Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46–61)–Ak or HEL-(116–129)–Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.

The role of MHC class I glycoproteins in the regulation of induction of cell death in immunocytes by malignant melanoma cells

A deranged expression of MHC class I glycoproteins, characteristic of a variety of malignancies, contributes to the ability of cancer to avoid destruction by T cell-mediated immunity. An abrogation of the metastatic capacity of B16 melanoma cells has been achieved by transfecting an MHC class I-encoding vector into class I-deficient B16 melanoma clones [Gorelik, E., Kim, M., Duty, L. & Galili, U. (1993) Clin. Exp. Metastasis 11, 439–452]. We report here that the deranged expression of class I molecules by B16 melanoma cells is more than a mere acquisition of the capacity to escape immune recognition. Namely, cells of the B16 melanoma prompted splenic lymphocytes to commit death after coculture. However, a class I-expressing and nonmetastatic CL8-2 clone was found to be less potent as an inducer of apoptosis than class I-deficient and metastatic BL9 and BL12 clones. Both Thy1.2+ and Thy1.2− splenocytes underwent cell death when exposed to the class I-deficient BL9 clone. A proportion of CD4+ and CD8+ cells among splenocytes exposed to the BL9 clone was lower than that observed in a coculture with cells of the CL8-2 clone. Consistently, none of the melanoma clones studied produced a ligand to the FAS receptor (FAS-L). Thus, our results provide evidence that (i) the production of FAS-L may not be the sole mechanism by which malignant cells induce apoptosis in immunocytes, and (ii) absence of MHC class I glycoproteins plays an important role in preventing the elimination of potential effector immunocytes by tumor cells.

Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

Major histocompatibility complex (MHC) class I KbDb −/− deficient mice possess functional CD8+ T cells and natural killer cells

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb −/− mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although β2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb −/− mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by β2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb −/− mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb −/− mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb −/− animals also have natural killer cells that retain their cytotoxic potential.

Specific T cell recognition of kinetic isomers in the binding of peptide to class II major histocompatibility complex

Helper T cells are triggered by molecular complexes of antigenic peptides and class II proteins of the major histocompatibility complex . The formation of stable complexes between class II major histocompatibility complex proteins and antigenic peptides is often accompanied by the formation of a short-lived complex. In this report, we describe T cell recognition of two distinct complexes, one short-lived and the other long-lived, formed during the binding of an altered myelin basic protein peptide to I-Ak. One myelin basic protein-specific T cell clone is triggered by only the short-lived complex, and another is triggered by only the stable complex. Thus, a single peptide bound to a particular class II molecule can activate different T cells depending on the conditions of the binding reaction.

Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-2M complexes

By combining two previously generated null mutations, Ii° and M°, we produced mice lacking the invariant chain and H-2M complexes, both required for normal cell-surface expression of major histocompatibility complex class II molecules loaded with the usual diverse array of peptides. As expected, the maturation and transport of class II molecules, their expression at the cell surface, and their capacity to present antigens were quite similar for cells from Ii°M° double-mutant mice and from animals carrying just the Ii° mutation. More surprising were certain features of the CD4+ T cell repertoire selected in Ii°M° mice: many fewer cells were selected than in Ii+M° animals, and these had been purged of self-reactive specificities, unlike their counterparts in Ii+M° animals. These findings suggest (i) that the peptides carried by class II molecules on stromal cells lacking H-2M complexes may almost all derive from invariant chain and (ii) that H-2M complexes edit the peptide array displayed on thymic stromal cells in the absence of invariant chain, showing that it can edit, in vivo, peptides other than CLIP.

Experimental hemochromatosis due to MHC class I HFE deficiency: Immune status and iron metabolism

The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE α1 and α2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.

Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.

Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with different in vivo half-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+ T cells.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-γ, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-γ-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4+ human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4+ T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1–20. DR3.Ab0 mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Vα transcripts (Vα1 and Vα2) and one TCR Vβ transcript (Vβ13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Vα1Vβ13 combination specifically recognized both the HLA-A2+ and HLA-DR1+ target cells, whereas the Vα2Vβ13 combination did not result in a TCR on the cell surface. Thus a single TCRαβ complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.