Takako Matsuoka

Monocytes Are Differentially Activated Through HLA-DR, -DQ, and -DP Molecules Via Mitogen-Activated Protein Kinases

When HLA-DR, -DQ, and -DP were cross-linked by solid-phase mAbs, monocytes produced monokines and only anti-DR markedly activated mitogen-activated protein (MAP) kinase extracellular signal-related kinase, whereas anti-DR, anti-DQ, and anti-DP all activated MAP kinase p38. Activation of extracellular signal-related kinase was not inhibited by neutralizing Ab to TNF-α. Anti-DR and DR-restricted T cells stimulated monocytes to produce relatively higher levels of proinflammatory monokines, such as IL-1β, whereas anti-DQ/DP and DQ-/DP-restricted T cells stimulated higher levels of anti-inflammatory monokine IL-10. IL-10 production was abrogated by the p38 inhibitor SB203580, but rather enhanced by the MAP/extracellular signal-related kinase kinase-I-specific inhibitor PD98059, whereas IL-1β was only partially abrogated by SB203580 and PD98059. Furthermore, DR-restricted T cells established from PBMC, which are reactive with mite Ags, purified protein derivative, and random 19-mer peptides, exhibited a higher IFN-γ:IL-4 ratio than did DQ- or DP-restricted T cells. These results indicate that HLA-DR, -DQ, and -DP molecules transmit distinct signals to monocytes via MAP kinases and lead to distinct monokine activation patterns, which may affect T cell responses in vivo. Thus, the need for generation of a multigene family of class II MHC seems apparent.

Ligation of HLA-DR Molecules on B Cells Induces Enhanced Expression of IgM Heavy Chain Genes in Association with Syk Activation*

Signals transmitted by class II major histocompatibility complex are important regarding cell function related to antigen presentation. We examined effects of DR-mediated signaling on Ig production from B cells. Cross-linking HLA-DR molecules on B cells by solid-phase anti-HLA-DR monoclonal antibodies, led to an increased production of IgM, without proliferation or apoptosis. This event was accompanied by an enhanced expression of both membrane- and secretory-type IgM heavy chain mRNA. When peptide-pulsed B cells were co-incubated with an HLA-DR-restricted T cell clone treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. CD40-CD154 interaction was not involved in IgM enhancement, in such a system. The protein-tyrosine kinase inhibitors and the Syk inhibitor piceatannol, but not the Src inhibitor PP2 had a marked inhibitory effect on IgM secretion. Furthermore, ligation of HLA-DR on B cells using the F(ab′)2 fragment of anti-DR monoclonal antibody, enhanced Syk activity. Our data suggest that HLA-DR on B cells not only present antigenic peptides to T cells, but also up-regulate IgM production, in association with Syk activation and without the involvement of Src kinases, hence the possible physiological relevance of Src-independent Syk activation.

Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-dependent kinase inhibitor p27Kip1

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-gamma and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27(Kip1) but not p21(Cip1) increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27(Kip1). We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27(Kip1). The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide.

Peptide length‐dependent TCR antagonism on class II HLA‐restricted responses of peripheral blood mononuclear cells and T cell clones

T cell clonal recognition of peptide ligands is highly diverse. To investigate how peptide mixtures with diverse sequences affect polyclonal responses of peripheral blood mononuclear cells (PBMC), we synthesized X n ( n = 9 – 19) peptides that consist of 9 to 19 residues with random sequences. We found that: (1) in antagonism assays, X n peptides inhibit polyclonal responses of PBMC induced by purified protein derivative (PPD) and crude mite extracts as well as mixed lymphocyte reaction (MLR), in which intermediate‐length peptides ( n = 13 or 15) show the largest inhibitory effects; and (2) a high‐affinity HLA‐DR4‐binding peptide is devoid of inhibitory activity against MLR to DR4, indicating that these effects are not caused by inhibition of peptide binding to HLA. Furthermore, X15 did not abrogate PBMC proliferation induced by phorbol 12‐myristate 13‐acetate + ionomycin, or anergize PBMC by pre‐culture. All these observations indicate that TCR antagonism does exist at peripheral T cell levels in humans, and that X n peptides, depending on peptide length, are capable of antagonizing T cell polyclonal responses. Indeed, even with cloned T cells, certain non‐agonistic peptides shorter (but not too short) than the wild type in their C termini, function as TCR antagonists, findings which corroborate the observation that X13 –15 antagonizes T cell responses more efficiently than does X17–19 or X9 –11.

Clonal expansion of freshly isolated CD4T cells by randomized peptides and identification of peptide ligands using combinatorial peptide libraries

We synthesized Xn (n = 9 – 19) peptides that consist of 9 to 19 residues with random sequences. X19 is considered to deliver antigenic stimuli to CD4 T cells, because: (a) X19 induces proliferation of peripheral blood mononuclear cells (PBMC), in the presence of IL‐2, which is abrogated by monoclonal antibodies to class II HLA; (b) X19 + IL‐2 induces proliferation of CD4 T cell clones of distinct specificities; and (c) T cell clones recognizing the same TCR ligands with distinct Vβ  usage are equally stimulated by X19 + IL‐2. We next co‐cultured single peripheral CD4 T cells with X19 and mitomycin‐treated autologous PBMC. Indeed, single T cells of CD45RA– memory phenotype exhibited clonal expansion, with variable rates of proliferation, when IL‐4, IL‐7, IL‐9, IL‐15 and agonistic antibody to CD29 were included in the culture. These T cell clones showed heterogeneous proliferation patterns against KGXXXXXXXXXGK‐based and KGXXXXXXXXXGKGKK‐based combinatorial peptides libraries, in the presence of IL‐2. Pattern‐match search on a T cell clone resulted in peptide ligand candidates, one of which induced proliferation, as did protein molecules carrying the corresponding sequence. These results indicate that X19 can induce proliferation of peripheral memory T cells, the peptide ligands of which can be determined using combinatorial peptide libraries.