Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus

Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PK8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ

Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response

Cell signaling for T-cell growth, differentiation, and apoptosis is initiated in the cholesterol-rich microdomains of the plasma membrane known as lipid rafts. Herein, we investigated whether enrichment of membrane cholesterol in lipid rafts affects antigen-specific CD4 T-helper cell functions. Enrichment of membrane cholesterol by 40–50% following squalene administration in mice was paralleled by an increased number of resting CD4 T helper cells in periphery. We also observed sensitization of the Th1 differentiation machinery through co-localization of IL-2Rα, IL-4Rα, and IL-12Rβ2 subunits with GM1 positive lipid rafts, and increased STAT-4 and STAT-5 phosphorylation following membrane cholesterol enrichment. Antigen stimulation or CD3/CD28 polyclonal stimulation of membrane cholesterol-enriched, resting CD4 T-cells followed a path of Th1 differentiation, which was more vigorous in the presence of increased IL-12 secretion by APCs enriched in membrane cholesterol. Enrichment of membrane cholesterol in antigen-specific, autoimmune Th1 cells fostered their organ-specific reactivity, as confirmed in an autoimmune mouse model for diabetes. However, membrane cholesterol enrichment in CD4+ Foxp3+ T-reg cells did not alter their suppressogenic function. These findings revealed a differential regulatory effect of membrane cholesterol on the function of CD4 T-cell subsets. This first suggests that membrane cholesterol could be a new therapeutic target to modulate the immune functions, and second that increased membrane cholesterol in various physiopathological conditions may bias the immune system toward an inflammatory Th1 type response

Double Negative (CD3+4−8−) TCRαβ Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes

-reactive T-cells. Herein, we analyzed the function and phenotype of DNCD3 splenic cells in young NOD mice predisposed to several autoimmune disorders among which, the human-like autoimmune diabetes. with a predominant Vβ13 gene usage.T-regulatory cells. DNCD3 splenic cells could be potentially manipulated towards the development of autologous cell therapies in autoimmune diabetes

Humanized DRAGA mice immunized with Plasmodium falciparum sporozoites and chloroquine elicit protective pre-erythrocytic immunity

Abstract Background Human-immune-system humanized mouse models can bridge the gap between humans and conventional mice for testing human vaccines. The HLA-expressing humanized DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice reconstitute a functional human-immune-system and sustain the complete life cycle of Plasmodium falciparum. Herein, the DRAGA mice were investigated for immune responses following immunization with live P. falciparum sporozoites under chloroquine chemoprophylaxis (CPS-CQ), an immunization approach that showed in human trials to confer pre-erythrocytic immunity. Results The CPS-CQ immunized DRAGA mice (i) elicited human CD4 and CD8 T cell responses to antigens expressed by P. falciparum sporozoites (Pfspz) and by the infected-red blood cells (iRBC). The Pfspz-specific human T cell responses were found to be systemic (spleen and liver), whereas the iRBCs-specific human T cell responses were more localized to the liver, (ii) elicited stronger antibody responses to the Pfspz than to the iRBCs, and (iii) they were protected against challenge with infectious Pfspz but not against challenge with iRBCs. Conclusions The DRAGA mice represent a new pre-clinical model to investigate the immunogenicity and protective efficacy of P. falciparum malaria vaccine candidates

The humanized DRAGA mouse (HLA-A2. HLA-DR4. RAG1 KO. IL-2R g c KO. NOD) establishes inducible and transmissible models for influenza type A infections

We have engineered a Human Immune System (HIS)-reconstituted mouse strain (DRAGA mouse: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD) in which the murine immune system has been replaced by a long-term, functional HIS via infusion of CD34+ hematopoietic stem cells (HSC) from cord blood. Herein, we report that the DRAGA mice can sustain inducible and transmissible H1N1 and H3N2 influenza A viral (IAV) infections. DRAGA female mice were significantly more resilient than the males to the H3N2/Aichi infection, but not to H3N2/Hong Kong, H3N2/Victoria, or H1N1/PR8 sub-lethal infections. Consistently associated with large pulmonary hemorrhagic areas, both human and murine Factor 8 mRNA transcripts were undetectable in the damaged lung tissues but not in livers of DRAGA mice advancing to severe H1N1/PR8 infection. Infected DRAGA mice mounted a neutralizing anti-viral antibody response and developed lung-resident CD103 T cells. These results indicate that the DRAGA mouse model for IAV infections can more closely approximate the human lung pathology and anti-viral immune responses compared to non-HIS mice. This mouse model may also allow further investigations into gender-based resilience to IAV infections, and may potentially be used to evaluate the efficacy of IAV vaccine regimens for humans

Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD)

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope (180WGIHHPPNSKEQ QNLY195) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use

Co-localization of cytokine receptors with lipid rafts of resting CD4 T-cells before and after squalene treatment.

<p>Resting CD4 T-cells from individual F1 mice (n = 5/group) before and 7 days after squalene treatment (180 µg/mouse) were analyzed for interleukin receptor expression and distribution at the single-cell level by CLSM. Cells were stained with IL-4Rα-, IL-12Rβ2-, or IL-2Rα-PE conjugates, and co-stained for GM1 ganglioside by CTB- FITC conjugate and for nuclei with DAPI. First column indicates single-channel color for DAPI staining (blue), second column indicates GM1 staining (green), third column indicates interleukin receptor (IL-Rs) staining (red), and last column indicates merged channels at X63 magnification. <i>Top-two rows</i>, indicate cells from untreated (<i>upper row</i>) and squalene treated mice (<i>lower row</i>) stained for IL-4Rα. <i>Middle-two rows</i>, indicate cells from untreated (<i>upper row</i>) and squalene treated mice (<i>lower row</i>) stained for IL-12Rβ2. <i>Bottom-two rows</i>, indicate cells from untreated (<i>upper row</i>) and squalene treated mice (<i>lower row</i>) stained for IL-2Rα. Arrows indicate presence of IL-Receptor co-expression with the GM1 resident of lipid rafts. Enlargements of the merged channels are depicted to the <i>right</i> along with two different angles of the membrane for each IL-Receptor at X220 magnification. Shown are representative images in one of three experiments.</p

Alteration in cytokine receptors mRNA expression after squalene enrichment of membrane cholesterol in resting lymphocytes.

<p>(<b>A</b>) Quantitative real-time RT-PCR of IL-4Rα, IL-12Rβ2, and IL-2Rα mRNA extracted from peripheral blood lymphocytes of individual F1 mice (n = 5/group) analyzed before squalene treatment (dark bars) and 7 days after squalene injection (180 µg/mouse) (light bars). Y axis indicates the mean fold increase in mRNA expression level relative to the endogenous 18S rRNA expression level (control ± SD). Shown are two combined separate experiments (*<i>p</i> values<0.05). (<b>B</b>) Aliquots samples in panel A were stained with CD4-FITC conjugate, co-stained either with IL-4Rα-PE or IL-12Rβ2-PE or IL-2Rα-PE conjugates, and analyzed by FACS at the single-cell level for the surface IL-Rs expression level based on MFI measurements. Shown are the IL-Rs MFI values ± SD measured in individual mice before and after squalene treatment. Of note, no significant changes occurred in the IL-Rs expression on cell surface after squalene treatment (*<i>p</i> values>0.05).</p