CD4+Thy1- thymocytes with a Th-type 2 cytokine response.

We have identified a small subset of CD3(+), CD4(+), CD8(-) thymocytes that do not express Thy1 (CD90). This Thy1(-) subset represents 1-3.7% of the total number of thymocytes in a naive mouse. CD4(+)Thy1(-) thymocytes express high levels of CD3, intermediate to high levels of heat-stable antigen (HSA), and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They produce high titers of IL-4 and no IFN-gamma upon stimulation in vitro, a response characteristic of T(h)2 cells. In the thymi of mice infected neonatally with a high dose of the retrovirus Cas-Br-E MuLV, the frequency of CD4(+)Thy1(-) cells increased approximately 10-fold. High-dose virus infection resulted in decreased HSA and increased CD44 expression on CD4(+)Thy1(-) cells relative to cells from naive mice. CD4(+)Thy1(-) cells from high-dose infected mice also secreted IL-4 and not IFN-gamma upon in vitro stimulation. We previously reported that infection of newborn mice with a high dose of murine retrovirus results in the induction of a non-protective anti-viral T(h)2 T cell response; CD4(+)Thy1(-) thymocytes with a T(h)2-like cytokine profile may play a role in determining the cytokine bias of this anti-viral response.

Graded deletion and virus-induced activation of autoreactive CD4+ T cells.

We have examined factors governing the negative selection of autoreactive CD4(+) T cells in transgenic mice expressing low (HA12 mice) vs. high (HA104 mice) amounts of the influenza virus hemagglutinin (HA). When mated with TS1 mice that express a transgenic TCR specific for the I-Ed-restricted determinant site 1 (S1) of HA, thymocytes expressing high levels of the clonotypic TCR were deleted in both HA-transgenic lineages. However, through allelic inclusion, thymocytes with lower levels of the clonotypic TCR evaded deletion in TS1 x HA12 and TS1 x HA104 mice to graded degrees. Moreover, in both lineages, peripheral CD4(+) T cells could be activated by the S1 peptide in vitro, and by influenza virus in vivo. These findings indicate that allelic inclusion can allow autoreactive CD4(+) thymocytes to evade thymic deletion to varying extents reflecting variation in the expression of the self peptide, and can provide a basis for the activation of autoreactive peripheral T cells by viruses bearing homologues of self peptides ("molecular mimicry").

Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice.

The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.

In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection.

To examine the role of germinal centers (GCs) in the generation and selection of high affinity antibody-forming cells (AFCs), we have analyzed the average affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific AFCs and serum antibodies both during and after the GC phase of the immune response. In addition, the genetics of NP-binding AFCs were followed to monitor the generation and selection of high affinity AFCs at the clonal level. NP-binding AFCs gradually accumulate in bone marrow (BM) after immunization and BM becomes the predominant locale of specific AFCs in the late primary response. Although the average affinity of NP-specific BM AFCs rapidly increased while GCs were present (GC phase), the affinity of both BM AFCs and serum antibodies continued to increase even after GCs waned (post-GC phase). Affinity maturation in the post-GC phase was also reflected in a shift in the distribution of somatic mutations as well as in the CDR3 sequences of BM AFC antibody heavy chain genes. Disruption of GCs by injection of antibody specific for CD154 (CD40 ligand) decreased the average affinity of subsequent BM AFCs, suggesting that GCs generate the precursors of high affinity BM AFCs; inhibition of CD154-dependent cellular interactions after the GC reaction was complete had no effect on high affinity BM AFCs. Interestingly, limited affinity maturation in the BM AFC compartment still occurs during the late primary response even after treatment with anti-CD154 antibody. Thus, GCs are necessary for the generation of high affinity AFC precursors but are not the only sites for the affinity-driven clonal selection responsible for the maturation of humoral immune responses.

A major T cell determinant from the influenza virus hemagglutinin (HA) can be a cryptic self peptide in HA transgenic mice.

Transgenic (Tg) mice expressing the influenza virus PR8 hemagglutinin (PR8 HA) were infected with PR8 virus and analyzed for their ability to generate T cell responses to individual MHC class II-restricted T cell determinants from the HA. HAmemb and HAtrunc mice each express HA transgene mRNA in many tissues (including the thymus), but differ in the form and amount of the HA that is expressed: HAmemb mice express the entire viral HA as a membrane-bound neo-self antigen, whereas HAtrunc mice express lower levels of a truncated HA polypeptide. HAmemb mice were found to mediate efficient negative selection of autoreactive T cells directed to the major I-Ed-restricted and I-Ad-restricted determinants from the HA (designated S1 and S2 respectively). S1-specific T cell responses were similarly undetectable in PR8-infected HAtrunc mice. However, S2-specific T cells were only partially eliminated in HAtrunc mice; indeed, even though their frequency was reduced relative to non-Tg mice, S2-specific T cells still constituted a sizable population in PR8-infected HAtrunc mice. Moreover, the S2-specific T cells from HAtrunc and non-Tg mice appeared to be equally sensitive to stimulation with S2 peptide, and in each case utilized highly diverse T cell receptors to recognize S2-I-Ad. The findings demonstrate that an individual class II-restricted T cell determinant can be recognized as a cryptic self peptide during T cell repertoire formation and as an immunodominant peptide in the context of an anti-viral T cell response, providing a basis for the induction of autoreactive T cells by viruses containing homologs of self antigens.

Transgenic mice that express different forms of the influenza virus hemagglutinin as a neo-self-antigen.

We have generated transgenic mouse lineages that express the influenza virus hemagglutinin in different physical forms. One kind expresses the full-length hemagglutinin molecule as a cell surface glycoprotein and can be recognized by hemagglutinin-specific B and T cells. The other expresses a truncated polypeptide corresponding to the N-terminal third of the hemagglutinin molecule. This polypeptide encodes known hemagglutinin-specific T-cell determinants; however, it contains no native B-cell epitopes, since these depend on the conformation of the fully folded protein. In each case, the hemagglutinin transgenic mice display ubiquitous expression of transgenic messenger RNA and induce T-cell tolerance to the transgene-encoded T-cell determinant site 1. Thus, the hemagglutinin is a neo-self-antigen in both kinds of hemagglutinin transgenic mice and should provide a useful system for understanding the factors and mechanisms that govern tolerance and autoimmunity to self-antigens.

Low avidity recognition of a class II-restricted neo-self peptide by virus-specific T cells.

The specificity with which CD4+ T cells recognize self peptides in vivo was examined in transgenic mice that express an influenza virus PR8 hemagglutinin (HA) polypeptide in many tissues, including the thymus (HA Tg mice). HA Tg and non-Tg mice were analyzed for their T cell responses to the major PR8 HA I-E(d)-restricted CD4+ T cell determinant S1. Negative selection eliminated S1-specific T cells from HA Tg mice. Nevertheless, HA Tg mice retained the ability to mount a T cell response to a closely related analog of the S1 determinant [S1(K113)], and some S1(K113)-specific TCRs displayed a partial reactivity with S1 as indicated by their ability to transmit signals for IL-3 but not IL-2 secretion in response to the neo-self peptide. Moreover, the neo-self S1 peptide antagonized the ability of these TCRs to signal IL-2 secretion in response to the foreign S1(K113) determinant. Thus, TCRs that exhibit a partial reactivity with a self peptide are present in the peripheral T cell repertoire and can be activated by a virus containing an analog of the self peptide. These findings provide a model for the induction of autoimmunity by viruses that are close homologs of self peptides, and suggest a way in which TCRs could react with self peptides during positive selection of developing thymocytes.

T cells from unprimed mice respond to the self antigen heme, in a class II restricted manner, at a frequency similar to alloresponses.

Heme is a non-protein autoantigen which stimulates potent proliferative responses by T cells from unprimed mice of some strains. These studies show that T cells responding to heme in primary responses are predominantly CD4+, classically I-A restricted, and use diverse TCR characterized by the expression of distinct V, D and J gene segments. These characteristics distinguish heme from superantigens and mitogens which exhibit degenerate MHC restriction and, in the case of superantigens, restricted V gene usage. Using limiting dilution analysis these studies also show that the potent primary response of H-2s mice reflects a high frequency (0.26-0.45%) of heme responsive T cells in the periphery, comparable to the frequency of alloresponsive T cells reported by others in primary mixed lymphocyte reactions. In contrast, heme responsive T cells occur at -10-fold lower frequency in unprimed H-2d mice (0.03%). To determine the antigen recognized by heme reactive T cells, the mass spectra of peptides eluted from the high responder haplotype, I-A(s), were examined. These indicated a markedly different molecular weight distribution of peptides isolated from cells grown in the presence of heme, compared with those from cells grown in its absence. This suggests that heme mediates the expansion of diverse T cells in the peripheral repertoire by a mechanism similar to that for allogeneic responses in which the profile of naturally processed peptides bound to the MHC class II molecule is changed.