How the immune system protects the host from infection.

The immune system is made up of sets of interacting cells. The first to respond in all cases are the antigen-presenting cells (APCs), which are equipped with receptors for microbial patterns. Engagement of these receptors induces co-stimulatory molecules on the surface of the APCs, and allows it to stimulate potent CD4 T-cell responses, and also CD8 T-cell responses. This in turn leads to B-cell-derived antibody responses. The entire response is controlled by suppressor T cells, as predicted many years ago by Richard Gershon.

The source of early IFN-gamma that plays a role in Th1 priming.

When naive CD4 T cells are primed, they rapidly differentiate into polarized Th1 and/or Th2 phenotypes. A major factor in producing such polarization is the early production of cytokines (IL-12 and IFN-gamma in the case of Th1 cells and IL-4 in the case of Th2 cells). One issue that remains unresolved is the source of the early IFN-gamma that synergizes with IL-12 to fully polarize CD4 T cells into Th1 cells. We have examined this question by injecting mice with anti-CD3 and examining cells from normal and various MHC-knockout mice. We found that IFN-gamma is induced rapidly in a small subset of CD8 T cells. This subset is absent in mice that lack beta2-microglobulin, but not in K(b)D(b)-double-knockout mice, indicating that these CD8 T cells are dependent on nonclassical MHC class Ib molecules. The early burst of IFN-gamma polarizes CD4 T cells toward Th1 cells, in part by stimulating the release of IL-12 from APC. We also use TAP- and CD1-knockout mice to show that such cells are not CD1-restricted NK T cells, nor are they dependent on TAP-1 transport for surface expression of the relevant MHC class Ib molecule. Therefore, they arise on MHC class Ib molecules that do not depend on TAP-1 transporters.

Paradoxical intrathymic positive selection in mice with only a covalently presented agonist peptide.

The Y-Ae mAb and the 1H3.1 alphabeta T cell antigen receptor (TCR) are both specific for the I-Ealpha52-68 peptide bound to the I-A(b) major histocompatibility complex (MHC) class II molecule. Antigen-presenting cells (APCs) from I-A(b+) mice with a natural or transgenic (Tg) I-Ealpha chain activate mature 1H3.1 T cells and cause the deletion of 1H3.1 TCR Tg thymocytes. However, 1H3.1 T cells were neither activated nor inactivated by confrontation with APCs from I-Ab-Ep mice in which I-A(b) molecules are occupied only by the covalently associated Ealpha52-68 peptide. Instead, immature 1H3.1 TCR Tg thymocytes were efficiently positively selected into the CD4 lineage in the I-Ab-Ep thymus. This selection relied on specific recognition of the Ealpha52-68/I-A(b) complex because it was blocked by Y-Ae. 1H3.1 TCR Tg T cells maturing in the I-Ab-Ep thymus efficiently populated the periphery, displayed a naive phenotype, and were specifically reactive to the Ealpha52-68 peptide or to I-A(b+)I-Ealpha(+) APCs, indicating that 1H3.1 T cells were not antagonized in I-Ab-Ep mice. The data identify major histocompatibility complex class II molecules with only a covalently attached self-peptide as a ligand for in vivo positive selection of T cells specific for the same peptide.

Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers.

T cell receptor (TCR) allelic exclusion is believed to be primarily mediated by suppression of further recombination at the TCR locus after the expression of a functional TCR protein. Genetic allelic exclusion has been shown to be leaky for the beta chain and, more commonly, for the alpha chain. Here, we demonstrate an additional mechanism by which T cells can maintain monoclonality. T cells from double TCR transgenic mice express only one or the other of the two available TCRs at the cell surface. This "functional allelic exclusion" is apparently due to control of the TCR assembly process because these T cells express RNA and protein for all four transgenic TCR proteins. Lack of cell surface expression of the second TCR may be controlled by a failure to assemble the TCR heterodimer.

CD62L is required on effector cells for local interactions in the CNS to cause myelin damage in experimental allergic encephalomyelitis.

Adhesion molecules are believed to facilitate infiltration of leukocytes into the CNS of mice with experimental allergic encephalomyelitis (EAE). The role of the adhesion molecule CD62L (L-selectin) in the immunopathology of EAE is not known. To study this, we crossed CD62L-deficient mice with myelin basic protein-specific TCR (MBP-TCR) transgenic mice. CD62L-deficient MBP-TCR transgenic mice failed to develop antigen-induced EAE, and, despite the presence of leukocyte infiltration, damage to myelin in the CNS was not seen. EAE could, however, be induced in CD62L-deficient mice upon adoptive transfer of wild-type macrophages. Our results suggest that CD62L is not required for activation of autoimmune CD4 T cells but is important for the final destructive function of effector cells in the CNS and support a novel mechanism whereby CD62L expressed on effector cells is important in mediating myelin damage.

A role for accessibility to self-peptide-self-MHC complexes in intrathymic negative selection.

Whether intrathymic-positive and -negative selection of conventional alpha beta T cells occur in anatomically distinct sites is a matter of debate. By using a system composed of two distinct immune receptors, the Y-Ae mAb and the 1H3.1 (V alpha 1/V beta 6) TCR, both directed against the 52--68 fragment of the I-E alpha-chain (E alpha 52--68) bound to I-A(b), we examined the occurrence of negative selection imposed in vivo by a self-peptide-self-MHC class II complex with differential tissue expression. 1H3.1 TCR-transgenic (Tg) mice were bred to mice having an I-E alpha transgene with expression directed to all MHC class II-positive cells, restricted to thymic epithelial cells, or restricted to B cells, dendritic cells, and medullary thymic epithelial cells. All 1H3.1 TCR/I-E alpha double-Tg mice revealed a severely diminished thymic cellularity. Their lymph node cells were depleted of V beta 6(+)CD4(+) cells and were unresponsive to E alpha 52--68 in vitro. The absolute number of CD4(+)CD8(+) thymocytes was drastically reduced in all combinations, indicating that negative selection caused by an endogenously expressed self-determinant can effectively occur in the thymic cortex in vivo. Moreover, both cortical epithelial cells and, interestingly, the few cortical dendritic cells were able to support negative selection of CD4(+)CD8(+) thymocytes, albeit with a distinct efficiency. Collectively, these observations support a model where, in addition to the avidity of the thymocyte/stromal cell interaction, in vivo negative selection of autoreactive TCR-Tg T cells is determined by accessibility to self-peptide-self-MHC complexes regardless of the anatomical site.

The T cell activation molecule H4 and the CD28-like molecule ICOS are identical.

The recently cloned CD28-like molecule ICOS displays striking similarities with H4, characterized some years ago in the mouse and recently in humans. Both molecules are selectively expressed by activated and germinal center T cells, display similar structure, and display co-stimulatory activities. H4 displays lateral association with the CD3/TCR and is expressed by mature thymocytes. In the mouse, H4 is also expressed at high levels by thymic NKT cells that are resistant to negative selection. The aim of this work was to evaluate whether H4 and ICOS are the same molecule using the C398.4A (binding human and mouse H4) and F44 (binding human ICOS) monoclonal antibody (mAb) in parallel experiments on human T cells. ICOS and H4 displayed the same expression pattern in a panel of T cell lines and the same expression kinetics in phytohemagglutinin-activated T cells. C398.4A completely blocked cell staining by F44, whereas F44 partially blocked C398.4A. H4 and ICOS immunoprecipitates displayed identical SDS-PAGE patterns and H4 immunoprecipitation completely removed ICOS from cell lysates. Finally, the C398.4A mAb specifically stained cells transfected with the human or mouse ICOS. These data prove that H4 and ICOS are the same molecule and that F44 and C398.4A bind partially different epitopes.

Qa-2-dependent selection of CD8alpha/alpha T cell receptor alpha/beta(+) cells in murine intestinal intraepithelial lymphocytes.

Murine intestinal intraepithelial lymphocytes (iIELs) are made up of a heterogeneous mix of T cells with unique phenotypes. Whereas CD8(+) T cells in peripheral lymphoid organs use CD8alpha/beta and are selected on MHC class Ia molecules, a majority of iIELs use CD8alpha/alpha. Here, we report that the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is independent of classical MHC class I molecules K(b) and D(b), as illustrated by their presence in K(b)/D(b) double-knockout mice and in mice lacking a nonclassical MHC class I molecule, CD1d. Most strikingly, their presence is decreased by approximately 70% in mice lacking transporter associated with antigen processing (TAP). The TAP-dependent nonclassical MHC class I molecule Qa-2 is strongly implicated in the presence of these cells, as inferred from the low numbers of CD8alpha/alpha TCR-alpha/beta T cells in mice deficient in Qa-2 genes. Second, a Qa-2-transgenic mouse made in a Qa-2(-) strain showed an increase in the numbers of CD8alpha/alpha cells among its iIELs. Thus, the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is mainly dependent on the nonclassical MHC class I molecule Qa-2.

On the self-referential nature of naive MHC class II-restricted T cells.

The use of mutant mice expressing a normal MHC class II molecule surface level but a severely restricted self-peptide diversity (H-2Malpha(-/-)) previously revealed that T cells carrying the Ealpha(52-68)-I-A(b) complex-specific 1H3.1 TCR rely on self-peptide(s) recognition for both their peripheral persistence in irradiated hosts and their intrathymic positive selection. Here, we identify Ealpha(52-68) structurally related self-peptide(s) as a major contributor to in vivo positive selection of 1H3.1 TCR-transgenic thymocytes in I-A(b+)/I-Ealpha(-) mice. This is demonstrated by the drastic and specific reduction of the TCR high thymocyte population in 1H3.1 TCR-transgenic (Tg) mice treated with the Ealpha(52-68)-I-A(b) complex-specific Y-Ae mAb. Self-peptide(s) recognition is also driving the maturation of T cells carrying a distinct MHC class II-restricted specificity (the Ealpha(6) alphass TCR), since positive selection was also deficient in Ealpha(6) TCR Tg H-2Malpha(-/-) thymi. Such a requirement for recognition of self-determinants was mirrored in the periphery; Ealpha(6) TCR Tg naive T cells showed an impaired persistence in both H-2Malpha(-/-) and I-A(b)ss(-/-) irradiated hosts, whereas they persisted and slowly cycled in wild-type recipients. This moderate self-peptide(s)-dependent proliferation was associated with a surface phenotype intermediate between those of naive and activated/memory T cells; CD44 expression was up-regulated, but surface expression of other markers such as CD62L remained unaltered. Collectively, these observations indicate that maturation and maintenance of naive MHC class II-restricted T cells are self-oriented processes.

Differential sensitivity to mutations in a single peptide by two TCRs having identical beta-chains and closely related alpha-chains.

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.

A NK1.1+ thymocyte-derived TCR beta-chain transgene promotes positive selection of thymic NK1.1+ alpha beta T cells.

As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCR beta-chain derived from conventional alphabeta T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companion alpha-chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing a beta-chain derived from nonclassical, NK1.1+ alphabeta T cells, the thymus-derived, CD1. 1-specific DN32H6 T cell hybridoma. Results of the sequence analysis of genomic DNA from developing DN32H6 beta Tg thymocytes revealed that the frequency of the parental alpha-chain sequence, in this instance the Valpha14-Jalpha281 canonical alpha-chain, is specifically and in a CD1.1-dependent manner, increased in the postselection thymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, but interestingly not peripheral, NK1.1+ alphabeta T cells in DN32H6 beta Tg mice, possibly indicating a thymic determinant-dependent maintenance. Thus, in vivo expression of the rearranged TCR beta-chain from a thymus-derived NK1.1+ Valpha14+ T cell hybridoma promotes positive selection of thymic NK1.1+ alphabeta T cells. These observations indicate that the strong influence of productive beta-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selection on self-determinants, applies to both classical and nonclassical alphabeta T cells and therefore represents a general phenomenon in intrathymic alphabeta T lymphocyte development.

Relapsing and remitting experimental autoimmune encephalomyelitis in B cell deficient mice.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for the human autoimmune central nervous system (CNS) disease multiple sclerosis (MS). To examine the role of B cells in EAE with a relapsing and remitting disease course (R-EAE) we generated (B10.PL x SJL/J)F1 mice deficient in B cells by disrupting their mu heavy chain transmembrane region (B10.PL x SJL/J)F1 muMT-/-. By immunizing (B10.PL x SJL/J)F1 and (B10.PL x SJL/J)F1 muMT-/- mice with the encephalitogenic N-terminal peptide Acl-11 of myelin basic protein (MBP), we observed that B-cell deficient mice exhibited a relapsing and remitting disease course. Since a similar day of onset and day of first relapse were observed these data suggest that B cells do not play a vital role in the activation of T cells leading to the initiation of EAE, nor in the reactivation of T cells resulting in R-EAE.

Alteration at a single amino acid residue in the T cell receptor alpha chain complementarity determining region 2 changes the differentiation of naive CD4 T cells in response to antigen from T helper cell type 1 (Th1) to Th2.

To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR alpha chain of the D10.G4.1 (D10) TCR and bred them to D10 beta chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by approximately 100-fold. A mutation in the reference peptide CA134-146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex-bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.

Variability of invariant mouse CD3epsilon chains detected by anti-CD3 antibodies.

Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the alpha beta (or gamma delta) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4(+) T lymphocytes and T cell lines are bound with different avidity by anti-CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3epsilon delta to CD3epsilon gamma chain pairing. Rather, they are linked to heterogeneity of the N-terminal region of CD3epsilon chains, as detected by peptide-specific antibodies. In turn, these differences among CD3epsilon chains correlate with variations in the strength of TCR / CD3 interaction. N-terminal CD3epsilon heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription-PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3epsilon N-terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.

Differential adhesion molecule requirements for immune surveillance and inflammatory recruitment.

Activated CD4 Th1 lymphocytes can enter the brain in the absence of an inflammatory focus. However, the molecular mediators that regulate this early migration of lymphocytes into the brain have remained unclear. We hypothesized that the entry of these 'pioneer' lymphocytes into the brain is regulated by a set of molecular events that are distinct from those used once inflammation has been established. Using cells fluorescently labelled with the lipophilic dye DiI, myelin basic protein (MBP)-specific CD4 lymphocytes that expressed low or high levels of very late antigen-4 (VLA-4) and non-antigen-specific activated splenocytes homed to mouse brain in similar quantities 2 h after adoptive transfer. However, antigen specificity and VLA-4 expression were required for more robust recruitment by 24 h. Immunocytochemistry revealed endothelial and microenvironmental upregulation of vascular cell adhesion molecule (VCAM), intercellular cell adhesion molecule 1 (ICAM-1), MHC class II and interferon-gamma 48 h after transfer of MBP-specific cells. In contrast, expression of meningeal and choroid plexus-associated P selectin was upregulated 2 h after adoptive transfer, but not at 48 h. Monoclonal antibody to P selectin, but not to VLA-4, inhibited early migration of high VLA-4-expressing MBP-specific lymphocytes. These results suggest that early migration occurs independent of the lymphocyte integrin VLA-4 and endothelial VCAM, but does require increased surface expression of endothelial P selectin.

Functional and phenotypic evidence for presentation of E alpha 52-68 structurally related self-peptide(s) in I-E alpha-deficient mice.

The Y-Ae mAb and the 1H3.1 TCR-alpha beta (V alpha 1/V beta 6) are two immune receptors specific for I-Ab MHC class II molecules complexed to the 52-68 fragment of the alpha-chain of I-E class II molecules (the E alpha 52-68 peptide). A profound intrathymic negative selection occurs in 1H3.1 TCR transgenic mice in the presence of an I-E alpha transgene. The administration of mAbs to 1H3.1/I-E alpha double-transgenic newborn mice reveals that Y-Ae, but not the isotype-matched anti-I-E Y17 mAb, rescues a significant number of mature (V beta 6highCD4+CD8-) thymocytes and allows the detection of E alpha 52-68-reactive T cells in the periphery. These observations indicate that deletion of autoreactive T cells can be specifically inhibited in vivo by an mAb specific for the deleting self-peptide:self-MHC class II complex. Similar inhibition experiments indicate that C57BL/6 (I-Ab+/I-E alpha-) mice constitutively express an E alpha-independent, Y-Ae-recognizable epitope(s). This finding is confirmed by the phenotypic analysis of mature (MHC class II high) C57BL/6 bone marrow-derived dendritic cells. Collectively, these observations further illustrate the peptide specificity of negative selection and demonstrate that MHC class II-positive cells from unmanipulated C57BL/6 mice that lack a functional I-E alpha gene can assemble one or more self-peptide:I-Ab complexes recognizable by the E alpha 52-68:I-Ab complex-specific Y-Ae mAb.

Expression of the novel T cell activation molecule hpH4 in HIV-infected patients: correlation with disease status.

We have described hpH4, a surface glycoprotein selectively expressed by activated T cells and mature thymocytes and displaying weak lateral association with CD4. The hpH4 expression pattern and biochemical features, together with analysis of its tryptic digest by peptide mass searching using MALDI-MS, suggested that it is a novel molecule. The aim of this work was to evaluate the peripheral blood T cell expression of hpH4 in HIV-infected patients and the interplay between HIV gp120 and hpH4, since both molecules interact with CD4. hpH4 expression during HIV-1 infection was evaluated by assessing 55 patients at various disease stages and following up 3 patients with primary infection and 3 patients with AIDS. hpH4 expression displayed a peak in the early phase of primary infection, dropped to control levels in the asymptomatic phase, and was newly expressed, at low levels, as AIDS developed. The expression kinetics were different than those shown by HLA-DR, CD25, and CD38. The most striking findings were the transient hpH4 expression peak displayed in the earliest stage, which was unique for hpH4. Incubation of T cells from normal donors with HIV gp120 induced transient hpH4 expression in resting CD4+ T cells and potentiated the hpH4 lateral association with CD4 in activated T cells. Moreover, hpH4 triggering inhibited gp120-induced death of CD4+ cells. Therefore, H4 expression may be a response to avoid apoptosis induced by HIV products.