Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19.

We report a Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2 RγcKO.NOD) for COVID-19 research. DRAGA mice express transgenically HLA-class I and class-II molecules in the mouse thymus to promote human T cell development and human B cell Ig-class switching. When infused with human hematopoietic stem cells from cord blood reconstitute a functional human immune system, as well as human epi/endothelial cells in lung and upper respiratory airways expressing the human ACE2 receptor for SARS-CoV-2. The DRAGA mice were able to sustain SARS-CoV-2 infection for at least 25 days. Infected mice showed replicating virus in the lungs, deteriorating clinical condition, and human-like lung immunopathology including human lymphocyte infiltrates, microthrombi and pulmonary sequelae. Among the intra-alveolar and peri-bronchiolar lymphocyte infiltrates, human lung-resident (CD103+) CD8+ and CD4+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, suggesting anti-viral cytotoxic activity. Infected mice also mounted human IgG antibody responses to SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathological mechanisms and testing the safety and efficacy of candidate vaccines and therapeutics.

A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19.

We report the first Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19 research. This mouse is reconstituted with human cord blood-derived, HLA-matched hematopoietic stem cells. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and TMPRSS2 serine protease co-localized on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing deteriorated clinical condition, replicating virus in the lungs, and human-like lung immunopathology including T-cell infiltrates, microthrombi and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103+) CD8+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice also developed antibodies against the SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.

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.

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

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.

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.

Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice.

Humanized mice expressing Human Leukocyte Antigen (HLA) class I or II transgenes have been generated, but the role of class I vs class II on human T and B cell reconstitution and function has not been investigated in detail. Herein we show that NRG (NOD.RagKO.IL2RγcKO) mice expressing HLA-DR4 molecules (DRAG mice) and those co-expressing HLA-DR4 and HLA-A2 molecules (DRAGA mice) did not differ in their ability to develop human T and B cells, to reconstitute cytokine-secreting CD4 T and CD8 T cells, or to undergo immunoglobulin class switching. In contrast, NRG mice expressing only HLA-A2 molecules (A2 mice) reconstituted lower numbers of CD4 T cells but similar numbers of CD8 T cells. The T cells from A2 mice were deficient at secreting cytokines, and their B cells could not undergo immunoglobulin class switching. The inability of A2 mice to undergo immunoglobulin class switching is due to deficient CD4 helper T cell function. Upon immunization, the frequency and cytotoxicity of antigen-specific CD8 T cells in DRAGA mice was significantly higher than in A2 mice. The results indicated a multifactorial effect of the HLA-DR4 transgene on development and function of human CD4 T cells, antigen-specific human CD8 T cells, and immunoglobulin class switching.

Nonobese Diabetic (NOD) Mice Lack a Protective B-Cell Response against the "Nonlethal" Plasmodium yoelii 17XNL Malaria Protozoan.

Background. Plasmodium yoelii 17XNL is a nonlethal malaria strain in mice of different genetic backgrounds including the C57BL/6 mice (I-Ab/I-Enull) used in this study as a control strain. We have compared the trends of blood stage infection with the nonlethal murine strain of P. yoelii 17XNL malaria protozoan in immunocompetent Nonobese Diabetic (NOD) mice prone to type 1 diabetes (T1D) and C57BL/6 mice (control mice) that are not prone to T1D and self-cure the P. yoelii 17XNL infection. Prediabetic NOD mice could not mount a protective antibody response to the P. yoelii 17XNL-infected red blood cells (iRBCs), and they all succumbed shortly after infection. Our data suggest that the lack of anti-P. yoelii 17XNL-iRBCs protective antibodies in NOD mice is a result of parasite-induced, Foxp3+ T regulatory (Treg) cells able to suppress the parasite-specific antibody secretion. Conclusions. The NOD mouse model may help in identifying new mechanisms of B-cell evasion by malaria parasites. It may also serve as a more accurate tool for testing antimalaria therapeutics due to the lack of interference with a preexistent self-curing mechanism present in other mouse strains.

Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

BACKGROUND: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. METHODS: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. RESULTS: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. CONCLUSIONS: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Humanized HLA-DR4 mice fed with the protozoan pathogen of oysters Perkinsus marinus (Dermo) do not develop noticeable pathology but elicit systemic immunity.

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.

Long-term silencing of autoimmune diabetes and improved life expectancy by a soluble pHLA-DR4 chimera in a newly-humanized NOD/DR4/B7 mouse.

Several human MHC class II (HLA) molecules are strongly associated with high incidence of autoimmune diseases including type 1 diabetes (T1D). The HLA-humanized mice may thus represent valuable tools to test HLA-based vaccines and therapeutics for human autoimmune diseases. Herein, we have tested the therapeutic potential of a soluble HLA-DR4-GAD65 271-280 (hu DEF-GAD65) chimera of human use in a newly-generated NOD/DR4/B7 double transgenic (dTg) mouse that develops spontaneously an accelerated T1D regardless the gender. The NOD/DR4/B7 dTg mice generated by a two-step crossing protocol express the HLA-DR*0401 molecules on 20% of antigen presenting cells, the human B7 molecules in pancreas, and HLA-DR4/GAD65-specific T-cells in the blood. Some 75% of pre-diabetic NOD/DR4/B7 dTg mice treated with hu DEF-GAD65 chimera remained euglycemic and showed a stabilized pancreatic insulitis 6 months after treatment. The 25% non responders developing hyperglycemia survived 3-4 months longer than their untreated littermates. T1D prevention by this reagent occurred by a Th2/TR-1 polarization in the pancreas. This study strongly suggests that the use of soluble pHLA reagents to suppress/stabilize the T1D progression and to extend the life expectancy in the absence of side effects is an efficient and safe therapeutic approach.

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-12Rß2 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.

Differential effect of CD4+Foxp3+ T-regulatory cells on the B and T helper cell responses to influenza virus vaccination.

The T-regulatory (T-reg) cells restrict the T-cell functions in various viral infections including influenza infection. However little is known about the effect of T-regs in influenza vaccination. Herein, we found that immunization of BALB/c mice with a prototype of UV-inactivated influenza PR8/A/34 virus vaccine expanded the CD4(+)Foxp3(+) T-reg pool and fostered the development of virus-specific CD4(+)Foxp3(+) T-reg cells. Increasing the size of Foxp3(+) T-reg pool did not alter the primary PR8-specific B-cell response, but it did suppress the primary and memory PR8-specific T helper responses induced by vaccination. In contrast, the vaccination-induced T helper cell response was augmented in the absence of CD4(+)Foxp3(+) T-reg cells. Since CD4 T helper cells contribute to anti-influenza protection, therapeutic "quenching" of T-reg function prior to vaccination may enhance the efficacy of influenza vaccination.

Double negative (CD3+ 4- 8-) TCR alphabeta splenic cells from young NOD mice provide long-lasting protection against type 1 diabetes.

BACKGROUND: Double negative CD3(+)4(-)8(-) TCR alphabeta splenic cells (DNCD3) can suppress the immune responses to allo and xenografts, infectious agents, tumors, and some autoimmune disorders. However, little is known about their role in autoimmune diabetes, a disease characterized by the reduction of insulin production subsequent to destruction of pancreatic beta-cells by a polyclonal population of self-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. METHODOLOGY/PRINCIPAL FINDINGS: DNCD3 splenic cells from young NOD mice (1) provided long-lasting protection against diabetes transfer in NOD/Scid immunodeficient mice, (2) proliferated and differentiated in the spleen and pancreas of NOD/Scid mice and pre-diabetic NOD mice into IL-10-secreting T(R)-1 like cells in a Th2-like environment, and (3) their anti-diabetogenic phenotype is CD3(+)(CD4(-)CD8(-))CD28(+)CD69(+)CD25(low) Foxp3(-) iCTLA-4(-)TCR alphabeta(+) with a predominant Vbeta13 gene usage. CONCLUSIONS/SIGNIFICANCE: These findings delineate a new T regulatory component in autoimmune diabetes apart from that of NKT and CD4(+)CD25(high) Foxp3(+)T-regulatory cells. DNCD3 splenic cells could be potentially manipulated towards the development of autologous cell therapies in autoimmune diabetes.

Reversal of type 1 diabetes by a new MHC II-peptide chimera: "Single-epitope-mediated suppression" to stabilize a polyclonal autoimmune T-cell process.

Polyclonality of self-reactive CD4(+) T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II-peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T-cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3: 383-391]. Since most of the glutamic acid decarboxylase 65 (GAD65)-specific CD4(+) T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T-regulatory cell population (GAD65(217-230)-specific CD4(+) T cells) using a soluble I-A(αß) (g7)/GAD65(217-230)/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T-cell process. Activated GAD65(217-230)-specific T cells and a fraction of the diabetogenic (B(9-23)-specific) T cells were polarized toward the IL-10-secreting T-regulatory type 1-like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post-therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the ß cells. These findings argue for the stabilization of a polyclonal self-reactive T-cell process by a single epitope-mediated bystander suppression. Dimeric MHC class II-peptide chimeras-like approach may provide rational grounds for the development of more efficient antigen-specific therapies in type 1 diabetes.

CD28 signaling in T regulatory precursors requires p56lck and rafts integrity to stabilize the Foxp3 message.

Naturally occurring CD4(+)25(high)Foxp3(+) T regulatory (T-reg) cells are critical for maintaining tolerance to self and non-self Ags. The Foxp3 master-regulatory gene and CD28 costimulation are both required for thymic development and suppressogenic function of CD4(+)25(high)Foxp3(+) T-regs. Herein, we show that the sole CD28 stimulation of T-reg thymic precursors augments Foxp3 expression through the increase in Foxp3 mRNA life span by a mechanism involving p56(lck) and its binding motif on CD28 cytosolic tail, as well as the lipid rafts. We found that 1) the glycosphingolipids and cholesterol components of lipid rafts were highly expressed and unusually partitioned in T-reg thymic precursors as compared with the conventional T cell precursors, 2) the CD28 receptor density on cell membrane is proportional with the content of cholesterol in lipid rafts and with the level of Foxp3 mRNA expression in T-reg precursors, and 3) the CD28-mediated increase of Foxp3 mRNA life span was paralleled by an increased proliferative and suppressogenic capacity of terminally differentiated CD4(+)25(high)Foxp3(+) T-reg precursors. Thus, the functional integrity of CD28 receptor p56(lck) and plasma membrane lipid rafts are all prerequisites for up-regulation and long-term expression of Foxp3 mRNA transcripts in CD4(+)25(high)Foxp3(+) T-reg precursors.

A peptide-major histocompatibility complex II chimera favors survival of pancreatic beta-islets grafted in type 1 diabetic mice.

BACKGROUND: Transplantation of pancreatic islets showed a tremendous progress over the years as a promising, new therapeutic strategy in patients with type 1 diabetes. However, additional immunosuppressive drug therapy is required to prevent rejection of engrafted islets. The current immunosuppressive therapies showed limited success in maintaining long-term islet survival as required to achieve insulin independence in type 1 diabetes, and they induce severe adverse effects. Herein, we analyzed the effects of a soluble peptide-major histocompatibility complex (MHC) class II chimera aimed at devising an antigen-specific therapy for suppression of anti-islet T cell responses and to improve the survival of pancreatic islets transplants. METHODS: Pancreatic islets from transgenic mice expressing the hemagglutinin antigen in the beta islets under the rat insulin promoter (RIP-HA) were grafted under the kidney capsule of diabetic, double transgenic mice expressing hemagglutinin in the pancreas and T cells specific for hemagglutinin (RIP-HA, TCR-HA). The recipient double transgenic mice were treated or not with the soluble peptide-MHC II chimera, and the progression of diabetes, graft survival, and T cell responses to the grafted islets were analyzed. RESULTS: The peptide-MHC II chimera protected syngeneic pancreatic islet transplants against the islet-reactive CD4 T cells, and prolonged the survival of transplanted islets. Protection of transplanted islets occurred by polarization of antigen-specific memory CD4 T cells toward a Th2 anti-inflammatory response. CONCLUSIONS: The peptide-MHC II chimera approach is an efficient and specific therapeutic approach to suppress anti-islet T cell responses and provides a long survival of pancreatic grafted islets.

Differential partitioning and trafficking of GM gangliosides and cholesterol-rich lipid rafts in thymic and splenic CD4 T cells.

The GM gangliosides and cholesterol components of plasma membrane lipid rafts play an important role in the recruitment and signaling of protein receptors in eukaryotic cells. Herein, we have analyzed at the single-cell level the partitioning and intracellular trafficking of GM gangliosides and cholesterol in quiescent (CD4+CD69-) and CD3-activated (CD4+CD69+) thymic and splenic T cells. First, regardless the gender and the quiescent or activated status of T cells, the GM and cholesterol content in cytosol and plasma membrane as well as the expression levels of GM synthase, Sphingomyelin phosphodiestarase 2 and HMG Co-A reductase genes involved in GM and cholesterol synthesis were constantly lower in CD4 thymocytes than in CD4 splenocytes. Second, we detected variations in the balance between GM and cholesterol in plasma membrane depending on aging, and found that deprivation of cellular cholesterol does not necessarily affect the GM content in both quiescent CD4 thymocytes and splenocytes. Third, CD3 stimulation up-regulated the GM and little if any the cholesterol content in both thymic and splenic CD4 T cells, suggesting a cross talk between the CD3 signaling and GM but not cholesterol biosynthesis pathway. Fourth, partitioning and trafficking of GM to the plasma membrane depended on the transport of ceramide precursors from endoplasmic reticulum to Golgi network, as well as on the synthesis, glycosylation and vesicular assembly in trans-Golgi, and less on the cytoskeleton architecture in both quiescent and activated CD4 thymic and splenic T cells. Together, these findings suggest that the differential partitioning and intracellular trafficking of GM and cholesterol in thymic and splenic CD4 T cells may account for the stage of functional maturation.

Down-regulation of autoreactive T-cells by HMG CoA reductase inhibitors.

The inhibitors of HMG CoA reductase (statins) are widely used as cholesterol-lowering drugs with excellent safety records in hypercholesterolemic patients. Statins exert pleiotropic effects on a variety of cells, and they were recently described as a new class of immune modulators. Depending on their structure, dose, and route of administration, statins regulate the function of both the antigen-presenting cells and T-cells by HMG CoA reductase-dependent and independent mechanisms. Herein, we describe these mechanisms leading to prevention, amelioration, and reversal of autoimmune diseases. We also present data from our laboratories showing for the first time that in a double transgenic mouse model for autoimmune diabetes, atorvastatin (lipitor) prevented the onset of disease when administered in the neonatal period, and stabilized the glucose levels when administered in mice developing a mild form of diabetes.

Soluble, dimeric HLA DR4-peptide chimeras: an approach for detection and immunoregulation of human type-1 diabetes.

Still there are no effective methods to predict or cure type 1 diabetes (T1D) in humans. Soluble, dimeric MHC class II-peptide (DEF) chimeras have potential for both early diagnosis and immunospecific therapy. DEF chimeras prevent and reverse diabetes in mice by stimulating antigen-specific type 1 T regulatory cell (Tr1)-like cells. We also showed that diabetes could be predicted by changes in the phenotype of autoreactive CD4 T cells in peripheral blood. Herein, we demonstrated that human DEF (HLA-DR*0401/Fcgamma1) chimeras expressing peptides of beta-cell antigens stimulate Tr1-like cells in blood of patients with T1D, non-diabetic relatives, and controls. Furthermore, the specific and stable binding of DEF chimeras to cognate TCR and CD4 coreceptor allowed quantification and phenotyping of autoreactive CD4 T cells in non-stimulated blood by FACS. Our results indicate that (1) autoreactive CD4 T cells to GAD65 autoantigen are commonly present in humans expressing diabetes-susceptible HLA-DR*0401 molecules; (2) these autoreactive T cells undergo avidity maturation upon encountering the self antigen early in life; (3) the disease is associated with an imbalance between autoreactive CD4+CD25+ and CD4+CD69+ T cells specific for GAD65. Based on this, we propose a model to explain the kinetics of autoreactive CD4 T cells in blood during the natural history of T1D.

Efficacy of clonal deletion vs. anergy of self-reactive CD4 T-cells for the prevention and reversal of autoimmune diabetes.

The self-reactive CD4 T-cells play an essential role in triggering and sustaining organ-specific autoimmune diseases. Silencing or elimination of these cells can prevent and reverse an autoimmune process. We have previously showed that a single dose-administration of a soluble dimeric MHC II-peptide chimera (DEF) in double-transgenic mice delayed the onset autoimmune diabetes, and restored the euglycemia in already diabetic mice for a period of 1 week. DEF dimer protection relied on induction of anergy of diabetogenic CD4 T-cells in spleen, and stimulation of IL-10-secreting T regulatory type 1 cells in pancreas. Herein, we show that an octameric form of DEF has doubled the period of protection and reversal of disease by clonal deletion of diabetogenic CD4 T-cells in both the thymic and peripheral compartments. Deletion occurred by activation-induced cell death subsequent to repartitioning and signaling of FAS-FADD apoptotic module in the plasma membrane lipid rafts. Our previous and present data indicated first, that DEF valence translates into various effects on the antigen-specific CD4 T-cells, i.e., Th2 immune deviation, anergy, and apoptosis. Second, the present findings argue for a better efficacy of clonal deletion than anergy of diabetogenic CD4 T-cells for the protection and reversal of autoimmune diabetes.