Longitudinal humoral response after SARS-CoV-2 vaccination in ocrelizumab treated MS patients: To wait and repopulate?

OBJECTIVE: The objective of this study was to measure humoral responses after SARS-CoV-2 vaccination in MS patients treated with ocrelizumab (OCR) compared to MS patients without disease modifying therapies (DMTs) in relation to timing of vaccination and B-cell count. METHODS: OCR treated patients were divided into an early and a late group (cut-off time 12 weeks between infusion and first vaccination). Patients were vaccinated with mRNA-1273 (Moderna). B-cells were measured at baseline (time of first vaccination) and SARS-CoV-2 antibodies were measured at baseline, day 28, 42, 52 and 70. RESULTS: 87 patients were included (62 OCR patients, 29 patients without DMTs). At day 70, seroconversion occurred in 39.3% of OCR patients compared to 100% of MS patients without DMTs. In OCR patients, seroconversion varied between 26% (early group) to 50% (late group) and between 27% (low B-cells) to 56% (at least 1 detectable B-cell/µL). CONCLUSIONS: Low B-cell counts prior to vaccination and shorter time between OCR infusion and vaccination may negatively influence humoral response but does not preclude seroconversion. We advise OCR treated patients to get their first vaccination as soon as possible. In case of an additional booster vaccination, timing of vaccination based on B-cell count and time after last infusion may be considered.

An optimized CFSE-based T-cell suppression assay to evaluate the suppressive capacity of regulatory T-cells induced by human tolerogenic dendritic cells.

In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T-cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC-induced-Treg activity. IL-10-generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T-cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL-10 DC-induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen-presenting cells (APC) as T-cell stimulation. Lowering the ratio between responder T-cells and stimulator mDC present in the coculture clearly improved the read-out of the suppression assay. Furthermore, mDC-primed T-cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T-cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity.

Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.

BACKGROUND: Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS: Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS: The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION: We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.

Melanoma-specific tumor-infiltrating lymphocytes but not circulating melanoma-specific T cells may predict survival in resected advanced-stage melanoma patients.

PURPOSE: To study the effect of autologous tumor cell vaccinations on the presence and numbers of circulating CD8+ T cells specific for tumor-associated antigens (TAA) in metastatic melanoma patients. To investigate the correlation between the presence of tumor-infiltrating lymphocytes (TIL) and circulating TAA-specific CD8+ T cells before and after autologous tumor cell vaccination with overall survival. EXPERIMENTAL DESIGN: Twenty-five stage III and resected stage IV metastatic melanoma patients were adjuvantly treated with a series of intracutaneously injected autologous tumor cell vaccinations, of which the first two contained BCG as an immunostimulatory adjuvant. Tumor samples and blood samples obtained before and after vaccination of these patients were studied for the presence of TAA-specific T cells using HLA-tetramers and results were correlated with survival. RESULTS: In 5 of 17 (29%) melanoma patients, circulating TAA-specific T cells were detectable prior to immunizations. No significant changes in the frequency and specificity were found during the treatment period in all patients. Presence of circulating TAA-specific T cells was not correlated with survival (log rank, P=0.215). Inside melanoma tissue, TAA-specific TIL could be detected in 75% of 16 available tumor samples. In case of detectable TAA-specific TIL, median survival was 22.5 months compared to median survival of 4.5 months in case of absence of TAA-specific T cells (log rank, P=0.0094). In none of the patients, TAA-specific T cells were found both in tumor tissue and blood at the same time. CONCLUSIONS: These data suggest that the presence of TAA-specific TILs forms a prognostic factor, predicting improved survival in advanced-stage melanoma patients. The absence of TAA-specific T cells in the circulation suggests that homing of the tumor-specific T cell population to the tumor site contributes to the effectiveness of antitumor immunity.

The fimbria gene cluster of nonencapsulated Haemophilus influenzae.

The occurrence of fimbria gene clusters in nonencapsulated Haemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of hifA to hifE inserted in the chromosome between purE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the -10 and -35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity. hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA and purE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes purE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element.

Interaction between HLA-DM and HLA-DR involves regions that undergo conformational changes at lysosomal pH.

Antigenic peptide loading of major histocompatibility complex class II molecules is enhanced by lysosomal pH and catalyzed by the HLA-DM molecule. The physical mechanism behind the catalytic activity of DM was investigated by using time-resolved fluorescence anisotropy (TRFA) and fluorescence binding studies with the dye 8-anilino-1-naphthalenesulfonic acid (ANS). We demonstrate that the conformations of both HLA-DM and HLA-DR3, irrespective of the composition of bound peptide, are pH sensitive. Both complexes reversibly expose more nonpolar regions upon protonation. Interaction of DM with DR shields these hydrophobic domains from the aqueous environment, leading to stabilization of the DM and DR conformations. At lysosomal pH, the uncovering of additional hydrophobic patches leads to a more extensive DM-DR association. We propose that DM catalyzes class II peptide loading by stabilizing the low-pH conformation of DR, favoring peptide exchange. The DM-DR association involves a larger hydrophobic surface area with DR/class II-associated invariant chain peptides (CLIP) than with stable DR/peptide complexes, explaining the preferred association of DM with the former. The data support a release mechanism of DM from the DM-DR complex through reduction of the interactive surface, upon binding of class II molecules with antigenic peptide or upon neutralization of the DM-DR complex at the cell surface.

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading.

BACKGROUND: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide 'editor', favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive. RESULTS: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM. CONCLUSIONS: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.

Human histocompatibility leukocyte antigen (HLA)-DM edits peptides presented by HLA-DR according to their ligand binding motifs.

Human histocompatibility leukocyte antigen (HLA)-DM is a facilitator of antigen presentation via major histocompatibility complex (MHC) class II molecules. In the absence of HLA-DM, MHC class II molecules do not present natural peptides, but tend to remain associated with class II-associated invariant chain peptides (CLIP). Recently, DM was shown to catalyze the release of CLIP from HLA-DR. We have investigated which peptides bound to HLA-DR are vulnerable to release upon encountering DM. By directed substitution of allele-specific anchor residues between CLIP and DR3-cognate peptides and the application of recombinant DM we show that DM catalyzes the release of those peptides bound to HLA-DR3 that do not have appropriate anchor residues and, hence, no optimal ligand binding motif. Thus, HLA-DM acts as a peptide editor, facilitating selection of peptides that stably bind to class II molecules for eventual presentation to the immune system from the pool of available peptides.

Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes.

Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by using mutants that were inactivated in distinct fimbrial genes. Both the major and minor subunits were required for adherence of H. influenzae to oropharyngeal epithelial cells and human erythrocytes carrying the AnWj antigen. Cloning of defined H. influenzae fimbrial genes in an Escherichia coli strain with type 1 fimbriae yielded recombinants expressing high amounts of HifA-containing H. influenzae fimbriae either with or without coexpression of both H. influenzae minor subunits. Both clones exhibited the specific adherence properties of H. influenzae fimbriae, implying that the minor H. influenzae subunits are dispensable for adherence and that the adhesive domain resides in the major subunit, HifA. In H. influenzae itself, the minor subunits probably affect adherence by raising the number of fimbriae above the minimal level required to establish adherence.

The fimbrial gene cluster of Haemophilus influenzae type b.

Haemophilus influenzae infections are preceded by airway colonization, a process facilitated by fimbriae. Here, we identified the complete fimbrial gene cluster of H. influenzae type b. HifA forms the major subunit. HifB, a periplasmic chaperone, and HifC, an outer membrane usher, are typical assembly genes; their inactivation abolished fimbriae formation. HifD and HifE are putative minor subunits, both participating in fimbriae biogenesis. Inactivation of either one drastically reduced fimbriae expression. HifD represents a novel type of fimbrial subunit with lipoprotein characteristics, pointing to a membrane-associated function of HifD. Transcription of all fimbrial genes is coregulated through two clustered promoters. The flanking of the fimbrial gene cluster by repetitive extragenic palindromic sequences together with a partial duplication of an adjacent unrelated operon indicated that the cluster was once inserted in the H. influenzae genome as a mobile virulence unit.

Phase variation of H. influenzae fimbriae: transcriptional control of two divergent genes through a variable combined promoter region.

The expression of H. influenzae fimbriae is subject to reversible phase variation between three expression levels. This phenomenon is controlled at the transcriptional level of two divergently orientated genes, hifA and hifB, encoding the major fimbrial subunit and the fimbrial chaperone, respectively. The hifA and hifB promoter regions were found to be clustered through an almost complete divergent overlap with a variable DNA backbone of repetitive TA units. Variation in the number of units changes the normally strictly constrained spacing between the -35 and -10 sequences and controls the bidirectional transcription initiation, thus forming a novel mechanism directing multiple gene transcription.

Fimbria-mediated adherence and hemagglutination of Haemophilus influenzae.

The gram-negative bacterium Haemophilus influenzae expresses morphologically and functionally distinct types of fimbriae, of which the LKP fimbriae mediate hemagglutination and adherence to human epithelial cells but hamper mucosal invasion. Therefore, the both in vivo and in vitro observed fimbrial phase variation may contribute to the pathogenesis of the infection. The existence of greater than 14 LKP serotypes hampers vaccine development based on fimbriae, since a monovalent fimbria vaccine confers protection against only the homologous strain. Cloning of the fimbrial genes in Escherichia coli results in the expression of morphologically intact fimbriae. Analysis of the cloned DNA indicates that a fimbrial gene cluster is necessary for formation of complete fimbriae and for fimbria-mediated adherence. The gene encoding the subunit is highly conserved among H. influenzae and belongs to the family of E. coli fimbriae. The phase variation is transcriptionally regulated by variation of the length of the reiterated sequence that forms the promoter region of the subunit gene.

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.