Response to Listeria monocytogenes in mice lacking MHC class Ia molecules

MHC class Ia-deficient mice (H2 Kb-/- Db-/-) inoculated with the intracellular pathogen Listeria monocytogenes (LM) displayed a three- to fourfold expansion of splenic CD8+ T cells 6 days following infection. Culture of these spleen cells in vitro gave rise to CTL that recognized LM-infected target cells and were restricted by the class Ib molecules, Qa1b and M3. Exposure of target cells to heat-killed LM (HKLM) rather than live bacteria did not result in CTL-mediated lysis. Target cells pulsed with three LM peptides known to bind M3, f-MIGWII, f-MIVTLF, and f-MIVIL, were recognized by effector cells from both B6 and Kb-/- Db-/- animals. In vivo analysis showed that B6 and Kb-/- Db-/- mice clear LM from the spleen and liver rapidly with similar kinetics, whereas TAP.1-/- mice, which are deficient in class Ia and Ib molecules, clear LM slowly upon infection. To establish the in vivo role of CD8+ T cells in Kb-/- Db-/- animals, we showed that depletion of such cells from the spleens of immune mice prevented the adoptive transfer of protective immunity to syngeneic recipients. Spleen cells from Kb-/- Db-/- mice were also capable of generating responses directed against syngeneic as well as allogeneic class Ia molecules in vitro. Thus, class Ia-deficient animals have a CD8+ T cell repertoire capable of recognizing both class Ia and class Ib molecules and can generate protective immunity to LM.     

Distinct mechanisms regulate MHC class II gene expression in B cells and macrophages

In a previous series of studies, we had shown that the constitutive Ia expression in an immunoselected Ia-human B cell variant, RJ 2.2.5, could be restored by somatic cell hybridization with mouse B cells. These experiments allowed us to show the existence of a transacting activator factor(s) operating across species barriers and encoded by the aIr-1 locus located on mouse chromosome 16. The aim of the present study was to investigate whether the B cell constitutive Ia expression and the inducible Ia expression, as seen in macrophages treated with IFN-gamma, are controlled by similar intracellular factors. To this purpose, we constructed an interspecies somatic cell hybrid between the human Ia-RJ 2.2.5 B cells and the mouse Ia-P388 D1 macrophage cells. These murine cells transiently express Ia antigens when incubated with IFN-gamma. Our results show that RJ 2.2.5 X P388 D1 cell hybrids do not express either human or mouse class II gene products. Treatment with human recombinant IFN-gamma did not modify the MHC phenotype of either the hybrid cells or the human parental cells. On the other hand, treatment of the hybrid cells with murine recombinant IFN-gamma resulted in de novo expression of mouse Ia mRNA and corresponding cell surface antigens without, however, reinduction of the human class II-positive phenotype. Furthermore, treatment with the mouse lymphokine significantly increased the levels of human HLA class I mRNA and corresponding cell surface antigens in the hybrid cells, further reinforcing the notion of the existence of non-species-specific secondary mediators generated after receptor-ligand interaction in the IFN-gamma system. Together, these results indicate that in macrophages, the intracellular events taking place after binding of IFN-gamma with its own receptor and leading to the expression of a class II-positive phenotype do not operate via an activation of the aIr-1 locus and/or its products. Thus, at least in our experimental system, we can firmly establish a first, relevant distinction between constitutive and inducible class II gene expression. This difference, dictated by the specific differentiation program of each cell type, may be relevant for the understanding of the function of class II gene products.     

Novel regulation of MHC class II function in B cells

The presence of post-translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen-presenting cells (APCs). Here, we report that MARCH-I, an E3 ubiquitin ligase, plays a pivotal role in the post-translational regulation of MHC II in B cells. MARCH-I expression was particularly high in B cells, and the forced expression of MARCH-I induced the ubiquitination of MHC II. In B cells from MARCH-I-deficient mice (MARCH-I KO), the half-life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH-I-deficient B cells highly expressed exogenous antigen-loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH-I.     

Castration prevents suppression of MHC class II (Ia) expression on macrophages after trauma-hemorrhage.

Several studies indicate that cell-mediated immune responses, i.e., macrophage (MPhi) cytokine release capacities, myosin heavy chain (MHC) class II (Ia) expression, etc., are suppressed after trauma-hemorrhage in male mice. Testosterone has been shown to be responsible for the depression of MPhi cytokine responses in males after trauma-hemorrhage. Antigen presentation via MHC class II plays a key role in initiating and maintaining cell-mediated and humoral immune responses. It remains unknown, however, whether testosterone has any effect on MHC class II after trauma-hemorrhage. To study this, male C3H/HeN mice were castrated or sham castrated 2 wk before trauma (midline laparotomy) and hemorrhage (Hem; blood pressure 35 +/- 5 mmHg for 90 min and resuscitation) or sham operation. Four hours thereafter, MHC class II (Ia) expression was measured using flow cytometry. The results indicate that MHC class II (Ia) expression on peritoneal and splenic MPhi was significantly suppressed in male mice after trauma-hemorrhage. Prior castration, however, prevented the depression in MHC class II (Ia) expression on peritoneal and splenic MPhi after trauma-hemorrhage. Castration did not affect MHC class II (Ia) expression in MPhi from sham-castrated mice. Thus testosterone depresses MHC class II (Ia) expression on peritoneal and splenic MPhi after trauma-hemorrhage in males. Because MHC class II is necessary for an adequate immune response, our results suggest that depletion of male sex steroids or blockade of androgen receptors using agents such as flutamide might prevent immunosuppression via maintaining MHC class II (Ia) expression after trauma and severe blood loss.     

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.     

Characterization of the signaling function of MHC class II molecules during antigen presentation by B cells.

In addition to their role as peptide binding proteins, MHC class II proteins can also function as signal transducing molecules. Recent work using B cells expressing genetically engineered truncated MHC class II molecules has suggested that signaling through the cytoplasmic domains of these proteins plays an important role in the generation of signals required for the activation of some T cell hybrids. Treatment of truncated Ia-expressing B cells with cAMP-elevating agents corrects the deficiency in Ag presentation by these cells. We report that the MHC class II-mediated signal appears to act by a mechanism that increases the efficiency of Ag presentation by B cells thereby lowering the amount of specific Ag required for T cell activation. We further show that the induction of the cAMP-induced signal in B cells is inhibited by cycloheximide and cytochalasin A, implicating protein synthesis as well as cytoskeletal rearrangements in Ag presentation to accessory signal- dependent hybrids. In contrast, these agents do not block Ag presentation to a T cell hybrid previously shown not to require the cAMP-induced signal for activation. The signal-dependent T hybrid is additionally dependent on LFA-1-ICAM-1 interaction for activation, whereas the signal-independent hybrid is not. These observations suggest the existence of two types of T cell hybrid with respect to their requirements for activation: those that require only the recognition of MHC class II-peptide complexes without accessory signals, as shown by their ability to respond to purified Ia on planar membranes, and those that, in addition to recognition of MHC II/Ag, require LFA-1-ICAM-1 interaction and the delivery of additional signal(s) induced in the B cell via signal transduction through MHC class II molecules.     

Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.     

Murine thyroid follicular epithelial cells can be induced to express class II (Ia) gene products but fail to present antigen in vitro

To determine whether thyroid follicular epithelial cells (TFEC) might be involved in the induction of autoimmune thyroiditis, they were tested for their potential to express Ia antigens, and for their ability to present antigen in vitro. Results showed that Ia antigens, absent on normal TFEC, could be readily induced with interferon gamma, as detected by immunofluorescence. Maximal expression of Ia antigens in over 50% of TFEC was observed after 4 days of culture in the presence of IFN-gamma, and was quantitatively comparable to spleen cells by cytofluorometric analysis. Moreover, primary TFEC in culture secreted thyroglobulin (tg) and interleukin 1. However, TFEC consistently failed to stimulate various populations of T cells. These included lymph node cells sensitized to tg, a T-cell clone specific for azo-benzene-arsonate tyrosine (ABA), and a hybridoma specific for beef insulin. Likewise, Ia-positive TFEC did not stimulate T-cell hybridomas restricted to the class II alloantigen I-Ab, while stimulating a hybridoma specific for the class I alloantigen Kb. T-cell unresponsiveness could not be explained by inhibitory activity of TFEC, released either into the culture supernatant or exerted by cell contact. The data indicate that Ia-positive TFEC failed to serve as class II-restricted antigen-presenting cells (APC) in vitro and thus argue against a primary role for these cells in the inductive phase of thyroiditis.     

Induction and functional characterization of class II MHC (Ia) antigens on murine keratinocytes

To induce Ia molecules on the surface of murine keratinocytes (KC), healthy mice were treated daily with i.p. injections of rIFN-gamma at a dose of 50,000 U/day for 6 days. This resulted in strong Ia expression by KC as determined by immunofluorescence of epidermal sheets or cell suspensions with anti-class II mAb. To obtain a population of Ia-bearing KC devoid of Langerhans cells, a method of depleting Langerhans cells from such suspensions was developed. Although Ia+ KC were unable to stimulate allogeneic T cells in a primary epidermal cell-lymphocyte reaction (less than 5% control), they did induce a proliferative response in an allospecific T cell line. Ia+ KC were unable to present native peptide molecules to class II restricted, Ag-specific T cell hybridomas. However, Ia+ KC were able to present a peptide fragment of pigeon cytochrome c to a hybridoma, suggesting that although these cells cannot process native protein Ag, they can present antigenic peptides. Ia+ (but not Ia-) KC also served as targets for class II restricted cytolytic T cell clones. These data indicate that the Ia expressed by KC is a functional molecule, and that Ia+ KC can participate in some immunologic reactions.     

Inhibition of class II MHC gene expression by anti-sense RNA in transgenic mice.

We have established transgenic mice carrying the anti-sense DNA to the gene encoding beta chain of the class II major histocompatibility complex (I-A) molecule. The amount of I-A molecule on splenic B lymphocytes from the mice was reduced in the presence of a large amount of the exogenous anti-sense RNA. The amount of I-A beta chain RNA was selectively reduced and inversely correlated with the amount of anti-sense RNA in the spleens. These results suggest that the I-A beta chain RNA is rapidly degraded by duplex formation with the anti-sense RNA in splenic B cells from the transgenic mice.     

Increased expression of I-region-associated antigen (Ia) on B cells after cross-linking of surface immunoglobulin

Both surface Ig (sIg) surface Ia (sIa) have been shown to have important roles in B lymphocyte activation. In order to investigate a possible relationship between these molecules, we studied the effects of cross-linking of sIg on the expression of sIa, as measured by fluorescence-activated cell sorter analysis of lymphoid cells stained with conventional anti-Ia anti-serum or with fluorescein-labeled anti-Ia antibodies. Exposure of cells for 24 hr in vitro to anti-delta, anti-mu, anti-kappa antibodies, or their F(ab')2 fragments induced a very dramatic increase in expression of sIa. Similarly, i.v. injection of anti-delta antibodies into adult mice induced a 2- to 3-fold increase in expression of B cell sIa on spleen, lymph node, and Peyer's patch lymphocytes. There was no increase under these conditions in expression of other B lymphocyte surface antigens, including H-2, 4B9, and 17C9. Furthermore, exposure of B lymphocytes to antibodies directed to B lymphocyte surface antigens other than sIg did not result in an increase in expression of sIa. The anti-Ig-induced increase in sIa expression appeared to be T independent, required cellular protein synthesis, and required more time to occur than did the cross-linking and removal of sIg. This increase in expression of sIa did not occur on B lymphocytes obtained from mice younger than 3 wk old. This increase in expression of sIa may reflect a proximal event in B lymphocyte activation that occurs after cross-linking of sIg by antigen and that may enhance subsequent cellular interactions involving B lymphocytes.     

Localization of an antibody to CD74 (MHC class II invariant chain) to human B cell lymphoma xenografts in nude mice

The tumor-specific localization of an anti-CD74 Ab, LL1, was demonstrated in nude mice bearing xenografts of human B-cell lymphoma. This Ab, conjugated to radionuclides emitting Auger electrons, including ¹²⁵I and ¹¹¹In, was previously reported to kill tumor cells in vitro effectively and specifically. The cytotoxic potency of this Ab is due to its uptake and catabolism at a very high level, which also affected the Ab biodistribution experiments. Thus, Ab localization to the tumor was only detected if a “residualizing” radiolabel was used, meaning a label that is trapped within cells, usually within lysosomes, after catabolism of the Ab to which it was conjugated. Similar results were obtained with three different residualizing labels: ¹¹¹In conjugated via the chelators benzyl diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), or ¹³¹I-dilactitol-tyramine, a residualizing form of iodine. The Ab protein dose could be high, 0.5 mg/mouse, without causing a decrease in specific tumor uptake, probably reflecting the high capacity for uptake. Moreover, tumors of moderate size were found to cause rapid, specific removal of the Ab from the blood, also a result of catabolic processes. This induced blood clearance naturally affected the Ab localization experiments, but this factor could be circumvented by increasing the Ab protein dose. Using a different Ab, anti-(mature MHC class II), the ability of Ab to penetrate relatively large solid tumors was investigated. Complete saturation of antigenic sites was observed in tumors up to 0.3 g in size, but quite high Ab protein doses were required, 5.0 mg/mouse. These results provide a rationale for attempting therapy with radiolabeled LL1. Received: 4 November 1999 / Accepted: 19 January 2000