Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner.

Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines.

Cutting edge: innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli.

Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αß T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

H2 control of natural T regulatory cell frequency in the lymph node correlates with susceptibility to day 3 thymectomy-induced autoimmune disease.

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by natural regulatory T cells (nTregs) and development of autoimmune ovarian dysgenesis (AOD) and autoimmune dacryoadenitis (ADA) in A/J and (C57BL/6J × A/J) F(1) hybrids (B6A), but not in C57BL/6J (B6) mice. Previously, using quantitative trait locus (QTL) linkage analysis, we showed that D3Tx-AOD is controlled by five unlinked QTL (Aod1-Aod5) and H2. In this study, using D3Tx B6-Chr(A/J)/NaJ chromosome (Chr) substitution strains, we confirm that QTL on Chr16 (Aod1a/Aod1b), Chr3 (Aod2), Chr1 (Aod3), Chr2 (Aod4), Chr7 (Aod5), and Chr17 (H2) control D3Tx-AOD susceptibility. In addition, we also present data mapping QTL controlling D3Tx-ADA to Chr17 (Ada1/H2), Chr1 (Ada2), and Chr3 (Ada3). Importantly, B6-ChrX(A/J) mice were as resistant to D3Tx-AOD and D3Tx-ADA as B6 mice, thereby excluding Foxp3 as a susceptibility gene in these models. Moreover, we report quantitative differences in the frequency of nTregs in the lymph nodes (LNs), but not spleen or thymus, of AOD/ADA-resistant B6 and AOD/ADA-susceptible A/J, B6A, and B6-Chr17(A/J) mice. Similar results correlating with experimental allergic encephalomyelitis and orchitis susceptibility were seen with B10.S and SJL/J mice. Using H2-congenic mice, we show that the observed difference in frequency of LN nTregs is controlled by Ada1/H2. These data support the existence of an LN-specific, H2-controlled mechanism regulating the prevalence of nTregs in autoimmune disease susceptibility.

Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC class I in the endoplasmic reticulum.

Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC class I (MHC-I) are associated with several chaperones. Heat shock proteins 70, 90, and the TCP-1 ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study, we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly, we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC-I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC-I and hence presentation of the MHC-I-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC-I H chains and beta2-microglobulin as a trimolecular complex.

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex.

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rß/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of ß2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of ß2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.

SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation.

Previously, we showed that 2B4 is a dominant inhibitory receptor in SHIP-deficient NK cells that prevents efficient cytolysis of complex targets. We show in this study that 2B4 deficiency restores homeostatic control and cytolytic function to SHIP-deficient NK cells. However, 2B4(-/-)SHIP(-/-) NK cells still exhibit a profound disruption of their NK receptor repertoire and are compromised for induction of IFN-gamma by several NK-activating receptors, including NKp46, NK.1.1, and NKG2D. In addition, we find that 2B4(-/-) NK cells have an extensively disrupted repertoire, including a supernormal frequency of NKp46(+) NK cells. Consequently IFN-gamma is induced on a much higher percentage of 2B4(-/-) NK cells following engagement of NKp46. We also find that both SHIP and 2B4 are required to prevent expression of Ly49B, a myeloid lineage MHC class I receptor not normally expressed by the NK lineage. Finally, when SHIP-deficient NK cells are on an H-2(d) background, they exhibit supernormal levels of Ly49A and possess normal cytolytic function against MHC-matched tumor targets and enhanced cytolysis of MHC mismatched tumor targets. However, despite normal or elevated cytolytic function, H2d SHIP(-/-) NK cells exhibit poor induction of IFN-gamma like their H2b(+) or 2B4(-/-) counterparts, demonstrating a uniform requirement for SHIP in induction of IFN-gamma downstream of key NK activating receptors. These findings reveal a complex interplay of SHIP, 2B4, and MHC in the regulation of homeostasis, effector function, and repertoire formation in the NK cell lineage.

Microchimerism is strongly correlated with tolerance to noninherited maternal antigens in mice.

In mice and humans, the immunologic effects of developmental exposure to noninherited maternal antigens (NIMAs) are quite variable. This heterogeneity likely reflects differences in the relative levels of NIMA-specific T regulatory (T(R)) versus T effector (T(E)) cells. We hypothesized that maintenance of NIMA-specific T(R) cells in the adult requires continuous exposure to maternal cells and antigens (eg, maternal microchimerism [MMc]). To test this idea, we used 2 sensitive quantitative polymerase chain reaction (qPCR) tests to detect MMc in different organs of NIMA(d)-exposed H2(b) mice. MMc was detected in 100% of neonates and a majority (61%) of adults; nursing by a NIMA+ mother was essential for preserving MMc into adulthood. MMc was most prevalent in heart, lungs, liver, and blood, but was rarely detected in unfractionated lymphoid tissues. However, MMc was detectable in isolated CD4+, CD11b+, and CD11c+ cell subsets of spleen, and in lineage-positive cells in heart. Suppression of delayed type hypersensitivity (DTH) and in vivo lymphoproliferation correlated with MMc levels, suggesting a link between T(R) and maternal cell engraftment. In the absence of neonatal exposure to NIMA via breastfeeding, MMc was lost, which was accompanied by sensitization to NIMA in some offspring, indicating a role of oral exposure in maintaining a favorable T(R) > T(E) balance.

Protective role of major histocompatibility complex class II Ebd transgene on collagen-induced arthritis.

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.

HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.

Three different HLA-A2.1 monochains were engineered in which either the human or mouse beta2-microglobulin (beta2m) is covalently linked to the NH2 terminus of the heavy chain by a 15- amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db alpha3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse beta2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db-/- beta2m-/- double knockout mice. Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db-/- beta2m-/- mice generate only HLA-A2.1-restricted CD8(+) CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.

Ly49A transgenic mice provide evidence for a major histocompatibility complex-dependent education process in natural killer cell development.

The Ly49 natural killer (NK) cell receptors are class I MHC-specific inhibitory receptors that are distributed to overlapping NK cell subsets. The formation of the Ly49 receptor repertoire was examined with transgenic mice that express Ly49A in all NK cells. In MHC class I-deficient mice, the Ly49A transgene did not prevent expression of endogenous Ly49 genes. However, in H-2(d) mice that express a Ly49A ligand, the transgene caused clear alterations in the endogenous Ly49 repertoire. The frequency of NK cells expressing another H-2(d)-specific receptor, Ly49G2(+), was substantially reduced. Reduced numbers of cells expressing endogenous Ly49A was suggested by reduced endogenous Ly49A mRNA levels. These results support the existence of an MHC-dependent education process that limits the number of NK cells that coexpress multiple self-specific Ly49 receptors. Ligand-dependent downregulation of Ly49 cell surface levels was also examined. Cell-surface downregulation occurred even when the transgene was expressed at low levels. The results demonstrate that downregulation of Ly49A cell surface levels is a posttranscriptional event, and argue against a model in which Ly49 receptors are calibrated to specific cell surface levels depending on the available class I ligands.

Structural requirements for class I MHC molecule-mediated antigen presentation and cytotoxic T cell recognition of an immunodominant determinant of the human immunodeficiency virus envelope protein.

In H-2d mice, the immunodominant determinant of the HIV-1-IIIB gp160 envelope glycoprotein recognized by CD8+ CTL is represented by a 15-residue synthetic peptide (315-329: RIQRGPGRAFVTIGK). This peptide is seen in association with the Dd class I MHC molecule expressed on H-2k L cell fibroblast targets. We explored the structural requirements for CTL recognition of this peptide at the levels of both the peptide molecule and the class I MHC molecule. Using several transfectants expressing recombinant Dd/Ld molecules, we found that presentation of this epitope required both the alpha 1 and alpha 2 domains of the Dd molecule, in contrast to certain instances of allorecognition for which alpha 1 of Dd was sufficient in association with alpha 2 of Ld. Because this peptide derives from a hypervariable segment of the HIV envelope, substituted peptides could be used to define not only the structures affecting interaction of peptide with class I MHC molecule and with the TCR, but also the structural basis for the effect of naturally occurring viral variation on CTL recognition. The CTL-LINE specific for this HIV-1-IIIB-derived sequence could not recognize the HIV-1-RF variant-derived sequence from exactly the same site (315-329:--HIGPGRVIYATGQ). Peptides with single amino acid substitutions from the HIV-1-IIIB sequence toward the HIV-1-RF sequence were made to test the effect of each residue significantly affected recognition, and only one, 324(F), was obligatory. Moreover, both 322(R) and 324(F) substituted peptides failed to inhibit the binding of the wild type peptide to the MHC molecule. Therefore, the amino-acids 322(R) and 324(F) seem to be involved in regulating peptide interaction with the Dd class I MHC molecule. In contrast, 325(V) appeared to affect interaction with the TCR. We suggest that sequence variations among known HIV-1 isolates that affect peptide binding to MHC such as those described here, if occurring during the course of infection of an individual, could result in failure of the MHC molecules of that individual to present the peptide. If the number of dominant HIV CTL epitopes is indeed very limited, such a blind spot could allow the virus to escape immune control, proliferate rapidly, and cause AIDS.

Recognition of the major histocompatibility complex restriction element modulates CD8(+) T cell specificity and compensates for loss of T cell receptor contacts with the specific peptide.

Triggering of a T cell requires interaction between its specific receptor (TCR) and a peptide antigen presented by a self-major histocompatibility complex (MHC) molecule. TCR recognition of self-MHC by itself falls below the threshold of detection in most systems due to low affinity. To study this interaction, we have used a read-out system in which antigen-specific effector T cells are confronted with targets expressing high levels of MHC compared with the selecting and priming environment. More specifically, the system is based on CD8(+) T cells selected in an environment with subnormal levels of MHC class I in the absence of beta2-microglobulin. We observe that the MHC restriction element can trigger viral peptide-specific T cells independently of the peptide ligand, provided there is an increase in self-MHC density. Peptide-independent triggering required at least four times the natural in vivo level of MHC expression. Furthermore, recognition of the restriction element at expression levels below this threshold was still enough to compensate for lack of affinity to peptides carrying alanine substitutions in major TCR contact residues. Thus, the specificity in TCR recognition and T cell activation is fine tuned by the avidity for self-MHC, and TCR avidities for peptide and MHC may substitute for each other. These results demonstrate a functional role for TCR avidity for self-MHC in tuning of T cell specificity, and support a role for cross-reactivity on "self" during T cell selection and activation.

Differential ability of isolated H-2 Kb subsets to serve as TCR ligands for allo-specific CTL clones: potential role for N-linked glycosylation.

It is not known whether all forms of cell surface peptide-class I complexes, when bound with relevant peptide antigen, are recognized by T cells. We demonstrate herein that two distinct subsets of the murine H-2 Kb molecule can be separately isolated from H-2b-expressing cell lines using Y3 mAb immunoaffinity chromatography. Although both isolated Kb subsets were found to be strongly reactive with Y3 mAb by ELISA, one Kb subset is S19.8 mAb reactive (Ly-m11+Kb subset) and exhibits low reactivity with the M1/42 antibody, while the other subset is negative for the Ly-m11 epitope and highly reactive with the M1/42 antibody (M1/42high Kb subset). More importantly, whereas the M1/42high Kb subset is a very effective ligand for both TCR and CD8, the Ly-m11+ Kb subset could only function as a CD8 ligand, as determined in allo-specific CD8+ CTL clone adhesion and degranulation assays. Peptides acid-eluted from both Kb subsets sensitized Kb-transfected T2 cells expressing "peptide empty" Kb for lysis to a similar extent by allo-CTL clones, indicating that relevant endogenous peptide antigens are not limiting in the Ly-m11+ Kb subset. The major distinction identified between the two Kb subsets is that they differ substantially in their degree of N-linked glycosylation, with the Ly-m11+ subset containing Kb molecules with larger and more complex carbohydrate modifications than the M1/42high subset. The differences in glycosylation may explain the functional differences observed between the two Kb subsets. It is therefore possible that some forms of glycosylation on class I molecules interfere with TCR recognition and may limit CD8+ T cell responses, perhaps under circumstances where peptide antigen is limiting.

Physical and functional association of the major histocompatibility complex class I heavy chain alpha3 domain with the transporter associated with antigen processing.

CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain alpha3 domain. Substitution of a 15 amino acid portion of the H-2Db alpha3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) beta2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26 degrees C in the presence of excess peptide and beta2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the alpha3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.

Reactivity of V beta 17a+ CD8+ T cell hybrids. Analysis using a new CD8+ T cell fusion partner.

Tolerance to IE molecules leads to deletion of V beta 17a-bearing T cells. Both, the CD4+ as well as the CD8+ T cell subsets are affected. A large percentage of CD4+ V beta 17a+ T cell hybrids recognize IE molecules. We now have investigated the reactivity for IE antigens of CD8+ V beta 17a+ T cell hybrids. Using a transfection approach, we have introduced the murine CD8 molecule into different V beta 17a+ T cell hybrids. Furthermore, the CD8 cDNA was transfected into the BW5147 alpha-beta- fusion partner. This allowed us to generate a large number of V beta 17a+ T cell hybrids by fusion with the appropriate T cells. Only 6% of T cell hybrids were stimulated to produce IL-2 upon incubation with IE+ cells. However, in those, the CD8 molecule seemed not to contribute to the IE reactivity of the hybrid, since mAbs against the CD8 molecule failed to inhibit their reactivity. This low percentage of V beta 17a+ CD8+ IE-reactive T cell hybrids contrasts with the strong reduction of CD8+ V beta 17a+ T cells in IE+ mice, strongly suggesting that elimination of such cells in the thymus occurs when they are coexpressing CD4 and CD8. This view was confirmed by the occasional expression of CD4 in some hybrids in which case IE reactivity was detected. Furthermore, we demonstrated the functional integrity of the introduced CD8 molecule by: (a) reconstitution of the IL-2 response in a class I-restricted TNP-specific T cell hybrid; and (b) by generation of alloreactive class I-restricted T cell hybrids using the new CD8+ fusion cell line. This CD8+ fusion partner, BWLyt2-4, should prove useful to study antigen processing and antigen presentation requirements of class I-restricted T cells.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.

TAP, a novel T cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes.

Five mAbs have been generated and used to characterize TAP (T cell activating protein) a novel, functional murine T cell membrane antigen. The TAP molecule is a 12-kD protein that is synthesized by T cells. By antibody crossblocking, it appears to be closely associated with a 16-kD protein on the T cell membrane also identified with a novel mAb. These molecules are clearly distinct from the major well-characterized murine T cell antigens previously described. Antibody binding to TAP can result in the activation of MHC-restricted, antigen-specific inducer T cell hybridomas that is equivalent in magnitude to maximal antigen or lectin stimulation. This is a direct effect of soluble antibody and does not require accessory cells or other factors. The activating anti-TAP mAbs are also mitogenic for normal heterogeneous T lymphocytes in the presence of accessory cells or IL-1. In addition, these antibodies are observed to modulate specific immune stimulation. Thus, the activating anti-TAP mAbs synergise with antigen-specific stimulation of T cells, while a nonactivating anti-TAP mAb inhibits antigen driven activation. These observations suggest that the TAP molecule may participate in physiologic T cell activation. The possible relationship of TAP to known physiologic triggering structures, the T3-T cell receptor complex, is considered. TAP is expressed on 70% of peripheral T cells and therefore defines a major T cell subset, making it perhaps the first example of a murine subset-specific activating protein.

Modulation of thymic selection by expression of an immediate-early gene, early growth response 1 (Egr-1).

The potential involvement of early growth response (Egr)-1, a zinc-finger transcription factor belonging to the immediate-early genes, in positive/negative selection of thymocytes has been implicated by its expression in the population of CD4(+)CD8(+) double positive (DP) cells undergoing selection. To further investigate this possibility, transgenic mice overexpressing Egr-1 in thymocytes were bred with a transgenic mouse line expressing a T cell receptor (TCR) recognizing the H-Y male antigen in the context of H-2(b) class I major histocompatibility complex (MHC) molecules. In Egr-1/TCR H-Y double-transgenic mice, efficient positive selection of H-Y CD8(+) T cells occurred, even in mice on either a nonselecting H-2(d) background or a beta2-microglobulin (beta2m)-deficient background in which the expression of class I MHC heavy chains is extremely low; no positive selection was observed on a Kb-/-Db-/-beta2m-/- background where class I MHC expression is entirely absent. Similarly, when the Egr-1 transgene was introduced into a class II MHC-restricted TCR transgenic mouse line, Egr-1/TCR double-transgenic mice revealed increased numbers of CD4(+) T cells selected by class II MHC, as well as significant numbers of CD8(+) T cells selected by class I MHC (for which the transgenic TCR might have weak affinity). Thus, Egr-1 overexpression allows positive selection of thymocytes via TCR-MHC interactions of unusually low avidity, possibly by lowering the threshold of avidity required for positive selection. Supporting this possibility, increased numbers of alloreactive T cells were positively selected in Egr-1 transgenic mice, resulting in a strikingly enhanced response against allo-MHC. These results suggest that expression of Egr-1 and/or its target gene(s) may directly influence the thresholds required for thymocyte selection.

Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses.

The B cell receptor (BCR) triggers a variety of biological responses that differ depending upon the properties of the antigen. A panel of M13 phage-displayed peptide ligands with varying affinity for the 3-83 antibody was generated to explore the role of antigen-BCR affinity in cell activation studies using primary 3-83 transgenic mouse B cells. Multiple parameters of activation were measured. T cell-independent B cell proliferation, antibody secretion, induction of germline immunoglobulin gamma1 transcripts, and B cell production of interleukin (IL) 2 and interferon gamma responses were better correlated with antigen-BCR affinity than with receptor occupancy. In contrast, other responses, such as upregulation of major histocompatibility complex class II and B7.2 (CD86), secretion of IL-6, and B cell proliferation in the context of CD40 signaling were only weakly dependent on antigen affinity. Biochemical analysis revealed that at saturating ligand concentrations the ability of phage to stimulate some early signaling responses, such as Ca++ mobilization and tyrosine phosphorylation of syk or Igalpha, was highly affinity dependent, whereas the ability to stimulate Lyn phosphorylation was less so. These data suggest that the BCR is capable of differential signaling. The possibility that differential BCR signaling by antigen determines whether an antibody response will be T independent or dependent is discussed.

Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1-peptide complexes.

The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.