The role of C4d deposition in the diagnosis of antibody-mediated rejection after lung transplantation.

Antibody-mediated rejection (AMR) is an increasingly recognized form of lung rejection. C4d deposition has been an inconsistent finding in previous reports and its role in the diagnosis has been controversial. We conducted a retrospective single-center study to characterize cases of C4d-negative probable AMR and to compare these to cases of definite (C4d-positive) AMR. We identified 73 cases of AMR: 28 (38%) were C4d-positive and 45 (62%) were C4d-negative. The two groups had a similar clinical presentation, and although more patients in the C4d-positive group had neutrophilic capillaritis (54% vs. 29%, P = .035), there was no significant difference in the presence of other histologic findings. Despite aggressive antibody-depleting therapy, 19 of 73 (26%) patients in the overall cohort died within 30 days, but there was no significant difference in freedom from chronic lung allograft dysfunction (CLAD) or survival between the two groups. We conclude that AMR may cause allograft failure, but that the diagnosis requires a multidisciplinary approach and a high index of suspicion. C4d deposition does not appear to be a necessary criterion for the diagnosis, and although some cases may respond initially to therapy, there is a high incidence of CLAD and poor survival after AMR.

Peptide affinity and concentration affect the sensitivity of M3-restricted CTLs induced in vitro.

In vitro stimulation of mouse splenocytes with hemagglutinin (HA) 173-190, a peptide derived from influenza virus hemagglutinin (A/JAP/305/57, H2N2), induces CTLs that are directed to the MHC class Ib molecule, H2-M3. M3 preferably binds peptides bearing an N-terminal formylmethionine. In this study, we show that several related nonformylated peptides can induce anti-HA CTLs in vitro: MLIIW (the minimal epitope), derived from HA186-190 at the C-terminal end of HA173-190; MLIIWG; MLIIWGV; and MLIIWGI, as well as formylated MLIIW. The heptamer peptides correspond to a polymorphism of HA192 in H2 strains of influenza; they have the highest relative affinities for M3 of the nonformylated peptides and higher affinities than some formylated mitochondrial peptides. Depending on the affinity of the peptide, a range of concentrations can be used to induce CTLs. One nanomolar of the high affinity f-MLIIW peptide can induce anti-HA CTLs, whereas 100-fold more of the lower affinity MLIIW peptide is needed. Lines induced with high concentrations (1 microM or greater) of f-MLIIW recognize Ag poorly, and the most efficient CTLs are induced with the lowest concentrations of peptide. Analysis with a panel of anti-TCRVbeta Abs shows that different T cells respond to high vs low peptide; the repertoire of cells responding to higher concentrations is more diverse, consistent with the expansion of more, but less efficient, clones. Thus, peptide affinity and concentration should be considered together for generating efficient antipeptide CTLs in vitro.

H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro.

Most CTL responses to epitopes from influenza virus are restricted by MHC class Ia molecules. However, a synthetic peptide corresponding to residues 173 to 190 of influenza A/JAP/305/57 hemagglutinin (HA) can induce, in vitro, a CTL response to peptide presented by a mouse class Ib molecule encoded by a gene telomeric to H2-Q. Here, we identify the molecule as H2-M3 and show that the last five residues of HA173-190, MLIIW, is the minimal epitope for CTL recognition. Cells that express M3wt, from C57BL/6 or BALB/c mice, are sensitized by both MLIIW and the longer peptide HA173-190, whereas cells that express M3f, from A.CA or B10.M mice, are sensitized only by MLIIW; a single amino acid change at residue 31 (V-->M) of M3 accounts for this difference. Although M3-restricted CTLs preferably recognize N-formylated epitopes, i.e., those of mitochondrial or prokaryotic origin, our findings show that M3-restricted primary CTL responses can be generated in vitro against nonformylated epitopes.

H2-M3, a full-service class Ib histocompatibility antigen.

H2-M3 is an MHC class Ib molecule of the mouse with a unique preference for N-formylated peptides, which may come from the N-termini of endogenous, mitochondrial proteins or foreign, bacterial proteins. The crystal structure of M3 revealed a hydrophobic peptide-binding groove with an occluded A pocket and the peptide shifted one residue relative to class Ia structures. The formyl group is held by a novel hydrogen bonding network, involving His9 on the bottom of the groove, and the side chain of the P1 methionine is lodged in the B pocket. M3 is a full-service histocompatibility (H) antigen, i.e. self-M3 can present endogenous peptides as minor H antigens and foreign, bacterial antigens in a defensive immune response to infection; and foreign M3 complexed with endogenous self-peptides.

The evolution of red blood cell and lymphocyte Ro/SSA.

Recent studies have demonstrated that the Ro/SSA autoantigen is heterogeneous as is the corresponding autoimmune response. In addition the autoimmune responses is highly species specific and preferentially reactive with the human antigen. Quantitative ELISA study shows that red blood cell Ro/SSA evolves much more rapidly than lymphocyte Ro/SSA and Western Blot analysis shows that the quantitative ELISA results are mirrored by changes in the 60 kD Ro/SSA molecules but not the 52 kD and 54 kD Ro/SSA molecules. The 52 kD and 54 kD Ro/SSA molecules seem to be relatively conserved as indicated by the Western immunoblotting experiments. These studies add weight to the concept that the antigenic epitopes of these related proteins are under the control of separate genes which have undergone different rates of evolution.