Pathogenesis of an Infectious Mononucleosis-like Disease Induced by a Murine γ-Herpesvirus: Role for a Viral Superantigen?

The murine γ-herpesvirus 68 has many similarities to EBV, and induces a syndrome comparable to infectious mononucleosis (IM). The frequency of activated CD8⁺ T cells (CD62L^lo) in the peripheral blood increased greater than fourfold by 21 d after infection of C57BL/6J (H-2^b) mice, and remained high for at least a further month. The spectrum of T cell receptor usage was greatly skewed, with as many as 75% of the CD8⁺ T cells in the blood expressing a Vβ4⁺ phenotype. Interestingly, the Vβ4 dominance was also seen, to varying extents, in H-2^k, H-2^d, H-2^u, and H-2^q strains of mice. In addition, although CD4 depletion from day 11 had no effect on the Vβ4 bias of the T cells, the Vβ4⁺CD8⁺ expansion was absent in H-2IA^b–deficient congenic mice. However, the numbers of cycling cells in the CD4 antibody–depleted mice and mice that are CD4 deficient as a consequence of the deletion of MHC class II, were generally lower. The findings suggest that the IM-like disease is driven both by cytokines provided by CD4⁺ T cells and by a viral superantigen presented by MHC class II glycoproteins to Vβ4⁺CD8⁺ T cells.The murine γ-herpesvirus 68 (MHV-68)¹ is classified as a type 2 γ-herpesvirus (γHV; references 1, 2), along with Herpesvirus saimiri (3), and a novel γHV that has recently been implicated in Kaposi''s sarcoma (4–6). However, the disease process induced in mice infected intranasally with MHV-68 is much more similar to the syndrome associated with prototypic human type 1 γHV, EBV in people (7), than to that caused by the T lymphotrophic H. saimiri in nonhuman primates (8). The key characteristic is that MHV-68 replicates in the epithelial cells of the respiratory tract, with subsequent infection of B cells in lymphoid tissue (9–12). The productive growth phase in the lung cells is terminated by the CD8⁺ T cell response within 10–13 d. Little, if any, infectious virus can be recovered directly from homogenized lymphoid tissue, although reactivation of latent MHV-68 in B cells occurs readily after cocultivation on susceptible fibroblast monolayers (9–13).Infectious mononucleosis (IM) is a debilitating condition of adolescents resulting from primary infection with EBV. The disease is characterized by lymph node enlargement and the prolonged presence of greatly increased numbers of activated CD8⁺ T cells in peripheral blood, after an initial influenza-like phase reflecting the entry of EBV via the oropharyngeal/respiratory mucosa. Apart from the viral etiology, the pathogenesis of this selective lymphocytosis is not understood. Few of the circulating CD8⁺ T cells can be shown to be EBV-specific, while the virus persists as a latent infection in predominantly B, rather than T, lymphocytes (14–18). Analysis of the pathogenesis of MHV-68– induced IM described in this report suggests a mechanism involving both cytokines and a putative viral superantigen.     

Requirements for CD1d Recognition by Human Invariant Vα24+ CD4?CD8? T Cells

A subset of human CD4⁻CD8⁻ T cells that expresses an invariant Vα24-JαQ T cell receptor (TCR)-α chain, paired predominantly with Vβ11, has been identified. A series of these Vα24 Vβ11 clones were shown to have TCR-β CDR3 diversity and express the natural killer (NK) locus–encoded C-type lectins NKR-P1A, CD94, and CD69. However, in contrast to NK cells, they did not express killer inhibitory receptors, CD16, CD56, or CD57. All invariant Vα24⁺ clones recognized the MHC class I–like CD16 molecule and discriminated between CD1d and other closely related human CD1 proteins, indicating that recognition was TCR-mediated. Recognition was not dependent upon an endosomal targeting motif in the cytoplasmic tail of CD1d. Upon activation by anti-CD3 or CD1d, the clones produced both Th1 and Th2 cytokines. These results demonstrate that human invariant Vα24⁺ CD4⁻CD8⁻ T cells, and presumably the homologous murine NK1⁺ T cell population, are CD1d reactive and functionally distinct from NK cells. The conservation of this cell population and of the CD1d ligand across species indicates an important immunological function.The CD1 locus encodes a family of conserved nonpolymorphic proteins structurally related to MHC class I and II proteins (1–5). Human and murine CD1-restricted T cell lines and clones that recognize lipid antigens (6–8) or hydrophobic peptide antigens (9) have been identified, indicating that CD1 proteins can function as specialized antigen-presenting molecules. A distinct function for murine CD1d appears to be as a ligand or antigen-presenting molecule recognized by a population of T cells that express the NKR-P1C (NK1) cell surface C-type lectin (10–14). NK1 is otherwise restricted to NK cells and these NK1⁺ T cells have also been referred to as NK T cells or natural T cells (15, 16). Phenotypically, these cells are either CD4⁺CD8⁻ or CD4⁻CD8⁻ (double negative, DN¹), and forced expression of CD8 in transgenic mice results in the deletion of this population (12, 17). Most strikingly, the majority of these cells use an invariant TCR-α chain (Vα14-Jα281) that pairs preferentially with Vβ8, 7 or 2 (17–20). NK1⁺ T cells appear to play a role in regulating immune responses, based upon their ability to rapidly produce large amounts of IL-4 after stimulation with anti-CD3 in vivo (21–24). However, these cells also produce large amounts of IFN-γ, and production of this cytokine can be specifically induced by stimulation through NK1 (25). The immunological functions of these cells and the physiologically relevant CD1d-presenting cells mediating their activation remain to be determined.Analyses of the CD1 genes in humans and other species indicate that the proteins fall into two groups, CD1a-, b-, and c-like (group 1), and CD1d-like (group 2) (3, 26). The murine CD1 locus appears unique in that it has deleted the group 1 genes, and contains only a duplicated group 2 gene (27, 28). This observation suggests that there may be important functional differences between the CD1 proteins in mice and other species. Nonetheless, a human invariant TCR-α chain closely related to the murine invariant Vα14-Jα281 TCR has been identified as a predominant TCR used by TCR-α/β DN T cells from multiple normal donors (29). This human invariant TCR-α is generated by a rearrangement between Vα24 (TCRAV24) and JαQ with no N-region diversity. Subsequent studies have shown that the human invariant Vα24-JαQ TCR-α chain associates preferentially with Vβ11 (TCRBV11) (30–32), which is homologous to murine Vβ8. This invariant TCR may also be expressed by a small proportion of CD4⁺ T cells, but not CD8⁺ T cells (31). These observations suggest that human invariant Vα24⁺ T cells are homologous to murine NK1⁺ T cells.To better understand the function of these cells and their requirements for specific activation, a series of human invariant Vα24⁺Vβ11⁺ DN T cell clones were established and characterized. TCR-β sequence analysis demonstrated that these cells were derived from a polyclonal population with no evidence of a shared β chain CDR3 motif. Phenotypically, the clones expressed high levels of NKR-P1A, the only known human homologue of rodent NK1 (33). They also expressed CD94 and CD69, two other C-type lectins closely linked to NKR-P1A in a chromosomal region referred to as the NK locus. However, these cells did not express the NK cell-associated p58 or p70 HLA class I killer cell inhibitory receptors (KIRs; 34, 35) or other markers of NK cells including CD16, CD56, or CD57. Upon stimulation, the clones secreted cytokines associated with both Th1 and Th2 cells, including IFN-γ and IL-4, respectively. Of the four characterized human CD1 proteins, each clone specifically recognized only CD1d expressed on human or hamster cell transfectants. CD1d recognition was not dependent upon the specific TCR-β chain CDR3 sequence. Moreover, deletion of an endosomal targeting sequence motif in the cytoplasmic tail of CD1d (4, 5, 36) did not effect recognition, suggesting that recognition by these cells was not dependent upon efficient targeting of CD1d to a specialized endosomal compartment involved in antigen processing. These results demonstrate that human invariant Vα24⁺Vβ11⁺ DN T cells are a specialized population of CD1d-specific T cells. The results also indicate that the similarities between this T cell population, and presumably the homologous murine NK1⁺ T cell population, to NK cells may be limited to the expression of certain closely linked NK locus–encoded C-type lectins.     

��-Cell�CMediated Signaling Predominates Over Direct ��-Cell Signaling in the Regulation of Glucagon Secretion in Humans

OBJECTIVE

Given evidence of both indirect and direct signaling, we tested the hypothesis that increased β-cell–mediated signaling of α-cells negates direct α-cell signaling in the regulation of glucagon secretion in humans.

RESEARCH DESIGN AND METHODS

We measured plasma glucagon concentrations before and after ingestion of a formula mixed meal and, on a separate occasion, ingestion of the sulfonylurea glimepiride in 24 basal insulin-infused, demonstrably β-cell–deficient patients with type 1 diabetes and 20 nondiabetic, demonstrably β-cell–sufficient individuals; the latter were infused with glucose to prevent hypoglycemia after glimepiride.

RESULTS

After the mixed meal, plasma glucagon concentrations increased from 22 ± 1 pmol/l (78 ± 4 pg/ml) to 30 ± 2 pmol/l (103 ± 7 pg/ml) in the patients with type 1 diabetes but were unchanged from 27 ± 1 pmol/l (93 ± 3 pg/ml) to 26 ± 1 pmol/l (89 ± 3 pg/ml) in the nondiabetic individuals (P < 0.0001). After glimepiride, plasma glucagon concentrations increased from 24 ± 1 pmol/l (83 ± 4 pg/ml) to 26 ± 1 pmol/l (91 ± 4 pg/ml) in the patients with type 1 diabetes and decreased from 28 ± 1 pmol/l (97 ± 5 pg/ml) to 24 ± 1 pmol/l (82 ± 4 pg/ml) in the nondiabetic individuals (P < 0.0001). Thus, in the presence of both β-cell and α-cell secretory stimuli (increased amino acid and glucose levels, a sulfonylurea) glucagon secretion was prevented when β-cell secretion was sufficient but not when β-cell secretion was deficient.

CONCLUSIONS

These data indicate that, among the array of signals, indirect reciprocal β-cell–mediated signaling predominates over direct α-cell signaling in the regulation of glucagon secretion in humans.The regulation of pancreatic islet α-cell glucagon secretion is complex (1–10). It involves direct signaling of α-cells (1) and indirect signaling of α-cells by β-cell (2–6) and δ-cell (7) secretory products, the autonomic nervous system (8,9), and gut incretins (10).Appropriate glucagon secretory responses occur from the perfused pancreas (3,5) and perifused islets (2). Low plasma glucose concentrations stimulate glucagon secretion from the transplanted (i.e., denervated) human pancreas (11) and the denervated dog pancreas (12). Therefore, we have focused on the intraislet regulation of glucagon secretion. Furthermore, because selective destruction of β-cells results in loss of the glucagon response to hypoglycemia in type 1 diabetes (13), and partial reduction of the β-cell mass in minipigs results in impaired postprandial suppression of glucagon secretion (14), we have focused on the role of β-cell–mediated signaling in the regulation of glucagon secretion.Findings from studies of the perfused rat (3,4) and human (5) pancreas, rats in vivo (6), rat islets (2), isolated rat α-cells (2), and humans (15–18) have been interpreted to indicate that a β-cell secretory product or products tonically restrains basal α-cell glucagon secretion during euglycemia and that a decrease in β-cell secretion, coupled with low glucose concentrations at the α-cells, signals an increase in glucagon secretion in response to hypoglycemia. Parenthetically, the relative roles of the candidate β-cell secretory products (insulin, zinc, γ-aminobutyric acid, and amylin, among others) (2) that normally restrain α-cell glucagon secretion remain to be determined. However, that interpretation rests, in part, on results of studies in isolated rat α-cells (2), which are debated (1), and on the evidence that the islet microcirculation flows from β-cells to α-cells to δ-cells (4), which is also debated (19). Furthermore, it does not address the plausible possibility that a decrease in intraislet δ-cell somatostatin secretion might also signal an increase in α-cell glucagon secretion during hypoglycemia (7).Given that interpretation, it follows that an increase in β-cell secretion would signal a decrease in glucagon secretion in the postprandial state (14). The concept is an interplay of indirect reciprocal β-cell–mediated signaling of α-cells and of direct α-cell signaling in the regulation of glucagon secretion.There is, in our view, compelling evidence that, among other mechanisms, both indirect reciprocal β-cell–mediated signaling of α-cells (2–6) and direct α-cell signaling (1) are involved in the regulation of glucagon secretion by nutrients, hormones, neurotransmitters, and drugs. Given that premise, we posed the question: Which of these predominates in humans? Accordingly, we tested the hypothesis that increased β-cell–mediated signaling of α-cells negates direct α-cell signaling in the regulation of glucagon secretion in humans. To do so, we measured plasma glucagon responses to ingestion of a mixed meal and, on a separate occasion, to ingestion of the sulfonylurea glimepiride in patients with type 1 diabetes and in nondiabetic individuals. We conceptualized patients with type 1 diabetes as a model of α-cells isolated from β-cells because their β-cells had been destroyed but they have functioning α-cells. (Their α-cells are not, of course, isolated from other islet cells, including δ-cells.) Increased plasma amino acid and glucose levels after a mixed meal and sulfonylureas normally stimulate β-cell secretion; increased plasma amino acid and perhaps glucose (2) levels after a mixed meal and sulfonylureas (1) stimulate α-cell secretion. Our hypothesis predicts that such factors that normally stimulate both β-cells and α-cells would stimulate glucagon secretion in patients with type 1 diabetes but not in nondiabetic individuals, i.e., in the virtual absence and the presence of β-cell function, respectively. Indeed, a mixed meal (20,21) and the secretagogues tolbutamide (22), glyburide (23), and repaglinide (23) have been reported to raise plasma glucagon concentrations in patients with type 1 diabetes, but all of those studies lacked nondiabetic control subjects.     

Rifampin Increases Cytokine-Induced Expression of the CD1b Molecule in Human Peripheral Blood Monocytes

In recent years, it has been shown that a nonclassical, major histocompatibility complex-independent system (i.e., CD1-restricted T-cell responses) is involved in T-cell immunity against nonpeptide antigens. The CD1 system appears to function by presenting microbial lipid antigens to specific T cells, and the antigens so far identified include several known constituents of mycobacterial cell walls. Among the four known human CD1 isoforms, the CD1b protein is the best characterized with regard to its antigen-presenting function. Expression of CD1b is upregulated on human blood monocytes upon exposure to granulocyte/macrophage-colony stimulating factor, alone or in combination with interleukin-4 (IL-4) (S. A. Porcelli, Adv. Immunol. 59:1–98, 1995). Rifampin (RFP) and its derivatives are widely used for chemoprophylaxis or chemotherapy against Mycobacterium tuberculosis. However, this agent was found to reduce the mitogen responsiveness of human B and T lymphocytes, chemotaxis, and delayed-type hypersensitivity. The present study extends the immunopharmacological profile of RFP by examining its effects on CD1b expression by human peripheral blood monocytes exposed to GM-CSF plus IL-4. The results showed that clinically attainable concentrations (i.e., 2 or 10 μg/ml for 24 h) of the agent produced a marked increase in CD1b expression on the plasma membrane, as evaluated by fluorescence-activated cell sorter analysis, whereas it had no effect on cytosolic fractions, as indicated by Western blot analysis. This was found to be the result of increased CD1b gene expression, as shown by Northern blot analysis of CD1b mRNA. These results suggest that RFP could be of potential value in augmenting the CD1b-restricted antigen recognition system, thereby enhancing protective cellular immunity to M. tuberculosis.The incidence of mycobacterial infections has rapidly increased in recent years. One of the principal causes of this phenomenon appears to be the high susceptibility of human immunodeficiency virus-positive persons to mycobacterial pathogens (3, 11, 17).A large amount of experimental and clinical evidence showing that T-cell-mediated immune responses play a significant role in resistance against mycobacteria is now available (18, 28). Subpopulations of T cells that are involved in antimycobacterial immunity include CD3⁺ lymphocytes bearing the αβ T-cell receptor (TCR), predominantly of the CD4⁺ phenotype (18), and γδ TCR T lymphocytes (28). Effector CD4⁺ T cells are sensitized with mycobacterium-derived peptides presented by antigen-presenting cells in association with the class II major histocompatibility complex (MHC, 25). Immune lymphocytes show a Th1-like response pattern, being cytotoxic for mycobacterial targets and capable of secreting gamma interferon upon challenge with the relevant antigen (18, 28).In recent years, growing interest has been elicited by a nonclassical, MHC-independent system that appears to be additionally involved in T-cell responses against mycobacteria. In this case, the human antigen-presenting molecule is the group I, nonpolymorphic CD1b protein (1, 2, 15, 19) expressed by cytokine-activated macrophages (20). The antigens presented by the CD1b molecule belong to a variety of nonpeptide macromolecules (20). Among them, mycolic acids, lipoarabinomannan, and other lipid structures associated with the mycobacterial cell wall are believed to be involved in CD1-dependent host resistance against tuberculosis. Lipoarabinomannan is taken up by a macrophage mannose receptor that carries the antigen to macrophage endosomes, where it is loaded onto CD1b molecules (21).In this system, many of the responder cells come from the CD4⁻ 8⁻ phenotypic subset of CD3⁺, αβ TCR T cells. These cells, referred to as double-negative αβ T lymphocytes (20), proliferate and generate cytotoxic clones following interaction with mycobacterial glycolipids presented by CD1b⁺ monocytes preactivated with granulocyte/macrophage-colony stimulating factor (GM-CSF), alone or in combination with interleukin-4 (IL-4) (20). More recently, CD8⁺, αβ TCR T-cell clones with similar properties have also been demonstrated (26).Rifampin (RFP) and a number of its derivatives (e.g., rifabutin) are widely used for therapy against Mycobacterium tuberculosis in immunocompromised patients (16) or to treat various types of mycobacterial infections provoked by atypical strains (i.e., M. avium; 4). However, previous studies showed that RFP reduces humoral and cell-mediated immunity (9, 10, 14, 29). These observations suggested the possibility that antitubercular chemotherapy with RFP would attenuate the functional activity of the immune system, with possible negative effects on resistance against mycobacterial infections. To study the possible effects of this antibiotic on macrophage function relative to antigen presentation by CD1b molecules, CD1b molecule expression has been studied in vitro in human peripheral blood monocytes exposed to GM-CSF plus IL-4, alone or in the presence of RFP. The results showed that clinically attainable concentrations of the agent increased CD1b expression, thus suggesting that the antibiotic could be of potential value in improving the CD1b-restricted antigen recognition system.