Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigen

CSR and related terms in SME owner-managers' mental models in six European countries: national context matters

As a contribution to the emerging field of corporate social responsibility (CSR) cognition, this article reports on the findings of an exploratory study that compares SME owner–managers’ mental models with regard to CSR and related concepts across six European countries (Belgium, Italy, Norway, France, UK, Spain). Utilising Repertory Grid Technique, we found that the SME owner–managers’ mental models show a few commonalities as well as a number of differences across the different country samples. We interpret those differences by linking individual cognition to macro-environmental variables, such as language, national traditions and dissemination mechanisms. The results of our exploratory study show that nationality matters but that classifications of countries as found in the comparative capitalism literature do not exactly mirror national differences in CSR cognition and that these classifications need further differentiation. The findings from our study raise questions on the universality of cognition of academic management concepts and warn that promotion of responsible business practice should not rely on the use of unmediated US American management terminology

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Characterization of high avidity CD4+ T cells in HIV Controllers

Background: HIV Controllers spontaneously control HIV replication to very low levels (< 50 copies HIV RNA/mL) in the absence of antiretroviral treatment. We previously reported that HIV Controllers harbor a pool of memory CD4+ T cells with the capacity to recognize the immunodominant Gag293 peptide with a particularly high TCR avidity. Since this property may allow the persistence of an activated cellular response against minimal amounts of virus, we set to further characterize the nature of these high avidity memory CD4+ T cells. Methods: HIV Controllers from the ANRS CO18 cohort (n=8) with a viral load < 50 copies/mL for over 10 years were compared to efficiently treated patients with a similarly undetectable viral load for over 10 years (HAART group, n=8). Primary CD4+ T cell lines were generated by stimulating PBMC with decreasing doses of the Gag293 peptide. Cytokine production was monitored by IFN-γ ELISPOT and intracellular cytokine staining assays. TCR diversity was evaluated by staining with a panel of 24 anti-TCR Vβ chain antibodies and by Immunoscope analysis. Results: After stimulation with a high dose of Gag293 peptide (10-5 M), IFN-γ-positive CD4+ T cell lines were obtained for all patients studied (response rate of 8/8 in both groups). However, at low peptide doses, specific cell lines were obtained only for HIV Controllers (response rate of 6/8 at 10-9 M and 3/8 at 10-11 M) but not for treated patients. The Vβ repertoire appeared more restricted in cell lines stimulated with low peptide doses, suggesting that only a subset of HIV Controller memory CD4+ T cells were of high avidity. Conclusion: The presence of a high avidity subset within the pool of Gag293-specific memory CD4+ T cells is unique to HIV Controllers

Molecular characterization of high avidity CD4+ T cells in HIV Controllers

Background: HIV Controllers spontaneously control HIV replication to levels undetectable by standard assays in the absence of antiretroviral treatment. We previously reported that HIV Controllers harbour a pool of memory CD4+ T cells able to respond to the immunodominant Gag293 peptide with particularly high TCR avidity. We set to functionally analyze high avidity CD4+ T cells and to characterize their TCRs at the molecular level. Methods: HIV Controllers from the ANRS CODEX CO21 cohort (n=8) were compared to efficiently treated patients (HAART group, n=8). Primary CD4+ T cell lines were generated by stimulating PBMCs with decreasing doses of Gag293 peptide. Next, Gag293-specific CD4+ T cells were analyzed for cytokine production by IFNγ ELISPOT and for Gag293/MHC-classII tetramer binding ability. TCR diversity in sorted Gag293/Tetramer+ cells was evaluated by immunoscope analysis. Results: Stimulation with low Gag293 peptide doses generated IFNγ-positive CD4+ T cell lines in HIV Controllers (response rate: 6/8 at 10-9M, 2/8 at 10-11M), but not in HAART patients, confirming the presence of high-avidity Gag-293-specific cells in the HIC group. The immunoscope analysis revealed major amplifications of certain TCR Vα and Vβ chains in the sorted tetramer+ Gag293-specific population, indicating the presence of dominant clonotypes. Conclusion: We identified a number of TCR Vα and Vβ chains preferentially expressed by the high-avidity population of Gag293-specific CD4+ T cells. Transfer of these TCRs to heterologous cells will help determine whether they are sufficient to confer the efficient Gag-specific responses characteristic of HIV Controllers

Microbiota stimulation generates LCMV-specific memory CD8+ T cells in SPF mice and determines their TCR repertoire during LCMV infection

International audienceBoth mouse and human harbour memory phenotype CD8+ T cells specific for antigens in hosts that have not been previously exposed to these antigens. The origin and the nature of the stimuli responsible for generation of CD44hi CD8+ T cells in specific pathogen-free (SPF) mice remain controversial. It is known that microbiota plays a crucial role in the prevention and resolution of systemic infections by influencing myelopoiesis, regulating dendritic cells, inflammasome activation and promoting the production of type I and II interferons. By contrast, here we suggest that microbiota has a direct effect on generation of memory phenotype CD44hiGP33+CD8+ T cells. In SPF mice, it generates a novel GP33+CD44hiCD8+ T cell sub-population associating the properties of innate and genuine memory cells. These cells are highly enriched in the bone marrow, proliferate rapidly and express immediate effector functions. They dominate the response to LCMV and express particular TCRβ chains. The sequence of these selected TCRβ chains overlaps with that of GP33+CD8+ T cells directly selected by microbiota in the gut epithelium of SPF mice, demonstrating a common selection mechanism in gut and peripheral CD8+ T cell pool. Therefore microbiota has a direct role in priming T cell immunity in SPF mice and in the selection of TCRβ repertoires during systemic infection. We identify a mechanism that primes T cell immunity in SPF mice and may have a major role in colonization resistance and protection from infection

HIV Controller CD4+ T cells preferentially express a public TCR clonotype that confers high avidity responses against Gag

Background: HIV Controllers are rare patients who spontaneously control HIV replication to levels below 50 copies viral RNA/ml in the absence of antiretroviral therapy. We previously reported that HIV Controllers harbor a pool of memory CD4+ T cells able to respond to minimal amounts of virus, due to the expression of T cell receptors (TCRs) with high avidity for immunodominant Gag epitopes. We set to characterize these high avidity TCRs at the molecular and functional levels. Methods: HIV Controllers from the ANRS CO21 CODEX cohort (n=8) were compared to efficiently treated patients (n=8). Primary CD4+ T cell lines were generated by stimulating PBMCs with decreasing doses of the immunodominant Gag293 peptide. Specific CD4+ T cells were labeled with Gag293-loaded MHC class II tetramers and sorted. The TCR repertoire of tetramer+ cells was evaluated by CDR3 length polymorphism analysis (Immunoscope) and sequencing. Highly represented TCR Vα and Vβ chains were cloned in bicistronic lentiviral vectors and tested for CD69 induction and cytokine production. Results: Stimulation with low Gag293 peptide doses generated IFNγ-positive CD4+ T cell lines for HIV Controllers, but rarely for treated patients, confirming the predominance of high avidity cells in the Controller group. Immunoscope analysis revealed a major amplification of TCR Vα24 chains with a 10 a.a. CDR3 in sorted tetramer+ cells from Controllers, while this amplification was rarely detected in treated patients (P< 0.05). Sequencing revealed the presence of a public TCR Vα24-J17 clonotype shared by 6 Controllers and 2 treated patients. TCRs comprised of the public Vα24 chain and of highly expressed Vβ chains conferred high avidity Gag293 recognition and polyfunctionality to transduced T cells. One of these TCRs could achieve Gag293 recognition in the context of 4 distinct HLA-DR molecules, possibly explaining its public nature. Conclusions: We identified a highly prevalent TCR sequence preferentially expressed by Gag-specific CD4+ T cells from HIV Controllers. This public clonotype confers high avidity polyfunctional responses to Gag, raising the possibility that it could be used as molecular marker of efficient responses against HIV

HLA-A*01:03, HLA-A*24:02, HLA-B*08:01, HLA-B*27:05, HLA-B*35:01, HLA-B*44:02, and HLA-C*07:01 Monochain Transgenic/H-2 Class I Null Mice:Novel Versatile Preclinical Models of Human T Cell Responses

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α(1)α(2) H chain domains fused with a mouse α(3) domain and covalently linked to human β(2)-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8(+) T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ–producing CD8(+) T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8(+) T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell–based vaccines

Public T cell receptors confer high-avidity CD4 responses to HIV controllers.

International audienceThe rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure