Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing

Summary: During antigen processing, peptides are generated and displayed in the context of major histocompatibility complex (MHC) class II molecules on the surface of antigen‐presenting cells (APCs) to modulate immune responses to foreign antigens and guide self‐tolerance. Exogenous and cytoplasmic antigens are processed by distinct routes within APCs to yield class II ligands. Exogenous antigens are internalized, processed, and bound to class II molecules within endosomal and lysosomal compartments of APCs. Studies reviewed here demonstrate the importance of reduction in regulating exogenous antigen presentation. The differential expression of a γ‐interferon‐inducible lysosomal thiol reductase in professional APCs and melanomas is discussed in the context of tumor immune evasion. Cytoplasmic autoantigens, by contrast, are degraded by the proteasome and other enzymes in the cytosol, with the resulting peptides translocating to endosomal and lysosomal compartments for intersection with class II molecules. Processing and editing of these antigenic peptides within endosomes and lysosomes may be critical in regulating their display via class II proteins. Multiple pathways may regulate the transit of cytosolic peptides to class II molecules. The role of lysosome‐associated membrane protein‐2a and heat‐shock cognate protein 70 in promoting cytoplasmic peptide presentation by MHC class II molecules is discussed.     

HLA-DM negatively regulates HLA-DR4-restricted collagen pathogenic peptide presentation and T cell recognition

Rheumatoid arthritis, an autoimmune disease, is significantly associated with the HLA class II allele HLA-DR4. While the etiology of rheumatoid arthritis remains unknown, type II collagen (CII) is a candidate autoantigen. An immunodominant pathogenic epitope from this autoantigen, CII(261-273), which binds to HLA-DR4 and activates CD4+ T cells, has been identified. The non-classical class II antigen, HLA-DM, is also a key component of class II antigen presentation pathways influencing peptide presentation by HLA-DR molecules expressed on professional antigen-presenting cells (APC). Here, we investigated whether the HLA-DR4-restricted presentation of the pathogenic CII(261-273) epitope was regulated by HLA-DM expression in APC. We show that APC lacking HLA-DM efficiently display the CII(261-273) peptide/epitope to activate CD4+ T cells, and that presentation of this peptide is modulated dependent on the level of HLA-DM expression in APC. Mechanistic studies demonstrated that the CII(261-273) peptide is internalized by APC and edited by HLA-DM molecules in the recycling pathway, inhibiting peptide presentation and T cell recognition. These findings suggest that HLA-DM expression in APC controls class II-mediated CII(261-273) peptide/epitope presentation and regulates CD4+ T cell responses to this self epitope, thus potentially influencing CII-dependent autoimmunity.     

Autophagy proteins in antigen processing for presentation on MHC molecules

Autophagy describes catabolic pathways that deliver cytoplasmic constituents for lysosomal degradation. Since major histocompatibility complex (MHC) molecules sample protein degradation products and present them to T cells for adaptive immunity, it is maybe not too surprising that autophagy contributes to this protein antigen processing for MHC presentation. However, the recently recognized breath of pathways, by which autophagy contributes to MHC antigen processing, is exciting. Macroautophagy does not only seem to deliver intracellular but facilitates also extracellular antigen processing by lysosomal hydrolysis for MHC class II presentation. Moreover, even MHC class I molecules that usually display proteasomal products are regulated by macroautophagy, probably using a pool of these molecules outside the endoplasmic reticulum, where MHC class I molecules are loaded with peptide during canonical MHC class I antigen processing. This review aims to summarize these recent developments and point out gaps of knowledge, which should be filled by further investigation, in order to harness the different antigen-processing pathways via autophagy for vaccine improvement.     

Exogenous CLIP localizes into endocytic compartment of cells upon internalization: implications for antigen presentation by MHC class II molecules

We have previously shown that exogenous CLIP (class II associated invariant chain peptide) downregulated MHC class II expression on antigen presenting cells (APC) and modulated T cell mediated immune responses. The present study was undertaken to investigate the mechanism of uptake of exogenously added CLIP peptide by APC. We found that exogenous CLIP is rapidly internalized by APC and it co-localize with MHC class II in intracellular compartments including early-, late-endosomes and lysosomes. We suggest that exogenous CLIP acts as an in vivo regulator of immune response by internalization and passage through the intracellular compartments where it interferes in peptide loading and recycling of MHC class II molecules to the APC surface. Therefore, exogenous CLIP regulates immune responses by modulation of antigen presentation by the APC.     

Right place, right time, right peptide: DO keeps DM focused

Summary: Peptide loading of major histocompatibility class II molecules is catalyzed in late endosomal and lysosomal compartments of cells by the catalytic action of human leukocyte antigen (HLA)‐DM (H‐2M in mice). In B cells, dendritic cells and thymic epithelial cells, the peptide loading of class II molecules is modified by the expression of the non‐classical class II molecule, HLA‐DO (H‐2O in mice). Collectively, studies to date support that DO/H‐2O expression inhibits the presentation of antigens acquired by cells via fluid phase endocytosis. However, in B cells, the expression of H‐2O promotes the presentation of antigens internalized by the B‐cell receptor. In this review, we summarize the literature pertaining to DO assembly, transport, and function, with an emphasis on the function of DO/H‐2O.     

I-Aq and I-Ap bind and present similar antigenic peptides despite differing in their ability to mediate susceptibility to autoimmune arthritis

Susceptibility to collagen induced arthritis (CIA) in the murine model is linked to expression of the MHC class II alleles, I-Aq and I-Ar. We have examined the molecular basis for this MHC-linked susceptibility by studying the antigen presentation function of two class II molecules, I-Aq and I-Ap, that are closely related yet differ in mediating susceptibility to CIA. These class II molecules differ by only 4 amino acids, yet only mice expressing I-Aq develop CIA. Although the I-Ap molecule can bind the same immunodominant determinant from type II collagen as I-Aq, H-2p APC have difficulty generating I-Ap:CII peptide complexes when processing of CII is required. Immunization of H-2p mice with type II collagen (CII) generated only a weak T cell response when compared to H-2q mice, whereas immunization with the a CII peptide containing the dominant determinant induced a strong T cell response in both strains. In antigen presentation assays, H-2p APC were very inefficient in stimulating T cells when native CII was used as antigen, however they presented CII synthetic peptides with similar efficiency as H-2q APC. Processing and presentation of other antigens by H-2p APC was not affected. Using soluble class II binding assays, the affinity of I-Ap for the CII dominant peptide was 10 to 50 fold lower than I-Aq, however, this reduced affinity was not a general defect in I-Ap function. I-Aq and I-Ap had virtually identical affinities for binding other antigenic peptides. These data indicate that MHC-based susceptibility to autoimmunity may involve more than simple determinant selection and that the successful generation of an antigenic peptide by processing may be related to the overall affinity of the peptide for the MHC molecule.     

Antigen processing and presentation by a murine myoblast cell line

The ability of non-professional antigen-presenting cells (APC) lo process and present antigen to the immune system has been the subject of debate in autoimmunity and tumour immunology. The role of muscle cells in the processing and presentation of antigen to T cells via class 1 and class II MHC pathways is of increasing interest. Muscle cells are the targets of autoimmune attack in the inflammatory muscle diseases, and direct intramuscular injection of antigen-expressing DNA constructs is under scrutiny as a means of vaccination. Furthermore, the immunological properties of muscle cells are of relevance in attempts to transfer my oblasts as replacement cells in dystrophic diseases or as depot cells for the secretion of certain molecules in deficiency states. Using class I and class II MHC transfectant clones of the C2CI2 my oblast cell line, my oblasts have been shown to be capable of presenting antigen to. and stimulating secretion of IL-2 by, T cell hybridomas via both of these pathways. The epitopes which are dominantly presented by professional APC after processing of native antigens were also presented by the myoblast cell line after processing of either ovaibumin (class I) or hen egg lysozyme (class 11). Further, antigen processing and presentation via the class II pathway were enhanced by pretreatment of the my oblasts with interferon-gamma (IFN-γ), Up-regulation of invariant chain expression by this treatment may have contributed to this enhanced presentation, but an effect of IFN-γ on the expression of other molecules such as H-2 DM may have also played a role. The demonstration of the antigen-presenting properties of these my oblasts is of relevance to all three areas mentioned above. In each situation my oblasts comprise a significant population within muscle. In the case of inflammatory muscle diseases the process of muscle degeneration and regeneration is on-going, while in the vaccination procedure some muscle damage occurs, and vaccination is more effective when muscle damage has preceded inoculation.     

Identification and characterization of major histocompatibility complex class II compartments in cortical thymic epithelial cells

Previous studies have concentrated on elucidating the subcellular localization of major histocompatibility (MHC) class II molecules mainly in B cells, macrophages, and dendritic cells. Despite very rich cell-surface expression of MHC class II molecules by cortical thymic epithelial cells (cTECs), little is known regarding the expression of these molecules by cTECs at the subcellular level. In the present study we focused on the identification and characterization of MHC class II compartments (MIICs) in cTECs in situ by immunogold electron microscopy (IEM). We found that MHC class II molecules were located exclusively in the cytoplasmic vacuoles, and we identified these MHC class II molecule-containing cytoplasmic vacuoles as MIICs in cTECs. These MIICs were immunopositive for early endosomal, late endosomal, and lysosomal markers. Moreover, in these MIICs, MHC class II molecules were colocalized with cathepsin L, H2-DM, class II-associated invariant chain (Ii), and class II-associated invariant chain peptide (CLIP). Similarly, Ii molecules were colocalized with endosomal and lysosomal markers, cathepsin L, and H2-DM in the vacuoles. Taken together, these results suggest that MIICs in cTECs represent conventional endocytic compartments. The colocalization of MHC class II molecule or Ii with cathepsin L and H2-DM in the MIICs suggests that MIICs in cTECs may be sites of Ii degradation and peptide loading.     

H2-O influence on antigen presentation in H2-E-expressing mice

MHC class II molecules sample peptides generated in the endosomal/lysosomal system for cell surface presentation to CD4+ T cells. Peptide loading requires the endosomal/lysosomal resident HLA-DM (DM; H2-DM, mouse), but in B cells, DM is tightly associated with HLA-DO (DO; H2-O, mouse). We have previously shown that H2-O differentially modulates the processing and presentation of different antigenic epitopes on H2-Ab molecules. Using H2-Ead-transgenic mice, we here show that presentation of different epitopes by H2-Ed/b molecules is similarly influenced by H2-O after membrane immunoglobulin-mediated uptake of antigen. In addition, B cells from H2-Ead-transgenic mice (which co-express H2-Ab and H2-Ed/b molecules) show an altered pattern of presentation of H2-Ab-restricted epitopes. In H2-Ead-transgenic, H2-O-deficient mice, further changes in the peptide repertoire were observed. Thus, H2-Ed/b expression influences the epitopes presented by H2-Ab, and this effect is further altered by expression of H2-O.     

Differential expression of major histocompatibility complex class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic cells.

Blood monocyte derived antigen presenting cells (APC) such as dendritic cells and macrophages are considered as major promising tools for antitumoral immunotherapy. In order to contribute to their phenotype characterization, we have precisely investigated their levels of expression of MHC class Ia, Ib (HLA-G) and II molecules using mainly flow cytometry quantification assays. APC were generated from monocytes cultured for 7 days in the presence of GM-CSF and IL-4 or M-CSF. These cells, which exhibited known morphological and immunological features of dendritic cells and macrophages respectively, were evidenced to display high expression of MHC class Ia and class II antigens in comparison to that found in monocytes. Dendritic cells and macrophages thus expressed 2-fold more and 4-fold more MHC class Ia molecules and 5-fold and 3-fold more MHC class II DR molecules than parental monocytes. In addition, expression of MHC class II DP and DQ molecules, not or only barely detected in monocytes, was clearly demonstrated in the two kinds of APC. In contrast, monocytes, dendritic cells and macrophages failed to express MHC class Ib HLA-G antigen. The up-regulation in monocyte-derived APC of MHC class Ia and II molecules mediating the presentation of antigen peptides to lymphocytes fully supports the interest of such APC in antitumoral immunotherapy.     

Downmodulation of antigen presentation by H2-O in B cell lines and primary B lymphocytes

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigen-presenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.     

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.     

1142280946Effect of estradiol and PAMPs on class II mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

Problem:  Antigen presenting cells (APC) in the female reproductive tract play important roles in innate immune defense and activation of the adaptive immune responses. The objective of this study was to examine the effects of estradiol and PAMP on antigen presentation in the female reproductive tract.
Method of Study:  DO11.10 T cell antigen receptor transgenic mice specific for the MHC class II-restricted OVA323–339peptide were used to study the effects of estradiol and PAMP on antigen presentation of OVA by uterine epithelial (EC) and stromal cells as well as vaginal cells to OVA specific memory-T cells.
Results:  Estradiol inhibited antigen presentation of OVA by uterine EC, uterine stromal cells and vaginal cells to OVA specific memory-T cells. When ovariectomized animals were treated with estradiol for 1 or 3 days, antigen presentation decreased by 20–80%. In contrast, incubation with TLR agonists increased antigen presentation by EC (Poly (I:C), Pam3Cys), stromal cells (PGN, Pam3Cys) and vaginal cells (LPS, Pam3Cys). Analysis of mRNA expression by real time RT-PCR indicated that estradiol inhibited CD40, CD80/86 and class II in the uterus and vagina. In contrast, stimulation of antigen presentation by PAMP did not correlate with changes in costimulatory molecule mRNA expression.
Conclusions:  These results indicate that APC in the uterus and vagina are responsive to estradiol, which inhibits antigen presentation and costimulatory molecule expression. These findings suggest that whereas APC in the uterus and vagina respond to TLR agonists with increased antigen presentation, which initiates an adaptive immune response, their effects appear to be at levels other than the expression of costimulatory molecules.
Acknowledgement:  Supported by AI-13541 from NIH.     

Diversity in MHC class II antigen presentation

Processing exogenous and endogenous proteins for presentation by major histocompatibility complex (MHC) molecules to T cells is the defining function of antigen-presenting cells (APC) as major regulatory cells in the acquired immune response. MHC class II-restricted antigen presentation to CD4 T cells is achieved by an essentially common pathway that is subject to variation with regard to the location and extent of degradation of protein antigens and the site of peptide binding to MHC class II molecules. These subtle variations reveal a surprising flexibility in the ways a diverse peptide repertoire is displayed on the APC surface. This diversity may have profound consequences for the induction of immunity to infection and tumours, as well as autoimmunity and tolerance.     

Non-covalent presentation of sulfamethoxazole to human CD4+ T cells is independent of distinct human leucocyte antigen-bound peptides

BACKGROUND: It has been shown that drugs comprise a group of non-peptide antigens that can be recognized by human T cells in the context of HLA class II and that this recognition is involved in allergic reactions. Recent studies have demonstrated a MHC-restricted but processing- and metabolism-independent pathway for the presentation of allergenic drugs such as lidocaine and sulfamethoxazole (SMX) to drug-specific T cells. However, there is little information so far on the precise molecular mechanisms of this non-covalent drug presentation. OBJECTIVE: The aim of this study was to evaluate the requirements for a specific peptide occupying the groove of the MHC class II molecule for the efficient presentation of non-covalently bound drugs to CD4+ T cells. METHODS: We analysed the effect of coincubation or prepulse of antigen presenting cells (APC) with different peptides on the proliferative responses of SMX-specific CD4+ T cell clones. In a second series of experiments, we eluted HLA-bound peptides from the surface of antigen presenting cells by mild acid treatment. Successful removal of peptides was tested directly using labelled peptides and functionally by monitoring activation and proliferation of peptide-specific T cell clones. Finally, the presentation of SMX to SMX-specific T cell clones before and after elution of MHC class II bound peptides was tested. RESULTS: We found that neither peptide coincubation nor peptide prepulse of APC altered the proliferative response of SMX-specific T cells. APC treated with the acid for a short time retained cell viability, MHC class II expression and antigen presenting cell function. However, defined peptides could be eluted from surface MHC class II molecules nearly quantitatively. Nevertheless, the chemically non-reactive drug SMX could still be presented to specific T cells independent of the presence of distinct self-peptides. CONCLUSION: Our data suggest that small molecules like drugs can bind to a multitude of HLA-bound peptides or that, similar to superantigens, they might bind directly to HLA.     

LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4⁺ T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide‐binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II‐mediated antigen presentation. Lysosome‐associated membrane proteins such as LAMP‐2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP‐2 were examined for changes in MHC class II‐restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4⁺ T cells was impaired in the LAMP‐2‐deficient B cells. Peptide‐binding to MHC class II on LAMP‐2‐deficient B cells was reduced at physiological pH compared with wild‐type cells. However, peptide‐binding and class II‐restricted antigen presentation were restored by incubation of LAMP‐2‐negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP‐2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP‐2‐deficient B cells. Consequently, LAMP‐2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.     

Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1-mediated nuclear export

Accumulating evidence suggests that intracellular antigens are endogenously presented on MHC class II, but it is still unknown whether antigens within different subcellular compartments are presented with similar efficiency, and via the same or different pathways. We have previously shown that endogenous MHC class II presentation of the cytosolic bacterial antigen neomycin phosphotransferase II (NeoR) is mediated by autophagy. Here, we addressed whether secluding NeoR from this cytoplasmic pathway by directing the protein into the cell nucleus (NucNeoR) would affect antigen presentation. Unexpectedly, NucNeoR was presented at least as efficiently as the cytosolic version of the antigen. Furthermore, presentation of NucNeoR was also dependent on autophagocytosis and lysosomal processing, indicating that both antigens were presented via the same pathway. Inhibition of CRM1-mediated nuclear export did not impede antigen presentation, indicating that NucNeoR gained access to this autophagy-dependent MHC class II presentation pathway by a CRM1-independent route. Thus, this endogenous presentation pathway broadens the spectrum of intracellular antigens surveyed by CD4(+) T cells by efficiently sampling cytoplasmic as well as nuclear antigens.     

HLA class II antigen presentation by melanoma

Constitutive expression of HLA II molecules is normally restricted to professional antigen presenting cells (APCs) of the immune system, although it also occurs in melanoma. Clinical evidence suggests that HLA II expression by melanoma is unfavorable, implicating a role for HLA II expression in the immunopathogenesis of the disease. As a first step, we asked whether human melanoma cells can process antigen or present peptide via HLA II molecules to a peptide specific CD4+ T cell clone. We compared the T cell response to melanoma vs. autologous B cells. In all cell lines tested, melanoma cells were able to process antigen and present peptide efficiently to CD4+ T cells, resulting in T cell proliferation increased 5 to 26 fold over no peptide controls. Blocking CD28 mediated signalling with CTLA-4lg had no effect on the T cell response to either melanoma or B cells. Blocking ICAM-1 resulted in a significant decrease in proliferation (>60%) in response to peptide presentation by melanoma but not B cells. These data demonstrate that (1) HLA II molecules on melanoma cells are functional and antigen processing pathways are intact, and (2) costimulatory signalling mediated via ICAM-1 but not B7 is important in the response of antigen experienced CD4+ T cells to HLA II mediated peptide presentation by melanoma.     

Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.     

Peptide-induced proliferation and lymphokine production in human T cells in the absence of antigen-presenting cells: role of T-cell activation state and costimulatory signals.

The role of T-lymphocytes as antigen-presenting cells (APCs) for other T cells was investigated. Activated rabies-virus-specific human T-cell clones were shown to present peptide to class II major histocompatibility complex (MHC)-restricted T cells of a different fine specificity, resulting in lymphokine production and cell proliferation. Furthermore, purified and activated antigen-specific T cells could produce lymphokines and proliferate as a result of the addition of antigenic peptide in the absence of APC. The functional response of T cells to peptide in the absence of APC was amplified by the addition of phorbol ester (PMA) and was inhibited with antibodies specific to class II MHC or to the CD2 molecule. Experiments performed in single-cell suspension cultures using semisolid medium prepared with 1% agar demonstrate that T-cell proliferative and lymphokine responses to peptide both in the presence and absence of APC require the interaction of T-cell antigen receptor (TCR) molecules with class II MHC-peptide complexes on different cell surfaces (cell-cell contact). On the other hand, peptide self-presentation, which occurs by the binding of TCR with class II MHC-peptide complexes on the same cell surface (at the single-cell level), resulted in T-cell activation (i.e., high expression of surface CD2, CD25, and HLA-DR molecules), without proliferation or lymphokine secretion, a pattern observed in the induction of T-cell anergy by antigen. The results are discussed in terms of the role of class II MHC molecules on activated T-lymphocytes, which enable these cells to function as "professional APC" in the development of T-cell regulatory networks.