Negative selection during the peripheral immune response to antigen.

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen-major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 A(u)-restricted, acetylated NH(2)-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for A(u). In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

Sedimentation equilibrium analysis of recombinant mouse FcRn with murine IgG1.

The interaction of mouse IgG1 or IgG1-derived Fc fragment with recombinant, insect cell expressed mouse FcRn has been analyzed using sedimentation equilibrium. This results in a model for the interaction in which the two binding sites for FcRn on Fc or IgG1 have significantly different affinities with macroscopic binding constants of < 130 nM and 6 microM. This data indicates the formation of an asymmetric FcRn:Fc (or IgG1):FcRn complex which is consistent with earlier suggestions that for this form of recombinant FcRn, binding to IgG1 or Fc does not result in a symmetric 2:1 complex in which both binding sites are equivalent.

PET imaging of the immune system: immune monitoring at the whole body level.

As newer immunotherapies are developed, the necessity to non-invasively and temporally assess the changes in the immune system will be more important. Currently, a variety of cytokine therapies, vaccines, adoptive cellular therapy, and immunoregulatory antibodies are being advanced in the preclinical and clinical arenas. These developments highlight the necessity to use non-invasive imaging techniques to follow the therapeutic site of action, duration of immune response and the response of the tumor. Positron emission tomography (PET) imaging has emerged as a flexible tool which allows the user to assess multiple aspects of the immune response, including the ability to monitor the primary and secondary immune response, particular effector subpopulations of the immune response, and with novel probes, to more selectively monitor the immune response versus the tumor. This review focuses on the use of PET imaging to monitor the dynamic, multicellular and distinct spatiotemporal aspects of immunotherapy for malignancy.

Detection of autoreactive T cells in H-2u mice using peptide-MHC multimers.

Myelin basic protein (MBP)-specific T cells play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity. In PL/J and B10.PL mice (H-2(u) haplotype), the immunodominant epitope of MBP is represented by an N-terminal nonameric peptide, MBP1-9. To date, the MBP1-9-specific T cell repertoire has not been analyzed in quantitative terms. In the present study we demonstrate, using MHC class II tetramers, that 15,000-70,000 self-antigen-specific T(h) cells accumulate in the draining lymph nodes following immunization with spinal cord homogenate or MBP1-9. In contrast, MBP1-9-specific T cells are undetectable in unimmunized H-2(u) mice and represent >60% of the CD4 cells in naive mice transgenic for a TCR specific for this epitope. The results suggest that the extremely low affinity of the N-terminal peptide for I-A(u) does not limit the MBP1-9-specific T cells from expanding into a sizeable pool of autoreactive T cells. Therefore, the primary immune response to MBP1-9 does not differ quantitatively from previously reported CD4(+) T cell responses to foreign antigens.

Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies.

Preclinical tests of therapeutic antibodies are frequently carried out in mice to evaluate pharmacokinetics and efficacy. However, the observation that mouse IgG are cleared rapidly from the human circulation suggests that mice may not always be an ideal model. The Fc receptor, FcRn, regulates the serum half-lives of IgG in mice and most likely has a similar function in humans. In the current study we have carried out an extensive analysis of the interaction of the human or mouse forms of FcRn with IgG from various species using surface plasmon resonance. We show that in contrast to mouse FcRn, human FcRn is surprisingly stringent in its binding specificity for IgG derived from different species. Human FcRn binds to human, rabbit and guinea pig IgG, but not significantly to rat, bovine, sheep or mouse IgG (with the exception of weak binding to mouse IgG2b). In contrast, mouse FcRn binds to all IgG analyzed. The lack of binding of human FcRn to mouse IgG1 has been confirmed using transfectants that have been engineered to express human FcRn on the cell surface. Our results provide a molecular explanation for the enigmatic observation that mouse IgG behave anomalously in humans. These studies have implications for the successful application of therapeutic antibodies.

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases.

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.

Kinetics and thermodynamics of T cell receptor- autoantigen interactions in murine experimental autoimmune encephalomyelitis.

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)-peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1-11) bound to the MHC class II protein, I-A(u), and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1-11:I-A(u) with a 4-5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR-pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.

A role for the region encompassing the c" strand of a TCR V alpha domain in T cell activation events.

The distinct strand topology of TCR V alpha domains results in a flatter surface in the region encompassing the c" strand than the corresponding region in Ig V domains. In the current study a possible role for this region in T cell activation has been investigated by inserting a potential glycosylation site at V alpha residue 82. This residue is in proximity to the c" strand and distal to the putative interaction site for cognate peptide:MHC ligand. An additional N-linked carbohydrate at this position would create a protrusion on the V alpha domain surface, and this may interfere with TCR aggregation and/or recruitment of signaling molecules. The modified TCR has been expressed in transfected T cells, and the phenotype following stimulation has been compared with that of cells expressing the wild-type TCR. The mutation has significant effects on activation-induced cell death and TCR internalization, but, unexpectedly, does not affect IL-2 secretion. Furthermore, analyses with tetrameric, peptide:MHC class II complexes suggest that the mutation decreases the ability of the TCR to aggregate into a configuration compatible with avid binding by these multivalent ligands.

Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.

The analysis of the pharmacokinetics of wild-type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half-life in mice. Reduced serum half-life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I-related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half-life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half-life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2-CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.