol cell activation during the immune response in the GCs.

Unitary circular code motifs in genomes of eukaryotes.

A set X of 20 trinucleotides was identified in genes of bacteria, eukaryotes, plasmids and viruses, which has in average the highest occurrence in reading frame compared to its two shifted frames (Michel, 2015; Arquès and Michel, 1996). This set X has an interesting mathematical property as X is a circular code (Arquès and Michel, 1996). Thus, the motifs from this circular code X, called X motifs, have the property to always retrieve, synchronize and maintain the reading frame in genes. The origin of this circular code X in genes is an open problem since its discovery in 1996. Here, we first show that the unitary circular codes (UCC), i.e. sets of one word, allow to generate unitary circular code motifs (UCC motifs), i.e. a concatenation of the same motif (simple repeats) leading to low complexity DNA. Three classes of UCC motifs are studied here: repeated dinucleotides (D+ motifs), repeated trinucleotides (T+ motifs) and repeated tetranucleotides (T+ motifs). Thus, the D+, T+ and T+ motifs allow to retrieve, synchronize and maintain a frame modulo 2, modulo 3 and modulo 4, respectively, and their shifted frames (1 modulo 2; 1 and 2 modulo 3; 1, 2 and 3 modulo 4 according to the C2, C3 and C4 properties, respectively) in the DNA sequences. The statistical distribution of the D+, T+ and T+ motifs is analyzed in the genomes of eukaryotes. A UCC motif and its comp lementary UCC motif have the same distribution in the eukaryotic genomes. Furthermore, a UCC motif and its complementary UCC motif have increasing occurrences contrary to their number of hydrogen bonds, very significant with the T+ motifs. The longest D+, T+ and T+ motifs in the studied eukaryotic genomes are also given. Surprisingly, a scarcity of repeated trinucleotides (T+ motifs) in the large eukaryotic genomes is observed compared to the D+ and T+ motifs. This result has been investigated and may be explained by two outcomes. Repeated trinucleotides (T+ motifs) are identified in the X motifs of low composition (cardinality less than 10) in the genomes of eukaryotes. Furthermore, identical trinucleotide pairs of the circular code X are preferentially used in the gene sequences of eukaryotes. These two results suggest that the unitary circular codes of trinucleotides may have been involved in the formation of the trinucleotide circular code X. Indeed, repeated trinucleotides in the X motifs in the genomes of eukaryotes may represent an intermediary evolution from repeated trinucleotides of cardinality 1 (T+ motifs) in the genomes of eukaryotes up to the X motifs of cardinality 20 in the gene sequences of eukaryotes.

Circular code motifs in genomes of eukaryotes.

A set X of 20 trinucleotides was identified in genes of bacteria, eukaryotes, plasmids and viruses, which has in average the highest occurrence in reading frame compared to its two shifted frames (Michel, 2015; Arquès and Michel, 1996). This set X has an interesting mathematical property as X is a circular code (Arquès and Michel, 1996). Thus, the motifs from this circular code X, called X motifs, have the property to always retrieve, synchronize and maintain the reading frame in genes. In this paper, we develop several statistical analyzes of X motifs in 138 available complete genomes of eukaryotes in which genes as well as non-gene regions are examined. Large X motifs (with lengths of at least 15 consecutive trinucleotides of X and compositions of at least 10 different trinucleotides of X among 20) have the highest occurrence in genomes of eukaryotes compared to its 23 large bijective motifs, its two large permuted motifs and large random motifs. The largest X motifs identified in eukaryotic genomes are presented, e.g. an X motif in a non-gene region of the genome Solanum pennellii with a length of 155 trinucleotides (465 nucleotides) and an expectation E=10(-71). In the human genome, the largest X motif occurs in a non-gene region of the chromosome 13 with a length of 36 trinucleotides and an expectation E=10(-11). X motifs in non-gene regions of genomes could be evolutionary relics of primitive genes using the circular code for translation. However, the proportion of X motifs (with lengths of at least 10 consecutive trinucleotides of X and compositions of at least 5 different trinucleotides of X among 20) in genes/non-genes of the 138 complete eukaryotic genomes is about 8. Thus, the X motifs occur preferentially in genes, as expected from the previous works of 20 years.

Circular code motifs near the ribosome decoding center.

A maximal C(3) self-complementary trinucleotide circular code X is identified in genes of bacteria, eukaryotes, plasmids and viruses (Michel, 2015; Arquès and Michel, 1996). A translation (framing) code based on the circular code was proposed in Michel (2012) with the identification of several X circular code motifs (X motifs shortly) in both ribosomal RNAs (rRNAs) and their decoding center, and transfer RNAs (tRNAs). We extended these results in two ways. First, three universal X motifs were determined in the ribosome decoding center: the X motif mAA containing the conserved nucleotides A1492 and A1493, the X motif mG containing the conserved nucleotide G530 and the X motif m with unknown biological function (El Soufi and Michel, 2014). Secondly, statistical analysis of X motifs of greatest lengths performed on different and large tRNA populations according to taxonomy, tRNA length and tRNA score showed that these X motifs have occurrence probabilities in the 5' and/or 3' regions of 16 isoaccepting tRNAs of prokaryotes and eukaryotes greater than the random case (Michel, 2013). We continue here the previous works with the identification of X motifs in rRNAs of prokaryotes and eukaryotes near the ribosome decoding center. Seven X motifs PrRNAXm conserved in 16S rRNAs of prokaryotes P and four X motifs ErRNAXm conserved in 18S rRNAs of eukaryotes E are identified near the ribosome decoding center. Furthermore, four very large X motifs of length greater than or equal to 20 nucleotides, 14 large X motifs of length between 16 and 19 nucleotides and several X motifs of length greater or equal to 9 nucleotides are found in tRNAs of prokaryotes. Some properties of these X motifs in tRNAs are described. These new results strengthen the concept of a translation code based on the circular code (Michel, 2012).

Circular code motifs in the ribosome decoding center.

A translation (framing) code based on the circular code was proposed in Michel (2012) with the identification of X circular code motifs (X motifs shortly) in the bacterial rRNA of Thermus thermophilus, in particular in the ribosome decoding center. Three classes of X motifs are now identified in the rRNAs of bacteria Escherichia coli and Thermus thermophilus, archaea Pyrococcus furiosus, nuclear eukaryotes Saccharomyces cerevisiae, Triticum aestivum and Homo sapiens, and chloroplast Spinacia oleracea. The universally conserved nucleotides A1492 and A1493 in all studied rRNAs (bacteria, archaea, nuclear eukaryotes, and chloroplasts) belong to X motifs (called mAA). The conserved nucleotide G530 in rRNAs of bacteria and archaea belongs to X motifs (called mG). Furthermore, the X motif mG is also found in rRNAs of nuclear eukaryotes and chloroplasts. Finally, a potentially important X motif, called m, is identified in all studied rRNAs. With the available crystallographic structures of the Protein Data Bank PDB, we also show that these X motifs mAA, mG, and m belong to the ribosome decoding center of all studied rRNAs with possible interaction with the mRNA X motifs and the tRNA X motifs. The three classes of X motifs identified here in rRNAs of several and different organisms strengthen the concept of translation code based on the circular code.