Propionibacterium acnes-induced hepatic granuloma formation is impaired in mice lacking tetraspanin CD9

The granuloma is a host defence response to persistent pathogenic irritants. In the process of granuloma formation, the activation, migration, and fusion of macrophages occur locally, but the mechanisms involved remain elusive. Tetraspanins regulate cell migration and fusion by organizing functional molecular complexes in membrane microdomains. Here we investigated the role of tetraspanin CD9 in hepatic granuloma formation. Immunostaining of the liver of untreated wild-type mice showed that CD9 was expressed by vascular endothelial cells and perivenular hepatocytes. When intrahepatic granulomas were induced by intravenous injection of Propionibacterium acnes, hepatocyte CD9 was extensively upregulated, while inflammatory cells constituting granulomas were mostly negative for CD9. Compared with wild-type littermates, CD9-knockout mice showed dissemination of Propionibacterium acnes and reduced number and size of granulomas after the injection. Moreover, production of granuloma-inducing cytokines, TNF-alpha and IFN-gamma, was delayed and chemotactic activity for macrophages was suppressed in the liver of mutant mice. These results suggest that CD9 is one of the proteins that promotes granuloma formation in the liver.     

Correction of defective host response to Mycobacterium bovis BCG infection in TNF-deficient mice by bone marrow transplantation

Tumour necrosis factor-alpha (TNF) plays a central role in the recruitment and activation of mononuclear cells in mycobacterial infection. In the absence of type 1 TNF receptor, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection of mice is not contained, leading to fatal disease. Because type 1 TNF receptor binds both TNF and lymphotoxin-a, we used TNF-deficient mice to determine the specific role of TNF in the host resistance to BCG infection. The bacterial burden of the lungs of TNF-deficient mice was substantially increased and the mice succumbed to pneumonia between 8 and 12 weeks with a defective granuloma response. Atypical granulomas developed by 4 weeks expressing low levels of MHC class II, intracellular adhesion molecule (ICAM-1), CD11b and CD11c. Macrophages showed little signs of activation and had low levels of acid phosphatase activity and inducible nitric oxide synthase (INOS) expression. Despite the defective cellular recruitment, the chemokines, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1alpha), were increased in broncho-alveolar lavage fluid of TNF-deficient mice. The defective host response was corrected by the transplantation of normal bone marrow cells into irradiated TNF-deficient mice. These results demonstrate that TNF derived from hemopoietic cells rather than from mesenchymal origin are essential for a normal host response to BCG infection. Furthermore, TNF dependent expression of adhesion molecules may be essential for the recruitment of mononuclear cells for the formation of bactericidal BCG granulomas.     

Mycobacterium bovis BCG-induced granuloma formation depends on gamma interferon and CD40 ligand but does not require CD28

Progressive granuloma formation is a hallmark of chronic mycobacterial infection. Granulomas are localized, protective inflammatory reactions initiated by CD4+ T cells, which contribute to control of bacterial growth and blockade of bacterial dissemination. In order to understand the costimulatory requirements that allow CD4+ T cells to directly or indirectly induce granulomas, we studied granuloma formation after 6 weeks in Mycobacterium bovis BCG-infected CD28- and CD40 ligand (CD40L)-deficient mice and compared it to granuloma formation in infected wild-type inbred mice and infected cytokine-deficient mice. We characterized granulomas morphologically in liver sections, analyzed granuloma infiltrating cells by flow cytometry, and measured cytokine production by cultured granuloma cells. CD28-deficient mice have no defect at the local inflammatory site, inasmuch as they form protective granulomas and control bacterial growth. However, there are fewer activated T cells in the spleen compared to infected wild-type animals, and quantitative differences in the cellular composition of the granuloma are observed by flow cytometry. In CD40L-deficient mice, the granuloma phenotype is very similar to the phenotype in gamma interferon (IFN-gamma)-deficient mice. Both IFN-gamma-deficient and CD40L-deficient mice form granulomas which prevent bacterial dissemination, but control of bacterial growth is significantly impaired. The relative proportion of CD4+ T cells in granulomas from both CD28(-/-) and CD40L(-/-) mice is significantly decreased compared with wild-type animals. Both models demonstrate that the phenotype and activation stage of systemic T cells do not always correlate with the phenotype and activation stage of the localized granulomatous response.     

Further DNA sequence microheterogeneity of the HLA-DR4/Dw13 haplotype group: Importance of amino acid position 86 of the DRβ1 chain for T-cell recognition

Using the polymerase chain reaction we have isolated and sequenced cDNA clones corresponding to the polymorphic first domain of the DRβ1 chain from the DR4, “Dw13” cell line, JHa. We have found that the JHa DRβ1 allele differs from previously reported Dw13 alleles by a single amino acid substitution at position 86. The functional relevance of this polymorphism is supported by the reactivity pattern of a T-cell clone, E38. E38 is an alloreactive T-cell clone which reacts with all Dw14 stimulator cells and all Dw13-positive cells tested except the “Dw13”- positive homozygous typing cell line JHa. Inhibition studies with monoclonal antibodies revealed the stimulating determinant to be on DR and not on DQ or DP molecules. These data indicate that position 86 of the DRβ1 chain can play an important role in the formation of determinants recognized by T cells.     

Genetic regulation of pristane‐induced oil granuloma responses

Oil granuloma (OG) induced by intraperitoneal injection of pristane represents a non-infectious granuloma. Oil granuloma has been characterized, but the regulation of its formation still remains unknown. To address this, we injected pristane into various mice deficient for genes including, linker for activation of T cells (LAT), μMT, LTα, TNFα, IL-6. T cell deficient mice (LAT^−/−) responded to pristane by developing serosal granuloma and mesenteric granuloma (MG) as in wild type mice. The absence of B cells blocked serosal granuloma (SG) formation and diminished MG development in response to pristane. However, even when a comparable number of B cells were present in the mesentery, the absence of TNFα resulted in similar defects in OG formation after pristane treatment, demonstrating that both B cells and TNFα are very crucial for pristane-induced OG formation. Interestingly, IL-6^−/− mice had intact MG formation; however, SG organization was impaired. These studies provide insight into granulomateous pathology induced by non-infectious substances for example, biomedical sutures.     

Schistosomal granuloma modulation. I. Schistosoma mansoni worm antigens CAA and CCA prime egg-antigen-induced hepatic granuloma formation

Adult Schistosoma mansoni worms can positively modulate soluble egg antigen (SEA)-induced granulomas formed around SEA-coupled beads implanted in the liver. In this study, our aim was to further unravel the immunopathological characteristics of S. mansoni-worm-derived antigens in vivo. (a) Adult worm antigen (AWA)-coupled Sepharose beads, implanted into the liver, induced granulomas, containing numerous eosinophilic granulocytes and elicited marked periparticular fibrosis (composed of interstitial matrix proteins and basement membrane components). (b) Quantitative morphological analysis demonstrated that in naive mice, AWA-induced hepatic granuloma formation peaked in volume 16 days after injection of the beads. An accelerated response against AWA-coupled particles (peak volume at 8 days) was observed in mice carrying a single-sex, male S. mansoni infection. (c) When the granuloma volume induced by SEA-coupled beads in unisexually S. mansoni infected mice was compared to granulomas induced by beads laden with both SEA and AWA in unsensitized mice, no significant differences in granuloma volume were seen, indicating the existence of in vivo egg/worm antigen cross-sensitization. (d) Naive mice, sensitized with the worm antigens circulating anodic antigen (CAA) or circulating cathodic antigen (CCA), mounted a strongly accelerated response towards SEA-coupled beads implanted in the liver. We infer that, in vivo, worm antigens cross-sensitize with egg antigens and have both granulomogenic and fibrogenic characteristics. The S. mansoni soluble worm antigens CCA and CAA prime hepatic egg-antigen-induced granuloma formation possibly through the presence of immunogenic carbohydrates. These mechanisms lead to an accelerated response against SEA. Received: 10 April 1998 / Accepted: 25 June 1998     

Natural killer T cells suppress zymosan A-mediated granuloma formation in the liver by modulating interferon-γ and interleukin-10

Wild-type (WT) and CD1d(-/-) [without natural killer (NK) T cells] mice were treated with zymosan A to induce granuloma formation in the liver. Increased granuloma formation was seen in NKT-less mice on days 7 and 14 after administration. WT mice showed limited granuloma formation, and zymosan A eventually induced NKT cell accumulation as identified by their surface marker (e.g. CD1d-tetramer). Zymosan A augmented the expression of Toll-like receptor 2 on the cell surface of both macrophages and NKT cells. One possible reason for accelerated granuloma formation in NKT-less mice was increased production of interferon- γ (IFN-γ); a theory that was confirmed using IFN-γ(-/-) mice. Also, zymosan A increased interleukin-10 production in WT mice, which suppresses IFN-γ production. Taken together, these results suggest that NKT cells in the liver have the potential to suppress zymosan A-mediated granuloma formation.     

Nasal Administration of Schistosoma mansoni Egg Antigen-Cholera B Subunit Conjugate Suppresses Hepatic Granuloma Formation and Reduces Mortality in S. mansoni-Infected Mice

Granulomatous inflammation in schistosomiasis is a delayed-type hypersensitivity reaction mediated by CD4+ T cells specific for parasite egg antigens (Ags). In an attempt to control T-cell responses leading to excessive harmful inflammation and granuloma formation, especially in the liver, BALB/c mice were intranasally (i.n.) treated with soluble Schistosoma mansoni egg Ags (SEA) conjugated to cholera toxin B subunit (CTB), a mucosa-binding protein with demonstrated capacity to suppress inflammatory T-cell functions after mucosal administration. Treatment with CTB-SEA significantly conjugate a reduced liver granuloma formation in infected mice associated with decreased SEA specific Th1- and Th2-type immune responses by liver leukocytes. Importantly, treatment with CTB-SEA conjugate also significantly reduced the mortality in chronically infected mice. In S. mansoni-infected large-granuloma forming CBA mice, i.n. treatment with purified Sm-p40, the major egg antigen, conjugated to CTB likewise significantly inhibited hepatic egg granuloma formation. A reduction of SEA-driven lymphoproliferation and of interferon (IFN)-gamma, interleukin (IL)-4 and IL-5 production, together with an increase in transforming growth factor (TGF)-beta1 production, were observed in splenic cells from CTB-Sm-p40-treated SEA-sensitized mice, as well as in liver leukocytes from CTB-Sm-p40-treated schistosome-infected mice. These results indicate that mucosal administration of SEA or purified Sm-p40 antigen in conjunction with CTB is highly effective in curtailing immunopathologic manifestations of schistosomiasis in vivo in infected hosts.     

Attenuated Host Resistance against Mycobacterium bovis BCG Infection in Mice Lacking Osteopontin

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria. The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load. The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production. BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect. These results indicate that osteopontin augments the host response against a mycobacterial infection and that it acts independently from other antimycobacterial resistance mechanisms.     

Comparative analysis of hepatic,pulmonary, and intestinal granuloma formation around freshly laidSchistosoma japonicum eggs in mice

Hepatic, pulmonary, and intestinal granuloma formation was comparatively studied in mice implanted with freshly laidSchistosoma japonicum eggs. The liver and the lung showed similar kinetics of tissue reactivity, with the magnitude in the lung being of a significantly lower degree. When the footpad-swelling test was performed, the delayed-type hypersensitivity (DTH) response during hepatic granuloma formation was found to be significantly increased from the 28th day after egg implantation onward, whereas pulmonary granuloma formation showed a peak response at 14–21 days, suggesting differing kinetics of granulomatous reaction. In histologic analysis, the temporal infiltration of monocytes was revealed to correspond to the increase in the DTH response. During intestinal granuloma formation, cosinophil infiltration was the most marked feature. The present study demonstrates that throughout the course of reaction, cellular components participating in egg-granuloma formation differ greatly according to the tissues involved.     

Intercellular adhesion molecule 1 is the major adhesion molecule expressed during schistosome granuloma formation.

Endothelial cell adhesion molecules play a key role in inflammation by initiating leukocyte trafficking. One of the most complex inflammatory responses is the formation of a cellular granuloma. Expression of adhesion molecules during granuloma formation was investigated by using the murine host reaction to schistosome parasite eggs deposited in the liver as a model. By both immunohistochemistry and lymphocyte adhesion assays, the predominant interaction identified was between intercellular adhesion molecule 1 (ICAM-1) and its cognate integrin, leukocyte functional antigen 1 (LFA-1). ICAM-1 expression on sinusoidal endothelium was induced when eggs were first deposited in the liver, peaked in parallel with granuloma size, and was downregulated with modulation of the granuloma. Polyacrylamide beads coated with soluble parasite egg antigens could induce ICAM-1 expression on endothelial cells in vitro only in the presence of tumor necrosis factor alpha, a cytokine previously shown to be key to granuloma formation. A role for ICAM-1 in recruiting lymphocytes to the hepatic granuloma was also supported by the observation that lymphocytes preincubated with anti-LFA-1 antibody did not bind to granulomas in tissue sections. While ICAM-1 is the predominant adhesion molecule in schistosome egg granuloma formation in wild-type mice, when the ICAM-1 gene is knocked out, vascular cell adhesion molecule 1 is upregulated and granuloma formation is preserved.     

In vitro and in vivo evidence that autoimmune reactivity to collagen develops spontaneously in Schistosoma mansoni-infected mice

Egg-induced granuloma formation in murine schistosomiasis mansoni results from vigorous anti-parasite reaction by activated T cells, macrophages, eosinophils, and fibroblasts. The present study suggests that strain-specific, autoimmune T-cell reactivity directed against host matrix proteins might also contribute to granulomatous hypersensitivity. T cells from infected C57B1/6, but not from CBA or BALB/c mice, proliferative in vitro in response to denatured collagen. T cells from uninfected mice, previously immunized with soluble egg antigen (SEA), did not respond in vitro to collagen. Spleen cells from acutely infected mice, but not chronically infected or uninfected animals, formed granulomas around collagen-coupled polyacrylamide beads in vitro. This response was blocked by anti-collagen antibodies that had no inhibitory effect on in vitro granuloma formation around SEA-coupled beads. In related in vivo studies, granuloma formation was quantitated after iv injection of SEA-, collagen-, or uncoated beads into normal or infected recipients. The mean diameter of lung granulomas induced by collagen-coupled beads in infected mice was significantly greater than the diameter of granulomas around either collagen beads in uninfected mice or uncoated beads in infected mice. these observations indicate that anti-collagen responses develop spontaneously in Schistosoma-infected mice and suggest that such reactivity might play a secondary role in granuloma formation and the pathogenesis of hepatic fibrosis.     

BALB/c.CBA/N mice carrying the defective Btk(xid) gene are resistant to pristane-induced plasmacytomagenesis.

The X-chromosome from the CBA/N mouse which carries the defective Bruton's tyrosine kinase (Btk) allele (Xxid) has been introgressively backcrossed onto the plasmacytoma (PCT) induction-susceptible BALB/cAN. Inbred BALB/c.CBA/N-xid/xid (C.CBA/N) mice raised and maintained in our conventional colony were given three 0.5 ml injections of pristane and were highly refractory to PCT induction. Only one PCT was found among 59 mice followed for > or =300 days. Twenty mice were examined at day 200 for foci of plasma cells in the oil granuloma. Ten mice had small foci of plasma cells, most of which were plasmacytotic, embedded in the inflammatory oil granuloma. In one there were multiple foci, but most of the mice had only one or two foci. F1 hybrid XxidY males derived from CBA/N females crossed to BALB/cAnPt were also resistant to PCT induction, while heterozygous and homozygous XY males were susceptible. C.CBA/N mice can develop extensive mucosal plasma cells as well as plasma cell accumulations in oil granuloma tissue, but the precursors of these plasma cells do not give rise to PCT in genetically susceptible hosts. The failure of C.CBA/N mice to develop PCT is probably due to the elimination of B cell clones that can be perpetuated by repeated exposure to thymus-independent type 2 antigens.     

Problems for discussion in the development of candidal granulomas in the liver

Controversial aspects of the development of infectious, in particular mycotic granulomas in the liver of laboratory animals (mice and rats) are discussed. Liver is the organ which is frequently used as a model for studying mechanisms and dynamics of the granulomatous inflammation. A high reactivity of the liver macrophagal system is noted and its ready response by granuloma formation to the bacterial cells, their walls and wall polysaccharide complexes. The expedience of revision of the criteria adopted for granuloma identification is doubted.     

Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice

Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp-Fas(lpr) (/) (lpr) (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJ-Fas(lpr) (/) (lpr) (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr x C3H/lpr)F(2) mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score =4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method "cell-free system" with wheat germ ribosomes, the MRL-OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF-alpha, IL-1beta and IFN-gamma in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis.     

Egg laying is delayed but worm fecundity is normal in SCID mice infected with Schistosoma japonicum and S. mansoni with or without recombinant tumor necrosis factor alpha treatment

Mice with severe combined immunodeficiency (SCID mice) lack functional B and T cells. Egg laying by Schistosoma mansoni and S. japonicum was delayed in SCID mice, but in a matter of weeks worm fecundity was equivalent to that in intact mice. SCID mice formed smaller hepatic granulomas and showed less fibrosis than did intact mice. The reduction in egg-associated pathology in SCID mice correlated with marked reductions in interleukin-4 (IL-4), IL-5, IL-13, and gamma interferon mRNA expression in the liver. S. mansoni infections were frequently lethal for SCID mice infected for more than 9 weeks, while S. japonicum-infected SCID mice died at the same rate as infected intact mice. We were unable to affect hepatic granuloma formation or egg laying by worms in SCID mice by administration of recombinant murine tumor necrosis factor alpha (TNF-alpha). In fact, SCID and BALB/c mice appeared to express nearly equivalent levels of TNF-alpha mRNA in their granulomatous tissues, suggesting that there is little or no deficit in TNF-alpha expression in infected SCID mice. The data indicate that TNF-alpha may be in large part derived from a non-T-cell source. Together, these findings provide little evidence that TNF-alpha alone can reconstitute early fecundity, granuloma formation, or hepatic fibrosis in schistosome-infected SCID mice.     

Implication of the Thy-1 antigen on mouse thymus cells in the recognition of 'self' structures

Thymus cells of mice form rosettes with autologous and allogeneic erythrocytes. The nature of the thymus cell receptors which mediate the binding of erythrocytes is not known. The aim of the present study was to determine the effect of various antisera to T-cell specific antigens on the formation of rosettes by mouse thymus cells. In all strains of mice tested, the exposure of thymus cells to rabbit anti-mouse brain serum (RABR) was found to inhibit autorosette formation. Similarly, monoclonal Thy-1 antibodies inhibited the formation of autorosettes by thymus cells in all strains tested. Monoclonal antibodies against Lyt-1, Lyt-2, and TL determinants had no such effect. Monoclonal Thy-1 antibodies inhibited the formation of rosettes with allogeneic erythrocytes only when the thymus contained the Lyt-2.2 allele (BALB/c, C57BL), but not when it contained the Lyt-2.1 allele (AKR/J, C3H, DBA/2). These results indicate that Thy-1 determinants on thymus cells are involved in the recognition of 'self' structures, shared by erythrocytes of all strains of mice. Lyt-2 determinants may play a role in the recognition of 'non-self', allogeneic determinants, but the thymus cell surface structures encoded by the two Lyt-2 alleles may differ in their affinity to allogeneic erythrocytes.     

Interferon-induced guanylate-binding proteins: mapping of the murine Gbp-1 locus to chromosome 3

GBP-1 is the predominant species of a family of guanylate-binding proteins synthesized in mouse cells in response to interferons (IFNs) alpha, beta, or gamma. IFN inducibility of this 65,000-Da protein is controlled by alleles at a single autosomal locus, Gbp-1, with allele a encoding inducibility and allele b noninducibility. Here, we present evidence suggesting that both alleles occur in outbred populations of wild mice. Using recombinant inbred strains and classical linkage analysis of offspring of two-point and three-point backcrosses we demonstrate that Gbp-1 is linked to Adh-3 (encoding alcohol dehydrogenase C2) and VaJ (varitintwaddler-Jackson) located on the distal part of chromosome 3. The relevant recombination frequencies (RFs) (+/- SE) were 3.5 (+/- 1.1) and 11.7 (+/- 2.8)%, respectively. We further show that strain B6.C-H-23c/By(HW 53), congenic for a small segment of chromosome 3, carries the BALB/c alleles at both the Gbp-1 and the Adh-3 locus and not the alleles of the B6 background strain confirming the chromosomal location and close linkage of the two loci.     

Population studies of the human VK A18 gene polymorphism in Caucasians, blacks and Eskimos

Immunoglobulin gene polymorphisms are interesting because they reflect differences in the available antibody repertoire which may affect the susceptibility to specific infections. Until recently, the human Vk gene, A18, was known as a nonfunctional gene only. In this study, we cloned and sequenced four apparently functional alleles and determined the gene frequencies in three well-defined populations: Danish Caucasians, eastern Greenland Eskimos and Mozambican blacks. The A 18b allele that was recently described in Native American Navajos by Atkinson et al. was found in all three populations with gene frequencies of 8%, 45% and 23% in Caucasians, Eskimos and blacks, respectively. Conversely, the frequencies of the nonfunctional A 18a allele were 92%, 55% and 57%. Further, three new A18 alleles, c, d, and e were found exclusively in blacks, among whom they had an total frequency of 19%. These data indicate that both the A 18a and A 18b alleles originated before the diversification of Africans and non-Africans 90,000 years ago, whereas the A 18c, A18d and A18e alleles may have a more recent origin. The functionality of the A 18b allele was documented by the demonstration of properly rearranged and somatically hypermutated A18b messenger RNA present in the blood lymphocytes of individuals carrying this allele. The expression clearly exceeded that of a known functional V gene, A2, indicating that functional A18 alleles contribute significantly to the available antibody repertoire. In this context, it is surprising that the functional A 18b allele apparently has been negatively selected in the Caucasian population, among whom 85% completely lack a functional gene.     

Dominant suppressive effect of the silent Eb alpha allele on an in vivo T helper cell response under Ed beta Ed alpha region-linked immune response gene control

Previous adoptive spleen cell transfer experiments have demonstrated that an immune response (Ir) gene linked to the Ed beta Ed alpha region allows BALB/c T helper lymphocytes (Th) to respond to an idiotope on the V lambda 2(315) fragment of isologous myeloma protein M315. BALB.K (H-2k) and BALB.B (H-2b) do not respond to V lambda 2(315). While (H-2d X H-2k)F1 hybrids have been shown to be responders, it is now demonstrated that (H-2d X H-2b)F1 hybrids are low responders. By crossing BALB/c with various H-2 recombinants on B10 background and probing Th responsiveness to V lambda 2(315) in these F1 hybrids, the dominant suppressive gene of the H-2b haplotype is mapped to Eb alpha Sb. It is argued that the suppressive gene is Eb alpha, which is a silent allele. A likely explanation for the suppressive effect of the Eb alpha allele is that reduced amounts of Ed beta: Ed alpha restriction elements are present on antigen-presenting cells of (H-2d X H-2b)F1 hybrids because only one E alpha gene is functional in such mice. The present report extends previous in vitro findings from other laboratories to the in vivo situation and suggests that silent alleles for class II molecule chains may profoundly affect certain immune responses of individuals heterozygous for the silent allele.