Production of alloantisera against class II bovine lymphocyte antigens (BoLA) by cross-immunization between class I matched cattle

This paper describes the production of alloantisera directed against bovine major histocompatibility complex (MHC) (BoLA) class II antigens in animals whose MHC phenotypes had been defined by one dimensional isoelectric focusing. Animals of closely matched BoLA class I types were selected by serology and subsequently typed for class I and class II by 1D-IEF of immunoprecipitated antigens. Those with similar class I type by both methods, but differing at the class II locus, were chosen for reciprocal immunization. Cross-immunization was by two skin implantations 6 weeks apart. The resulting antisera showed low titre after the first immunization and elevated titre 3 weeks after the second immunization. The sera reacted strongly with cells expressing specific BoLA class II antigens. The pattern of reactivity correlated well with IEF class II typing on a panel of animals representing all of the class II IEF types present in the Friesian population.     

Alloreactive T-cell recognition of bovine major histocompatibility complex class II products defined by one-dimensional isoelectric focusing

T-cell recognition of bovine MHC (BoLA) class II antigens was investigated in relation to BoLA class II polymorphisms defined by one-dimensional isoelectric focusing (1D-IEF). One-way mixed lymphocyte reactions (MLRs), and allospecific cell lines and clones were used. In general, T-cell responses correlated with the 1D-IEF defined haplotypes (EDF types). However, with MLRs some responses appeared to be associated with BoLA class I differences. All combinations of responder-stimulator pairs produced alloreactive T-cell responses both in MLR and in generation of allolines/clones. Thus allospecific lines and clones were generated to all EDF types tested. Splits in the IEF typing were observed with EDF6 and EDF3, indicating that distinct BoLA class II haplotypes are not necessarily distinguished by 1D-IEF alone. Furthermore, the patterns of reactivity with EDF3 expressing cells were complex with the T-cell specificities splitting EDF3 into several distinct types. Also, in some cases it was clear that more than one T-cell specificity per EDF type was detectable. Thus, allospecific lines and clones provide complementary and additional information to the 1D-IEF typing for polymorphism of the BoLA class II complex. This extra information is particularly important in terms of the functional significance of the BoLA complex for antigen presentation and immune response gene effects.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

Impaired ability of MHC class II-/- dendritic cells to provide tumor protection is rescued by CD40 ligation.

The contribution of CD4+ T cells to dendritic cell (DC) activation and to the induction of CD8+ T cell responses in vivo was investigated using a model of antitumor immune responses. Immunization with peptide-loaded MHC class II-deficient (MHC class II-/-) DC induced the activation of Ag-specific CD8+ T cells and their accumulation in the lymph nodes and spleens of immunized mice. The accumulation induced by MHC class II-/- DC immunization was lower than the accumulation observed after immunization with MHC class II+/+ DC. Similarly, immunization with peptide-loaded, MHC class II-/- DC induced some degree of protection against tumor challenge, but this protection was lower than the protection achieved after immunization with MHC class II+/+ DC. Incubation with a membrane-associated form of CD40 ligand resulted in the up-regulation of costimulatory molecules on MHC class II-/- DC and fully rescued their ability to induce antitumor immunity. We conclude that CD4+ T cells play a critical role in the generation of antitumor immune responses through their capacity to induce the activation of DC via CD40/CD40 ligand interaction, and thus maximize CD8+ T cell responses.     

Allo-Reactivity of Mesenchymal Stem Cells in Rhesus Macaques Is Dose and Haplotype Dependent and Limits Durable Cell Engraftment In Vivo

The emerging paradigm that MSCs are immune privileged has fostered the use of “off-the-shelf” allogeneic MSC-based therapies in human clinical trials. However, this approach ignores studies in experimental animals wherein transplantation of MSCs across MHC boundaries elicits measurable allo-immune responses. To determine if MSCs are hypo-immunogeneic, we characterized the immune response in rhesus macaques following intracranial administration of allogeneic vs. autologous MSCs. This analysis revealed unambiguous evidence of productive allo-recognition based on expansion of NK, B and T cell subsets in peripheral blood and detection of allo-specific antibodies in animals administered allogeneic but not autologous MSCs. Moreover, the degree of MHC class I and II mismatch between the MSC donor and recipient significantly influenced the magnitude and nature of the allo-immune response. Consistent with these findings, real-time PCR analysis of brain tissue from female recipients administered varying doses of male, allogeneic MSCs revealed a significant inverse correlation between MSC engraftment levels and cell dose. Changes in post-transplant neutrophil and lymphocyte counts also correlated with dose and were predictive of overall MSC engraftment levels. However, secondary antigen challenge failed to elicit a measurable immune response in allogeneic recipients. Finally, extensive behavior testing of animals revealed no main effect of cell dose on motor skills, social development, or temperament. Collectively, these data indicate that allogeneic MSCs are weakly immunogenic when transplanted across MHC boundaries in rhesus macaques and this negatively impacts durable engraftment levels. Therefore the use of unrelated donor MSCs should be carefully evaluated in human patients.     

Differences in cellular immune response to l-glutamic acid60, l-alanine30, l-tyrosine10 (GAT) in full sibling embryo transfer cattle: Examination of requirements for cell proliferation

Pairs of full sibling embryo transfer cattle that expressed identical MHC class I and II products were tested for their in vitro proliferative response to GAT. Peripheral blood mononuclear cells from these cattle were either high or low responders to GAT. Cells from certain pairs of MHC identical siblings gave opposite responses. Low responder animals were further tested to determine if they might respond to GAT with different kinetics, with secondary in vitro restimulation, or with exogenous help provided by interleukin 2. Also, the role of antigen presenting cells and suppressor T cells from low responder animals was investigated. Using appropriate in vitro conditions, cells from all animals tested could respond to GAT. However, MHC identical animals tested under similar conditions exhibited differences in their response to GAT which suggests the proliferative immune response was influenced by factors in addition to MHC coded products.     

Production of alloantibodies against bovine B-lymphocyte antigens

A series of alloimmunizations were carried out between BoLA class I antigen typed bulls, with the aim of generating class II specific reagents. Of the antisera produced, seven demonstrated exclusively B cell reactivity. Another 19 sera reacted with both T and B cells from some animals and with B cells only in other cases. Suitable buffy coat absorptions removed T cell reactivity from some sera and shortened broader reactivities in certain B cell specific sera. Typing of separated T and B cells from related and unrelated animals permitted clustering of the sera into four groups. These groups behave as allelic specificities. The class II nature of the recognized structures was strongly indicated by two further pieces of evidence. The presence or absence of particular B cell antigens correlated with reactivity of cells in one-way mixed lymphocyte cultures. In addition, a number of the B cell specific sera were characterized by immunoprecipitation of radiolabelled lymphocytes. The precipitated products corresponded in molecular weight to alpha and beta chains of MHC class II dimers, as has been found in this and other species.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

Discrimination between major histocompatibility complex class II DQ and DR locus products in cattle

We have used a panel of anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAbs) and have assessed their specificity for the products of the individual bovine MHC (BoLA) class II subregions. The mAbs identified two distinct class II molecules by affinity purification and ELISA. Two-dimensional immunoblotting confirmed these data and NH₂-terminal sequencing of the purified class II α chains of one member of each group identified the subregion specificity of the mAbs. The mAbs VPM36, TH22A and TH81A are specific for BoLA DQ, whereas VPM54, TH14B and J11 are specific for BoLA DR. SW73.2 reacts with both MHC subgroups of all cattle tested.     

Ubiquitin-dependent control of class II MHC localization is dispensable for antigen presentation and antibody production

Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production.     

Humoral immune response to equine chorionic gonadotropin in ewes: association with major histocompatibility complex and interference with subsequent fertility.

In dairy ewes, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). In this report we show the presence of anti-eCG antibodies in plasma of treated ewes. The major histocompatibility complex (MHC) was involved in the individual variability of the humoral immune responses to eCG. We found significant associations between the anti-eCG response phenotype and some MHC class II alleles. The low immune response phenotype was associated with one MHC class II allele only in Lacaune ewes, and the high immune response phenotype was associated with one MHC class II allele both in Manech and in Lacaune ewes. In herds, the impact of residual anti-eCG antibodies on subsequent fertility after AI seems minimal because of an indirect elimination of high-responder ewes from AI breeding. Therefore, the true magnitude of the association between residual anti-eCG antibody concentration and fertility has been underestimated. An additional experiment without any high-responder female elimination showed a significant correlation between high residual antibody concentrations and lower lambing rate after AI at a fixed time, possibly because of a delayed preovulatory LH surge. The results suggest that anti-eCG antibody concentration is one risk factor for infertility after AI.     

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.     

CD4-directed peptide vaccination augments an antitumor response, but efficacy is limited by the number of CD8+ T cell precursors

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8(+) T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8(+) T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4(+) T cell effector function, but did not directly prime CD8(+) T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8(+) T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4(+) Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8(+) DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8(+) T cells will ultimately dictate the success of immunotherapy.     

Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.     

A role for a melanosome transport signal in accessing the MHC class II presentation pathway and in eliciting CD4+ T cell responses

Melanosomal membrane proteins are frequently recognized by the immune system of patients with melanoma and vitiligo. Melanosomal glycoproteins are transported to melanosomes by a dileucine-based melanosomal transport signal (MTS). To investigate whether this sorting signal could be involved in presentation of melanosome membrane proteins to the immune system, we devised a fusion construct containing the MTS from the mouse brown locus product gp75/tyrosinase-related protein-1 and full-length OVA as a reporter Ag. The fusion protein was expressed as an intracellular membrane protein, sorted to the endocytic pathway, processed, and presented by class II MHC molecules. DNA immunization with this construct elicited CD4+ T cell proliferative responses in vivo. Ag presentation and T cell responses in vitro and in vivo required a functional MTS. Mutations of either the upstream leucine in MTS or elimination of the entire MTS negated in vitro Ag presentation and in vivo T cell responses. In a mouse melanoma model, DNA immunization with MTS constructs protected mice from tumor challenge in a CD4+ T cell-dependent manner, but complete deletion of MTS decreased tumor rejection. Therefore, MTS can target epitopes to the endocytic pathway leading to presentation by class II MHC molecules to helper T cells.     

Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.     

Cationization of protein antigens. II. Alteration of regulatory properties

Immunoregulatory effects of cationized bovine serum albumin (cBSA) and native bovine serum albumin (nBSA) have been investigated. Intravenous administration of nBSA to BDF1 mice substantially suppressed the antibody response to subsequent immunization with either nBSA or cBSA, whereas pretreatment with cBSA by the same route significantly enhanced the responses to both antigens. The functional properties of BSA-specific T and B cells from mice immunized with cBSA or nBSA were examined in reconstitution experiments in which splenic T populations together with B cells were transferred into irradiated syngeneic recipients. Transfer of splenic T cells from mice primed with nBSA caused profound suppression of the response to subsequent immunization with nBSA or cBSA, whereas transfer of either B or T cells from cBSA treated mice produced an enhanced response to both antigens. C57BL/6 mice, which are considered to be low responders to BSA, produced a significant antibody response to BSA when immunized with cBSA. In contrast, immunization with nBSA did not produce measureable amounts of antibody in mice of this strain. Our data clearly demonstrate that cationized BSA exhibits unique immunogenic properties due to alterations in the self-regulation of the immune response.     

Stimulation of enhanced CD8 T cell responses following immunization with a hyper-antigen secreting intracytosolic bacterial pathogen

The intracytosolic niche for replication of Listeria monocytogenes (Lm) facilitates delivery of bacteria-derived Ags into the MHC class I pathway for subsequent stimulation of CD8 effector T cells. Using Lm strains that are equivalent for in vivo virulence yet express marked differences in the level of secretion of a protective target Ag, we have evaluated how these specific differences in secretion levels influences the magnitude and effector function of Ag-specific CD8 T cell responses following Lm injection. Immunization with low doses of a hyperantigen-secreting Lm strain stimulated enhanced target-Ag specific CD8 T cell responses compared with the magnitude stimulated following immunization with the same dose of wild-type Lm. The enhanced determinant-specific response was also evident by in vivo CTL activity, increased numbers of memory cells 4 wk following immunization, and enhanced antilisterial protection following a challenge infection. Initiation of antibiotic treatment 24 h following infection with wild-type Lm markedly reduced the magnitude of the effector CD8 T cell response. In contrast, antibiotic treatment initiated 24 h following immunization with the hyperantigen secreting strain of Lm did not impact the frequency of the target-Ag specific CD8 T cells. Thus, immunization with a low dose of a hyperantigen secreting Lm strain, followed by antibiotic treatment to limit the extent of the infection, may represent a safe strategy for the stimulation of enhanced effector CD8 T cell responses to specific Ag by a rLm vaccine.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.     

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.