Murine Major Histocompatibility Complex Class Research Articles

A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes.

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits Tcell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in Tcells remains enigmatic. Here weinvestigated how PD-1 affects transcriptome changes induced by Tcell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of Tcell populations.

Natural Spleen Cell Ligandome in Transporter Antigen Processing-Deficient Mice.

Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using high-throughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P1 position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.

Sleep restriction alters the temporal expression of major histocompatibility complex class II molecules in murine lymphoid tissues

Inadequate sleep is a major health concern of modern societies in view of the increased morbidity and mortality rates from physiological disturbances, including compromised adaptive immune responses. Many studies investigated the effect sleep restriction (SR) on the normal immune response in terms of leukocyte number and circulating cytokine and T helper cell (Th) profiles, but none considered the major histocompatibility complex (MHC), namely class II molecules. As no information exists about the normal temporal expression of MHC class II, the present study aimed at understanding how SR affects the adaptive immune response via altering the 1) normal daily expression profile and 2) overall constitutive levels of murine MHC class II by leukocytes’ isolates from spleen and axillary lymph nodes. Male C57BL/6 mice were acclimatized to 12:12 light/dark cycle (lights on at 0700, corresponding to Zeitgeber time (ZT) 0) for a week before splitting into 2 groups: control (C) and SR (exposed to a 12 and 18 h of activity, respectively). SR was carried for one week before lymphoid tissues from both C and SR mice were sampled at the following time points: ZT0, ZT5, ZT10, ZT13, and ZT18. Spleen and lymph node cells were assessed for leukocyte number and MHC class II expression at the preselected time points using flow cytometry. SR resulted in a 21% decrease in granulocyte and 24% increase in agranulocyte numbers. MHC class II expression in both lymphoid tissues of C mice varied synchronously across the preselected times of day; they were relatively high just prior to activity onset and later in this period. Comparatively, the diurnal protein profile was altered in both lymphoid tissues of SR: 1) the rise of MHC class II expression during the rest period occurred 4–5 hours earlier and 2) the cyclical pattern during the activity period was blunted and protein expression was maintained at relatively high levels. MHC class II expression was higher in the lymph nodes and lower in the spleen of SR than C, though these differences did not reach statistical significance. In SR; however, the average protein level was significantly higher in lymph nodes than spleen (376.0 + 184.9 vs 188.6 + 42.2, respectively; p = .002) and higher in the granulocytes relative to agranulocytes. Our findings provide empirical evidence of a constitutive diurnal expression pattern for MHC class II molecules that is prone to upregulation upon SR, namely in lymph nodes, and specifically expressed by granulocytes. We speculate that, in mice, chronic sleep deprivation would further dysregulate MHC class II expression that might result in aberrant T cell activation with probable immune-associated pathological diseases such as allergies, autoimmunity, and tumors.

Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-β-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

A tissue-based draft map of the murine MHC class I immunopeptidome

The large array of peptides presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC class I immunopeptidome. Although the MHC class I immunopeptidome is ubiquitous in mammals and represents a critical component of the immune system, very little is known, in any species, about its composition across most tissues and organs in vivo. We applied mass spectrometry (MS) technologies to draft the first tissue-based atlas of the murine MHC class I immunopeptidome in health. Peptides were extracted from 19 normal tissues from C57BL/6 mice and prepared for MS injections, resulting in a total number of 28,448 high-confidence H2Db/Kb-associated peptides identified and annotated in the atlas. This atlas provides initial qualitative data to explore the tissue-specificity of the immunopeptidome and serves as a guide to identify potential tumor-associated antigens from various cancer models. Our data were shared via PRIDE (PXD008733), SysteMHC Atlas (SYSMHC00018) and SWATH Atlas. We anticipate that this unique dataset will be expanded in the future and will find wide applications in basic and translational immunology.

The Use of Reverse Vaccinology and Molecular Modeling Associated with Cell Proliferation Stimulation Approach to Select Promiscuous Epitopes from Schistosoma mansoni.

Schistosomiasis remains an important parasitic disease that affects millions of individuals worldwide. Despite the availability of chemotherapy, the occurrence of constant reinfection demonstrates the need for additional forms of intervention and the development of a vaccine represents a relevant strategy to control this disease. With the advent of genomics and bioinformatics, new strategies to search for vaccine targets have been proposed, as the reverse vaccinology. In this work, computational analyses of Schistosoma mansoni membrane proteins were performed to predict epitopes with high affinity for different human leukocyte antigen (HLA)-DRB1. Ten epitopes were selected and along with murine major histocompatibility complex (MHC) class II molecule had their three-dimensional structures optimized. Epitope interactions were evaluated against murine MHC class II molecule through molecular docking, electrostatic potential, and molecular volume. The epitope Sm141290 and Sm050890 stood out in most of the molecular modeling analyses. Cellular proliferation assay was performed to evaluate the ability of these epitopes to bind to murine MHC II molecules and stimulate CD4+ T cells showing that the same epitopes were able to significantly stimulate cell proliferation. This work showed an important strategy of peptide selection for epitope-based vaccine design, achieved by in silico analyses that can precede in vivo and in vitro experiments, avoiding excessive experimentation.

Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

High-resolution analysis of the murine MHC class II immunopeptidome.

The reliable identification of peptides bound to major histocompatibility complex (MHC) class II is fundamental for the study of the host immune response against pathogens and the pathogenesis of autoimmune conditions. Here, we describe an improved methodology combining immuno-affinity enrichment of MHC class II complexes, optimized elution conditions and quadrupole Orbitrap mass spectrometry-based characterization of the immunopeptidome. The methodology allowed the identification of over 1000 peptides with 1% false discovery rate from 10(8) murine A20 lymphoma cells. The study revealed the I-A(d) -specific motif in high resolution after multisequence alignment. The methodology was generally applied to the purification of MHC class II from cell lines and murine spleens. We identified 2963 peptides from BALB/c and 2712 from C57BL/6 mouse spleens. The identification of peptides bound to MHC class II in vitro and in vivo will facilitate the characterization of T-cell specificities, as well as the development of biotherapeutics and vaccines.

Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3

AbstractMice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific “humanized” mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.

Release from endoplasmic reticulum matrix proteins controls cell surface transport of MHC class I molecules.

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi-step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER-Golgi intermediate compartment or the cis-Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H-2D(b) and H-2K(b) . We find that a novel pre-ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.

The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

Delayed onset of graft-versus-host disease in immunodeficent human leucocyte antigen-DQ8 transgenic, murine major histocompatibility complex class II-deficient mice repopulated by human peripheral blood mononuclear cells

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non-obese diabetic (NOD)-Rag1(-/-) mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD-Rag1(-/-) γc(-/-) mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft-versus-host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)-DQ8] on a mouse class II-deficient background (Aβ(-/-) ). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype-matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3(+) T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.

Fish oil disrupts MHC class II lateral organization on the B-cell side of the immunological synapse independent of B-T cell adhesion

Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4+ T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MβCD). MβCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.

The ‘peptidome’ of tumour-derived chaperone-rich cell lysate anti-cancer vaccines reveals potential tumour antigens that stimulate tumour immunity

Tumour-derived chaperone-rich cell lysate (CRCL) when isolated from tumour tissue or when embedded with peptide antigens is a potent anti-cancer vaccine consisting of numerous chaperone/heat shock proteins, including the highly immunogenic Hsp70, Hsp90, glucose regulated protein 94, and calreticulin. We have previously documented that CRCL provides both a source of tumour antigens and danger signals triggering antigen presenting cell activation. In this report we describe the ‘peptidome’ of potential antigens extracted from CRCL prepared from a murine tumour. Using mass spectrometry techniques we identify almost 60 different proteins of origin for the CRCL peptides; we determine that the parental proteins come from essentially all parts of the cell, and are involved in a broad range of functions. Further in silico analysis demonstrates that the parental proteins are components of major signalling networks of vital importance for cancer cell survival, proliferation, and migration. In many instances the peptides identified possess amino acid sequences that would allow their putative binding and display by murine major histocompatibility complex class I and II molecules, and there are also predicted binding motifs for Hsp70-type chaperones. By mixing fractionated pools of peptides with antigen-free (normal liver) CRCL, we were able to reconstitute effective anti-tumour activity of the vaccine, showing that the peptides are indeed the major purveyors of CRCL vaccines’ efficacy.

Exogenous, TAP‐independent lysosomal presentation of a respiratory syncytial virus CTL epitope

Respiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell-mediated immune responses. CD8(+) cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249-258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule K(d). In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249-258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249-258 can be generated from non-infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus-specific CTLs.

Analysis of major histocompatibility complex class I folding: novel insights into intermediate forms

Folding around a peptide ligand is integral to the antigen presentation function of major histocompatibility complex (MHC) class I molecules. Several lines of evidence indicate that the broadly cross-reactive 34-1-2 antibody is sensitive to folding of the MHC class I peptide-binding groove. Here, we show that peptide-loading complex proteins associated with the murine MHC class I molecule K(d) are found primarily in association with the 34-1-2(+) form. This led us to hypothesize that the 34-1-2 antibody may recognize intermediately, as well as fully, folded MHC class I molecules. To further characterize the form(s) of MHC class I molecules recognized by 34-1-2, we took advantage of its cross-reactivity with L(d) . Recognition of the open and folded forms of L(d) by the 64-3-7 and 30-5-7 antibodies, respectively, has been extensively characterized, providing us with parameters against which to compare 34-1-2 reactivity. We found that the 34-1-2(+) L(d) molecules displayed characteristics indicative of incomplete folding, including increased tapasin association, endoplasmic reticulum retention, and instability at the cell surface. Moreover, we show that an L(d) -specific peptide induced folding of the 34-1-2(+) L(d) intermediate. Altogether, these results yield novel insights into the nature of MHC class I molecules recognized by the 34-1-2 antibody.

Toxoplasma gondii HLA-B*0702-restricted GRA720-28 peptide with adjuvants and a universal helper T cell epitope elicits CD8+ T cells producing interferon-γ and reduces parasite burden in HLA-B*0702 mice

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.

Human peripheral blood CD4 T cell-engrafted non-obese diabetic-scid IL2rγnull H2-Ab1 tm1GruTg (human leucocyte antigen D-related 4) mice: a mouse model of human allogeneic graft-versus-host disease

Graft-versus-host disease (GVHD) is a life-threatening complication of human allogeneic haematopoietic stem cell transplantation. Non-obese diabetic (NOD)-scid IL2rγ(null) (NSG) mice injected with human peripheral blood mononuclear cells (PBMC) engraft at high levels and develop a robust xenogeneic (xeno)-GVHD, which reproduces many aspects of the clinical disease. Here we show that enriched and purified human CD4 T cells engraft readily in NSG mice and mediate xeno-GVHD, although with slower kinetics compared to injection of whole PBMC. Moreover, purified human CD4 T cells engraft but do not induce a GVHD in NSG mice that lack murine MHC class II (NSG-H2-Ab1(tm1Gru), NSG-Ab°), demonstrating the importance of murine major histocompatibility complex (MHC) class II in the CD4-mediated xeno-response. Injection of purified human CD4 T cells from a DR4-negative donor into a newly developed NSG mouse strain that expresses human leucocyte antigen D-related 4 (HLA-DR4) but not murine class II (NSG-Ab° DR4) induces an allogeneic GVHD characterized by weight loss, fur loss, infiltration of human cells in skin, lung and liver and a high level of mortality. The ability of human CD4 T cells to mediate an allo-GVHD in NSG-Ab° DR4 mice suggests that this model will be useful to investigate acute allo-GVHD pathogenesis and to evaluate human specific therapies.

Lentiviral Vectors Targeted to MHC II Are Effective in Immunization

Abstract vectors (LVs) that are targeted to APC using a chimeric measles virus (MV) hemagglutinin (H). The MV H protein is mutated to prevent binding to MV receptors and incorporates a single-chain antibody that recognizes murine major histocompatibility complex class II (MHC II). This targeted LV is highly efficient in transduction of freshly isolated mouse B cells and dendritic cells. MHC II-positive cells in spleen are transduced after intravenous injection, and a robust immune response to an antigen transgene is generated.