T cell development in mice expressing CD1d directed by a classical MHC class II promoter

CD1d and nonclassical MHC molecules differ markedly from classical MHC ligands in their ability to promote the selection and differentiation of developing T cells. Whereas classical MHC-restricted T cells have a predominantly naive phenotype and a broad TCR repertoire, most other T cells have a memory and/or NKT phenotype with a restricted repertoire. Because the nonclassical ligands selecting these memory-type cells are expressed by bone marrow-derived cells, it has been suggested that the development of large repertoires of naive-type cells was dependent on the classical MHC expression pattern in the thymus cortex, high on epithelial cells and low on cortical thymocytes. We redirected CD1d expression using the classical MHC II Ealpha promoter. pEalpha-CD1d mice lacked memory-type NKT cells, but, surprisingly, they did not acquire the reciprocal ability to select a diverse population of naive CD1d-restricted cells. These findings suggest that, whereas the development of NKT cells is dependent on the pattern of CD1d expression, the absence of a broad, naive CD1d-restricted T cell repertoire may reflect intrinsic limitations of the pool of TCR genes or lipid Ags.     

Alloreactive and syngeneic CTL are comparably dependent on interaction with MHC class I alpha-helical residues.

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.     

Recognition of self and altered self by T cells in autoimmunity and allergy

T cell recognition of foreign peptide antigen and tolerance to self peptides is key to the proper function of the immune system. Usually, in the thymus T cells that recognize self MHC+ self peptides are deleted and those with the potential to recognize self MHC+ foreign peptides are selected to mature. However there are exceptions to these rules. Autoimmunity and allergy are two of the most common immune diseases that can be related to recognition of self. Many genes work together to lead to autoimmunity. Of those, particular MHC alleles are the most strongly associated, reflecting the key importance of MHC presentation of self peptides in autoimmunity. T cells specific for combinations of self MHC and self peptides may escape thymus deletion, and thus be able to drive autoimmunity, for several reasons: the relevant self peptide may be presented at low abundance in the thymus but at high level in particular peripheral tissues; the relevant self peptide may bind to MHC in an unusual register, not present in the thymus but apparent elsewhere; finally the relevant self peptide may be post translationally modified in a tissue specific fashion. In some types of allergy, the peptide+ MHC combination may also be fully derived from self. However the combination in question may be modified by the presence of other ligands, such as small drug molecules or metal ions. Thus these types of allergies may act like the post translationally modified peptides involved some types of autoimmunity.     

A Quantitative Theory of Affinity-driven T Cell Repertoire Selection

Binding of the T cell antigen receptor (TCR) to peptides presented on molecules encoded by major histocompatibility complex (MHC) genes is the key event driving T cell development and activation. Selection of the T cell repertoire in the thymus involves two steps. First, positive selection promotes the survival of cells binding thymic self-MHC-peptide complexes with sufficient affinity. The resulting repertoire is self-MHC restricted: it recognizes foreign peptides presented on self, but not foreign MHC. Second, negative selection deletes cells which may be potentially harmful because their receptors interact with self-MHC-peptide complexes with too high an affinity. The mature repertoire is also highly alloreactive: a large fraction of T cells respond to tissues harboring foreign MHC. We derive mathematical expressions giving the frequency of alloreactivity, the level of self-MHC restriction, and the fraction of the repertoire activated by a foreign peptide, as a function of the parameters driving the generation and selection of the repertoire: self-MHC and self-peptide diversity, the stringencies of positive and negative selection, and the number of peptide and MHC polymorphic residues that contribute to T cell receptor binding. Although the model is based on a simplified digit string representation of receptors, all the parameters but one relate directly to experimentally determined quantities. The only parameter without a biological counterpart has no effect on the model's behavior besides a trivial and easily preventable discretization effect. We further analyse the role of the MHC and peptide contribution to TCR binding, and find that their relative, rather than absolute value, is important in shaping the mature repertoire. This result makes it possible to adopt different physical interpretations for the digit string formalism. We also find that the alloreactivity level can be inferred directly from data on the stringency of selection, and that, in agreement with recent experiments, it is not affected by thymic selection.     

Early appearance of donor-type antigen-presenting cells in the thymuses of 1200 R radiation-induced bone marrow chimeras correlates with self-recognition of donor I region gene products

The thymus exerts a potent influence on the development of I region self-recognition and antigen recognition by T cells. The mechanism by which the thymus acts on nascent T cells is unknown. It is assumed, however, that a cell interaction between the developing T cell and an la antigen-bearing cell in the thymus is involved. There are several candidates for the critical thymic cell; thymic epithelial, nurse, and antigen-presenting cells (APC) or dendritic cells. Because thymic epithelial cells derive from the third pharyngeal pouch and thymic APC derive from bone marrow, radiation-induced bone marrow chimeras allow the artificial creation of a chimeric thymus gland in which thymic epithelial cells and APC can be genetically different. We made radiation-induced bone marrow chimeras (F1 leads to P) using supralethal radiation doses (1200 R) and found bone marrow donor- (F1) type APC in the thymuses 3 wk after radiation. When such mice fully reconstitute their immune systems, their T cells behave as donor F1 phenotype T cells. Thus, the I region self-restriction and antigen-recognition repertoire of the T cells correlates with the genotype of the bone marrow-derived thymic APC, not the thymic epithelial cell.     

The role of polymorphic amino acids of the MHC molecule in the selection of the T cell repertoire

Allelic variants of MHC molecules expressed on cells of the thymus affect the selection and the specificity of the T cell repertoire. The selection is based on either the direct recognition by the TCR of the MHC molecules, or the recognition of a complex determinant formed by self-peptides bound to MHC molecules. In an analysis of the T cell repertoire in bone marrow chimeras that express allelic forms of MHC class II molecules in the thymus epithelium, we find that amino acid substitutions that are predicted to affect peptide binding influence the selection of the T cell repertoire during thymic selection.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

The immunocompetence of murine stromal cell-associated thymocytes

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.     

Expression of antigen on mature lymphocytes is required to induce T cell tolerance by gene therapy

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2K(b) led to long-term expression of K(b) on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2K(b)-transduced wild-type bone marrow were tolerant to K(b). In contrast, expression of K(b) in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to K(b), demonstrating that expression of K(b) on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.     

Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

Quantitative analysis of peptides from myelin basic protein binding to the MHC class II protein,I-Au,which confers susceptibility to experimental allergic encephalomyelitis.

BACKGROUND: An important issue in autoimmune diseases mediated by T cells, such as experimental allergic encephalomyelitis (EAE), is the affinity of the disease-inducing determinants for MHC class II proteins. Tolerance, either due to clonal deletion or anergy induction, is thought to require high-affinity interactions between peptides and MHC molecules. Low-affinity binding is compatible with the hypothesis that breaking tolerance to self proteins does not have to occur for onset of disease. In contrast, a high-affinity interaction implies that an event leading to a breakdown of tolerance is central to the autoimmune process. MATERIALS AND METHODS: Detergent-solubilized and affinity-purified I-Au was incubated with varying concentrations of a set of peptides from myelin basic protein and a biotinylated peptide agonist. The specific complexes were separated from excess peptide by capture on antibody-coated plates, and the affinity of the peptides was measured by adding europium-labeled streptavidin and measuring the resultant fluorescence. RESULTS: The immunodominant and encephalitogenic determinant, Ac 1-11, was shown to bind to I-Au relatively poorly (IC50 = 100 microM), demonstrating that in this protein, immunodominance did not correlate with high-affinity binding. In contrast with the natural sequence, the ability of shorter analogs to induce EAE did correlate with their apparent affinity. CONCLUSIONS: The dominance of the natural determinant does not arise from a high-affinity interaction with the MHC class II molecule. This suggests that other mechanisms are operative and that the specific T cell for this peptide/MHC ligand is of high affinity.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Normal thymic cortical epithelial cells developmentally regulate the expression of a B-lineage transformation-associated antigen

Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A^– epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag¹ cells and within the thymus, this antigen is found exclusively on A^– epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.     

Development of diabetogenic T cells from NOD/Lt marrow is blocked when an allo-H-2 haplotype is expressed on cells of hemopoietic origin, but not on thymic epithelium

Diabetes is a T cell-mediated process in NOD/Lt mice, with a major genetically recessive component of susceptibility linked to homozygous expression of the unique H-2g7 MHC haplotype. Heterozygous expression of the H-2nb1 haplotype derived from the NON/Lt strain confers diabetes resistance both in (NOD x NON)F1 hybrids and in NOD mice congenic for the H-2nb1 haplotype. However, diabetes resistance is abrogated in F1 hybrids by NOD/Lt bone marrow reconstitution. To establish whether the generation of beta cell autoreactive T cells from NOD/Lt bone marrow-derived precursors required at least heterozygous expression of the H-2g7 haplotype on thymic epithelium, adolescent thymectomized (NOD x NON)F1 mice were implanted with neonatal NON/Lt thymus grafts before lethal radiation and reconstitution with NOD/Lt bone marrow. Peripheral T cells maturing through this ectopic thymic implant exclusively expressed the NOD H-2g7 haplotype and were tolerant to H-2nb1 skin grafts. Nevertheless, diabetes developed in 32% of the NON/Lt thymus-grafted chimeras vs 38% of the sham-thymectomized NOD bone marrow chimeras. Thus, homozygous expression of the diabetes-resistant H-2nb1 haplotype on thymic epithelium failed to block development of a diabetogenic T cell repertoire. To examine if expression of H-2nb1 on hemopoietically derived APC could alter the diabetogenic potential of NOD/Lt marrow, diabetes-resistant NOD.NON-H-2nb1 congenic mice were mated with NOD/Lt mice to produce NOD-H-2g7/H-2nb1 heterozygous recipients. These were lethally irradiated and reconstituted with either NOD/Lt marrow alone, NOD.H-2nb1 homozygous congenic marrow alone, or a 1:1 mixture of the two marrow populations. By 25 wk of age, all of the MHC heterozygous recipients of NOD.NON-H-2nb1 marrow remained diabetes-free whereas 75% of the MHC heterozygous recipients of NOD/Lt marrow developed diabetes. A striking decrease in diabetes was observed when T cell precursors derived from NOD/Lt marrow interacted with H-2nb1 gene products on hemopoietically derived APC, inasmuch as only 7% of the MHC heterozygous recipients reconstituted with a 1:1 mixture of NOD/Lt and NOD.NON-H-2nb1 marrow developed diabetes. Peripheral leukocytes in all reconstitution classes expressed the MHC phenotype(s) of the marrow donor(s). Skin grafting confirmed that all reconstitution classes of MHC heterozygous recipients were tolerant to the H-2nb1 haplotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat

Bio-Breeding rat T lymphocytes proliferate poorly in response to alloantigen. Transplantation of Bio-Breeding rats with fetal thymus tissue from diabetes resistant rats leads to an improvement in the T cell proliferative response, but only if the thymus contains bone marrow-derived, radiation-resistant thymic antigen presenting cells of the diabetes-resistant phenotype. The current study provides evidence that thymus transplantation leading to the restoration of Bio-Breeding T cell proliferative function can also significantly reduce the incidence of insulitis and prevent the development of diabetes. It appears that a defect in the bone marrow-derived thymic APC population contributes to an abnormal maturation of Bio-Breeding T lymphocytes which in turn predisposes animals to insulitis and diabetic disease.     

Self-reactive T cells selected on thymic cortical epithelium are polyclonal and are pathogenic in vivo

Positive selection of CD4+ T cells requires that the TCR of a developing thymocyte interact with self MHC class II molecules on thymic cortical epithelium. In contrast, clonal deletion is mediated by dendritic cells and medullary epithelium. We previously generated K14 mice expressing MHC class II only on thymic cortical epithelium. K14 CD4+ T cells were positively, but not negatively, selected and had significant in vitro autoreactivity. Here, we examine the function of these autoreactive CD4+ T cells in more detail. Analysis of a series of K14-derived T hybrids demonstrated that the autoreactive population of CD4+ T cells is phenotypically and functionally diverse. Purified K14 CD4+ T cells transferred into lethally irradiated wild-type B6 mice cause acute graft vs host disease with bone marrow failure. Further, these autoreactive CD4+ T cells cause hypergammaglobulinemia and the production of autoantibodies when transferred into unirradiated wild-type hosts. Thus, positive selection by normal thymic cortical epithelial cells, unopposed by negative selection, produces polyclonal CD4+ T cells that are pathologic.     

T-Cell Tolerance: Central and Peripheral

Somatic recombination of TCR genes in immature thymocytes results in some cells with useful TCR specificities, but also many with useless or potentially self-reactive specificities. Thus thymic selection mechanisms operate to shape the T-cell repertoire. Thymocytes that have a TCR with low affinity for self-peptide–MHC complexes are positively selected to further differentiate and function in adaptive immunity, whereas useless ones die by neglect. Clonal deletion and clonal diversion (Treg differentiation) are the major processes in the thymus that eliminate or control self-reactive T cells. Although these processes are thought to be efficient, they fail to control self-reactivity in all circumstances. Thus, peripheral tolerance processes exist wherein self-reactive T cells become functionally unresponsive (anergy) or are deleted after encountering self-antigens outside of the thymus. Recent advances in mechanistic studies of central and peripheral T-cell tolerance are promoting the development of therapeutic strategies to treat autoimmune disease and cancer and improve transplantation outcome.T cells recognize pathogen fragments in the context of surface MHC molecules on host cells. As such, they have the potential to do enormous damage to healthy tissue when they are not appropriately directed, that is, when they respond to self-antigens as opposed to foreign antigens. T lymphocyte tolerance is particularly important, because it impacts B-cell tolerance as well, through the requirement of T cell help in antibody responses. Thus, failure of T-cell tolerance can lead to many different autoimmune diseases. The tolerance of T cells begins as soon as a T-cell receptor is formed and expressed on the cell surface of a T-cell progenitor in the thymus. Tolerance mechanisms that operate in the thymus before the maturation and circulation of T cells are referred to as “central tolerance.” However, not all antigens that T cells need to be tolerant of are expressed in the thymus, and thus central tolerance mechanisms alone are insufficient. Fortunately, additional tolerance mechanisms exist that restrain the numbers and or function of T cells that are reactive to developmental or food antigens, which are not thymically expressed. These mechanisms act on mature circulating T cells and are referred to as “peripheral tolerance.”     

Activation-threshold tuning in an affinity model for the T-cell repertoire

Naive T cells respond to peptides from foreign proteins and remain tolerant to self peptides from endogenous proteins. It has been suggested that self tolerance comes about by a 'tuning' mechanism, i.e. by increasing the T-cell activation threshold upon interaction with self peptides. Here, we explore how such an adaptive mechanism of T-cell tolerance would influence the reactivity of the T-cell repertoire to foreign peptides. We develop a computer simulation model in which T cells are tolerized by increasing their activation-threshold dependent on the affinity with which they see self peptides presented in the thymus. Thus, different T cells acquire different activation thresholds (i.e. different cross-reactivities). In previous mathematical models, T-cell tolerance was deletional and based on a fixed cross-reactivity parameter, which was assumed to have evolved to an optimal value. Comparing these two different tolerance-induction mechanisms, we found that the tuning model performs somewhat better than an optimized deletion model in terms of the reactivity to foreign antigens. Thus, evolutionary optimization of clonal cross-reactivity is not required. A straightforward extension of the tuning model is to delete T-cell clones that obtain a too high activation threshold, and to replace these by new clones. The reactivity of the immune repertoires of such a replacement model is enchanced compared with the basic tuning model. These results demonstrate that activation-threshold tuning is a functional mechanism for self tolerance induction.     

Regulated recruitment of MHC class II and costimulatory molecules to lipid rafts in dendritic cells

T cell activation has long been associated with the partitioning of Ag receptors and associated molecules to lipid microdomains. We now show that dendritic cells (DCs) also accomplish the selective recruitment to lipid rafts of molecules critical for Ag presentation. Using mouse bone marrow-derived DCs, we demonstrate that MHC class II molecules become substantially localized to rafts upon DC maturation. Even more striking is the fact that CD86 is recruited to rafts upon T cell-DC interaction. Recruitment is Ag dependent and requires CD28 on T cells. Despite the regulated recruitment of MHC class II and CD86 to rafts, unlike the counter-receptors in T cells, DCs do not polarize these molecules to sites of DC-T cell contact. This difference may reflect the necessity for DCs to interact with multiple T cells simultaneously and emphasizes that the biochemical and morphological correlates of lipid rafts are not necessarily equivalent.