CD4+ T cells that evade deletion by a self peptide display Th1-biased differentiation.

We have examined factors governing the differentiation of autoreactive CD4+ T cells that have evaded deletion by a self peptide. Two lineages of transgenic mice (HA12 and HA104) expressing the influenza virus hemagglutinin (HA) were mated with TS1 mice that express a clonotypic T cell receptor (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 TS1xHA transgenic lineages. However, through allelic inclusion, thymocytes expressing low levels of the clonotypic TCR and high levels of endogenous TCR alpha-chains evaded deletion in TS1xHA12 and TS1xHA104 mice to graded degrees. When stimulated with S1 peptide in vitro, the non-autoreactive TS1 T cells were biased toward differentiation into Th2 effectors. By contrast, CD4+ T cells that evaded deletion in TS1xHA12 and TS1xHA104 mice were progressively biased toward Th1-like differentiation. Moreover, the effector cells from TS1xHA12 and TS1xHA104 mice secreted higher levels of IFN-gamma , on a per cell basis, than were secreted by their non-autoreactive counterparts. Thus, CD4+ T cells that evade deletion by a self peptide can exhibit an intrinsic bias toward differentiation into Th1 effector cells.

Virus-induced maturation and activation of autoreactive memory B cells.

We have examined B cell populations that participate in distinct phases of the immune response to the influenza virus A/PR/8/34 hemagglutinin (HA) for their susceptibility to negative selection in mice that express the HA as a neo-self-antigen (HA104 mice). We demonstrated previously that specificity for the neo-self-HA causes a population of immunoglobulin G antibody-secreting cells, which dominate the primary response to virus immunization in BALB/c mice, to be negatively selected in HA104 mice. We find here that in contrast to these primary response B cells, HA-specific memory response B cells developed equivalently in HA104 and nontransgenic (BALB/c) mice. Indeed, there was no indication that HA-specific B cells were negatively selected during memory formation in influenza virus-immunized HA104 mice, even though the neo-self-HA can be recognized by memory B cells. Furthermore, HA-specific autoantibodies were induced in the absence of virus immunization by mating HA104 mice with mice transgenic for a CD4(+) HA-specific T cell receptor. These findings indicate that specificity for a self-antigen does not prevent the maturation of autoreactive B cells in the germinal center pathway. Rather, the availability of CD4(+) T cell help may play a crucial role in regulating autoantibody responses to the HA in HA104 mice.

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").

Anergy and suppression regulate CD4(+) T cell responses to a self peptide.

To examine the role of cognate peptide in establishing CD4(+) T cell tolerance, we have mated transgenic mice that express the major I-E(d)-restricted determinant (S1) from the influenza virus PR8 hemagglutinin (HA28 mice) with mice expressing a S1-specific T cell receptor (TS1 mice). Surprisingly, S1-specific CD4(+) T cells were not substantially deleted in TS1xHA28 mice; indeed, lymph node cells expressing the S1-specific TCR were as abundant in TS1xHA28 mice as in TS1 mice. The S1-specific T cells in TS1xHA28 mice were, however, impaired in their ability to respond to S1 peptide both in vitro and in vivo, and contained two distinct populations. Approximately half expressed a unique cell surface phenotype (CD25(hi)/CD45RB(int)) and had been anergized by the neo-self S1 peptide. The remainder responded normally to the S1 peptide if purified away from the anergic T cells, but their proliferation was suppressed when the anergic T cells were also present in unfractionated lymphnode cells or in mixed cultures. These findings establish that anergy and suppression are coordinated mechanisms by which autoreactive CD4(+) T cells are regulated and that anergic/suppressor CD4(+) T cells can develop in response to self peptides.

Genetic basis for T cell recognition of a major histocompatibility complex class II-restricted neo-self peptide.

We have analyzed the genetic basis for T cell recognition of an endogenous major histocompatibility complex class II-restricted self peptide. Transgenic mice expressing the influenza virus PR8 hemagglutinin I-Ed-restricted determinant S1 (HA Tg mice) mediate negative selection of PR8 S1-specific T cells, but respond to immunization with a virus containing a closely related analogue, S1(K113). Sequence analysis of S1(K113)-specific T cell receptors (TCR) from nontransgenic mice revealed a dominant TCR clonotype that cross-reacts with PR8 S1. This clonotype is eliminated by negative selection in HA Tg mice; nonetheless, modified versions of this TCR that used altered junctional sequences and a novel V alpha/V beta pairing to evade negative selection by the S1 self peptide were identified. The remaining S1(K113)-specific TCRs from HA Tg mice were highly diverse; 13 of 15 S1(K113)-specific TCRs from HA Tg mice used unique V alpha/V beta pairings. Thus, tolerance to PR8 S1 as a self peptide does not limit the diversity of the T cell response to S1(K113).

Altered protein tyrosine phosphorylation in anergic Th1 cells.

Clonal anergy as a mechanism for tolerance in T lymphocytes can be studied using an in vitro culture system, in which cloned CD4+ Th1-type murine T cells are rendered anergic for IL-2 transcription. The long-lasting molecular changes in anergic cells that prevent the response to Ag restimulation are not yet known. To determine whether the TCR might be uncoupled from normal intracellular signaling pathways, we investigated the response of anergic T cells to Ag, to anti-CD3 antibodies, or to anti-CD4 antibody restimulation in terms of early protein tyrosine phosphorylation events. Tyrosine phosphorylation of the CD3 zeta chain was apparently normal. In contrast, defects in the induction of tyrosine phosphorylation of three major T cell protein substrates were demonstrated. Altered phosphorylation correlated with functional nonresponsiveness for proliferation and reversal of anergy by growth in exogenous IL-2 resulted in reversal of the phosphorylation defects as well as in recovery of Ag responsiveness. These results suggest that specific defects in tyrosine phosphorylation pathways required for the induction of IL-2 synthesis may help to explain nonresponsiveness to Ag in tolerant T cells.

Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn.

Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells. Here we report that such anergic Th1 cells express constitutively reduced amounts of the protein tyrosine kinase p56lck and constitutively elevated levels of the protein tyrosine kinase p59fyn. Because protein tyrosine phosphorylation is known to be important for the normal induction of IL-2 synthesis, these results suggest that T cell anergy may be maintained, at least in part, by alterations in tyrosine phosphorylation signaling events.