Absence of Epstein-Barr virus-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes in infectious mononucleosis.

Cytotoxic T lymphocytes (CTL) with the CD4+ phenotype that recognize major histocompatibility complex (MHC) class II antigens are detectable very frequently in cultures of human alloreactive or virus-specific T cells. The significance of these CD4+ CTL for an immune reaction in vivo is not clear. Since Epstein-Barr virus (EBV) transformed B cells express HLA-class I and class II antigens equally well both CD8+ and CD4+ CTL should be stimulated during an acute EBV infection. We analysed the MHC specificity and the phenotype of EBV-specific CTL from patients with infectious mononucleosis (IM). When tested directly without any previous culture, T cells from patients in the acute phase of IM showed specific MHC-restricted cytotoxicity against the autologous B cell line. Addition of a HLA class I specific monoclonal antibody (MoAb) but not of a HLA class II specific MoAb resulted in a complete blocking of the lytic activity. Cell sorting revealed that the entire cytotoxic activity was present in the CD8+ fraction whereas no specific CTL were detectable in the CD4+ fraction. The absence of cytotoxicity in CD4+ cells was not due to a lack of activation of these cells since both CD8+ and CD4+ cells were activated in situ, showing spontaneous growth in interleukin-2 (IL-2) and expressing the activation marker TP103. Frequency estimation revealed that 1/300-1/600 CD8+ but only 1/2000-1/4000 CD4+ T cells gave rise to a specific CTL colony after 10 days. If CD4+ colonies were tested repeatedly for cytotoxicity we found that CD4+ CTL acquired their cytotoxicity during in vitro culture. In addition, we isolated EBV-specific CD4+ T cell clones able to lyse their stimulator cells in the presence but not in the absence of lectin, even after a long period of culture. Taken together our results show that cytotoxicity mediated by CD4+ T cells does not play a role in an anti-viral immune response.     

Activation of CD8+ T cells by allogeneic class II-deficient B-cell lines derived from patients with bare lymphocyte syndrome

The major histocompatibility complex (MHC) class II antigens (Ags) are known to carry the major stimulating determinants of the primary mixed lymphocyte reactions (MLR). We investigated the mechanism of generating HLA class I-directed alloreactive T-cells in primary MLR. With the use of class II-deficient EBV-transformed B-lymphoblastoid cell lines (B-LCLs) derived from patients with bare lymphocyte syndrome (BLS), we have demonstrated in the present study that class I disparity alone can trigger primary MLR in the absence of exogenous IL-2. The CD8+ T cells were primary MLR-responsive cells, and the CD4+ T cells seem to play no role in primary MLR when class II alloantigens are not involved in stimulation. Addition of autologous macrophages did not influence the primary MLR response. The primary MLR was completely blocked by anti-class I or anti-CD8 antibodies but not by anti-class II or anti-CD4 antibodies. The MLC-generated CD8+ T cells exhibited cytolytic activity as well as proliferative responses. The proliferative response of the CD8+ T cells was specifically directed against class I antigens, demonstrated by proliferative assays; and the helper-independent CD8+ T cells were generated only when the activation of CD4+ T cells did not occur. This observation suggests that functional recruitment of T-cell receptor (TCR) repertoire is under active regulation, and the suppression of CD8+ T-cell helper recruitment appears to be dictated by the CD4+ T-cell subset. Further analysis of the primed T-cell specificities showed that alloreactivity of the CD8+ T cells was mostly accounted for by the HLA-B Ags.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of OKT3 hybridoma cells to clonally activate CD3+ human T lymphocytes

A simple and reliable method was developed to induce clonal growth of resting human T cells. In this limiting dilution (LD) culture system, responder cells (unseparated mononuclear cells, E rosette-purified T cells, or cell sorter-separated CD4+ and CD8+ subsets) were activated by irradiated anti-CD3-secreting (OKT3) hybridoma cells in the presence of exogenous IL-2 (crude culture supernatant or recombinant IL-2). Under these conditions, one out of 2-3 CD4+ and CD8+ T cells developed into a proliferating cell clone. Addition of recombinant IL-1 slightly enhanced the growth frequency and increased the clone size of CD4+ cells but did not affect the growth pattern of CD8+ cells.     

A comparison of gene transfer and antigen-loaded dendritic cells for the generation of CD4+ and CD8+ cytomegalovirus-specific T cells in HLA-A2+ and HLA-A2- donors.

Dendritic cells have been used effectively to select for human cytomegalovirus (CMV)-specific T cells for immunotherapy applications. The ability to process and present relevant major histocompatibility complex class I and II peptides to T cells makes them ideal for selecting CD4+ and CD8+ T cells regardless of HLA tissue type. This study compared the generation of CMV-specific T cells by using dendritic cells loaded with either CMV pp65495-503 peptide or CMV lysate or transduced with adenovirus encoding the pp65 gene (Ad5pp65GFP) for the generation of CD4+ and CD8+ CMV-specific T cells in HLA-A2+ and HLA-A2 - donors. In HLA-A2+ donors, CD8+ tetramer+ T cells increased with all antigens but were greatest in peptide- and Ad5pp65GFP-stimulated T cells. The CD4+ /CD8+ ratio in the stimulated T-cell cultures proved to be dependent on the antigen used. CMV lysate-stimulated cells were primarily CD4+, whereas peptide- and Ad5pp65GFP-stimulated cultures were mostly CD8+. Analysis of cells from lysate-stimulated or gene-transduced-stimulated cultures showed expansion of CMV-specific CD4+ T cells, indicating that major histocompatibility complex class II peptides were present in both antigens. Furthermore, CMV-specific T cells were generated from HLA-A2 - donors by using Ad5pp65GFP transduction or CMV lysate stimulation and were able to recognize a pp65 peptide restricted to the HLA-B35 allele. These data indicate that either CMV lysate or adenovirus encoding CMV antigenic genes may be useful for the generation of both CD4+ and CD8+ CMV-specific T cells in donors irrespective of HLA tissue type and may be applicable to clinical immunotherapy.     

Major histocompatibility complex class I-restricted alloreactive CD4+ T cells

Although it is well established that CD4+ T cells generally recognize major histocompatibility complex (MHC) class II molecules, MHC class I-reactive CD4+ T cells have occasionally been reported. Here we describe the isolation and characterization of six MHC class I-reactive CD4+ T-cell lines, obtained by co-culture of CD4+ peripheral blood T cells with the MHC class II-negative, transporter associated with antigen processing (TAP)-negative cell line, T2, transfected with human leucocyte antigen (HLA)-B27. Responses were inhibited by the MHC class I-specific monoclonal antibody (mAb), W6/32, demonstrating the direct recognition of MHC class I molecules. In four cases, the restriction element was positively identified as HLA-A2, as responses by these clones were completely inhibited by MA2.1, an HLA-A2-specific mAb. Interestingly, three of the CD4+ T-cell lines only responded to cells expressing HLA-B27, irrespective of their restricting allele, implicating HLA-B27 as a possible source of peptides presented by the stimulatory MHC class I alleles. In addition, these CD4+ MHC class I alloreactive T-cell lines could recognize TAP-deficient cells and therefore may have particular clinical relevance to situations where the expression of TAP molecules is decreased, such as viral infection and transformation of cells.     

CD4+ CD25+ [corrected] regulatory T cells render naive CD4+ CD25- T cells anergic and suppressive

CD4(+) CD25(+) Foxp3(+) naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4(+) CD25(-) T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG(-/-)) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4(+) CD25(-) T cells and CD8(+) T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-beta partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4(+) CD25(-) responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.     

CD4+T cells restricted by DQ not by DR support CD8+T cells to grow

Much attention has been paid whether there are any differences in regulating the human immune response between HLA-DR and -DQ molecules encoded by the genes within the HLA class II multigene family. Previous studies have suggested that HLA DQ molecules control low responsiveness through activating CD4 T cells which generate CD8 positive T cells, whereas HLA -DR molecules control high responsiveness through activating CD4 helper T cells. To examine this model we investigated the streptococcal cell wall antigen (SCW) specific T cell lines restricted by either DR or DQ molecule. To identify the restricting molecules, L cell transfectants expressing DQw1, DR2AB1 or DR2AB5 from Dw12 haplotype or DQw4, DR4 or DRw53 from DW15 haplotype were used. 1. From individuals with Dw12 which is a low responder haplotype to SCW, T cell clones specific to SCW and restricted by HLA-DQw1 or DR2 were identified, whereas from individuals with Dw15 which is a high responder haplotype, only DR4 or DRw53 restricted T cell clones were identified and DQw4 restricted T cells were never observed. 2. SCW specific CD4 T cells restricted by DQw1 were able to support the growth of CD8 positive cells, whereas those restricted by DR4 could not do so. 3. The CD8 T cells also required autologous antigen presenting cells and SCW to grow, and they completely blocked the immune response to SCW in vitro. These observations clearly demonstrated the distinct function of HLA-DQ and -DR molecules in regulating the human immune response to SCW.     

Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.     

Expression of HLA class I/II antigens and T cell immune response in human neuroendocrine tumors of the pancreas

Peptide presentation by HLA class I and II antigens regulates specific antigen recognition by T cells. The present study aimed to investigate T cell infiltration and its relation to HLA antigen expression in pancreatic neuroendocrine tumors. Fresh tissue samples were collected from five insulinomas and six other neuroendocrine tumors (one gastrinoma, one glucagonoma, two carcinoid, and two neuroendocrine carcinomas). Normal pancreatic and splenic tissue samples were used as controls. Investigation of infiltrating lymphocyte populations, as well as staining of HLA class I and II antigens, were performed by standard immunohistochemistry. The majority of investigated tumors demonstrated an intratumoral infiltration by CD3+, CD4+ and CD8+ T cells that was significantly higher than in normal pancreatic islets. Only a minority of tumor-infiltrating T cells showed the CD45RO+ phenotype. The expression of HLA class I antigen was altered in 10 of 11 tumors. A loss of beta-2microglobulin represented the most frequent type of alteration to HLA class I expression, although the total loss of HLA class I was found in only one case of neuroendocrine carcinoma. HLA class II molecules were expressed by endothelial and lymphoid cells and not by tumor cells. In conclusion most neuroendocrine pancreatic tumors induce a T cell mediated immune response resulting in an intratumoral infiltration with CD3+, CD4+ and CD8+ T cells. Loss of beta-2microglobulin is a frequent alteration in these tumors, which may influence the normal function of the HLA class I antigen complex. In contrast to malignant tumors of the exocrine pancreas, expression of HLA class II was absent in neuendocrine pancreatic tumor cells.     

Peptide dependency of alloreactive CD4+ T cell responses

Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.     

Interferons (IFNs) and tumor necrosis factors (TNFs) in T cell-mediated immune responses against alloantigens. I. Influence on the activation of resting and antigen-primed T cells

The aim of this study was to investigate the influence that endogenous IFNs released in response to antigenic or viral stimuli has on recognition of alloantigens in MLC. Results indicated that both the magnitude and the kinetics of response can be modified by IFNs. Neutralizing antibodies with specificity for IFN-gamma inhibit early and enhance late proliferative responses in MLC. Addition of physiological concentrations of IFN-gamma enhanced both early and peak proliferation, whereas IFN-alpha markedly inhibited alloantigen-induced lymphocyte proliferation. Further experiments revealed that IFN effects in MLC are not caused by direct interaction with responder cells: pretreatment with IFNs neither failed to alter their subsequent proliferative reactivity, nor did it influence production of IL 2 in MLC. IFN-gamma mainly affected MLC responses by direct interaction with stimulator cells. These influences on hemopoietic and non-hemopoietic stimulator cells were complex and could not simply be explained on the basis of an altered expression of class II MHC antigens. When induced by IFN-gamma to maximally express class II antigens, pbmnc, LCL or homogeneous populations of macrophages showed a marked deficiency to induce primary or secondary proliferative T cell responses. Resting unsensitized or sensitized T cells were not stimulated by class II MHC antigens constitutively expressed or induced by IFN-gamma on cell types other than dendritic cells or LCL. Class II antigens on the former cells were, however, readily recognized by T helper blasts, and this process involved the T4 epitope of the T cell receptor. IFN-gamma treatment also influenced the intrinsic suppressive capacity of macrophages or keratinocytes without involving prostaglandin synthesis or inducing expression of IL 2 receptors on non T cells.     

Existence of mature human CD4+ T cells with genuine class I restriction.

A human T cell receptor (TcR) alpha/beta CD4+CD8-T cell clone (R416) is reactive with the minor histocompatibility antigen H-Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so-called specificity association of mature TcR alpha/beta+ T cells. R416 displays H-Y-specific, HLA-A2-restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H-Y-reactive CD4-CD8+ MHC class I-restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cells clone, it was found that interaction of CD4 with non-TcR-bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor-mismatched T cell clone was not generated in vitro but obtained by expansion of CD8-depleted peripheral blood mononuclear cells of a female who had been immunized against H-Y. The existence of such MHC class I-restricted mature TcR alpha/beta+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.     

Human keratinocyte induction of rapid effector function in antigen-specific memory CD4+ and CD8+ T cells

The ability of human keratinocytes to present antigen to T cells is controversial and, indeed, it has been suggested that keratinocytes may promote T cell hyporesponsiveness. Furthermore, it is unclear whether keratinocytes can process antigen prior to MHC class I and class II presentation. We tested the ability of keratinocytes to induce functional responses in epitope-specific CD4+ and CD8+ memory T cells using peptides, protein and recombinant expression vectors as sources of antigen. Keratinocytes were able to efficiently process and present protein antigen to CD4+ T cells, resulting in cytokine secretion (Th1 and Th2). This interaction was dependent on keratinocyte expression of HLA class II and ICAM-1, which could be induced by IFN-gamma. In addition, keratinocytes could present virally encoded or exogenous peptide to CD8+ T cells, resulting in T cell cytokine production and target cell lysis. Finally, T cell lines grown using keratinocytes as stimulators showed no loss of function. These findings demonstrate that keratinocytes are able to efficiently process and present antigen to CD4+ and CD8+ memory T cells and induce functional responses. The findings have broad implications for the pathogenesis of cutaneous disease and for transcutaneous drug or vaccine delivery.     

Enhancement of MHC class I-stimulated alloresponses by TNF/TNF receptor (TNFR)1 interactions and of MHC class II-stimulated alloresponses by TNF/TNFR2 interactions

In vivo TNF inhibition has been observed to ameliorate the disease process attributed to T cell-dependent immune responses such as those generated during graft-vs.-host disease. The present studies were designed to evaluate whether TNF/TNF receptor (TNFR)1 and TNF/TNFR2 interactions were involved in the generation of allospecific T cell responses. Splenic lymphocyte populations were obtained from TNFR1- or TNFR2-deficient B6 mice and from control B6 mice. These responder cells were cultured with irradiated MHC class II-disparate B6.C-H-2bm12 (bm12) or MHC class I-disparate B6.C-H-2bm1 (bm1) or irradiated syngeneic stimulator cells for 3 days before assay of [3H]thymidine incorporation. IL-2 levels of the mixed lymphocyte culture (MLC) supernatants were assessed by enzyme-linked immunosorbent assay. With MHC class II-disparate bm12 stimulator cells, a significant reduction in T cell proliferation was observed utilizing TNFR2-deficient CD4+ responder T cells, but not when using TNFR1 -deficient CD4+ responder T cells. A significant decrease in proliferation of TNFR1-deficient CD8+ responder cells, but not of TNFR2-deficient CD8 responder T cells was observed after stimulation with MHC class I-disparate bm1 stimulator cells. IL-2 levels were lower in MLC utilizing MHC class I stimulators and TNFR1-deficient responders or MHC class II stimulators and TNFR2-deficient responders. These results indicate that TNF/TNFR2 interactions promote MHC class II-stimulated alloresponses, while TNF/TNFR1 interactions promote MHC class I-stimulated alloresponses.     

CD4+ CD45RA+ T cells modulate allergen-induced interleukin 2 responsiveness in human lymphocytes.

Peripheral blood lymphocytes from nonallergic individuals acquired responsiveness to interleukin 2 (IL2) after stimulation with ovalbumin (OVA) or Dermatophagoides farinae (Df) antigens when they were pretreated with the CD45RA antibody, which has been shown to define the suppressor inducer subset of CD4+ cells and also to block its suppressor activity. The effect provided by the CD45RA antibody was lost if the lymphocytes had initially been activated with the OVA of Df antigens. The magnitude of the responses was comparable to the allergen-induced responses observed in OVA- or Df-sensitized lymphocytes from allergic patients. The pre-existing IL2 responsiveness in the patients was not increased by the CD45RA antibody pretreatment. However, the CD45RA antibody pretreatment gave rise to Df-induced IL2 responsiveness in the lymphocytes of the patients sensitized with OVA but not with Df; conversely, OVA-induced IL2 responsiveness was enhanced in Df- but not in OVA-sensitized lymphocytes. The CD45RA antibody apparently acts on CD4+ T cells, but not on CD8+ T cells, to induce the IL2 response. A further dissection of normal CD4+ T cells indicated that CD4+45RA- T cells preferentially respond to IL2 after stimulation with OVA or Df antigens. Since normal CD4+45RA+ T cells did not show antigen-induced IL2 responsiveness even after pretreatment with the CD45RA antibody, it is unlikely that the CD45RA antibody stimulates CD4+45RA+ T cells to become responsive to IL2 after antigenic challenge. Alternatively, CD4+45RA+ T cells may modulate the activity of CD4+45RA- T cells, which are potentially responsive to IL2 by antigenic stimulation and thus provide tolerance in nonallergic lymphocytes. Collectively, a defective suppressor activity of CD4+45RA+ T cells may exist in patients with hen-egg allergy and/or bronchial asthma, which may cause lymphocytes to be hyperreactive to OVA or Df antigens.     

Regulation of T helper-B lymphocyte adhesion through CD4-HLA class II interaction

Antigen-independent adhesion of CD4+ T lymphocytes to Epstein-Barr virus (EBV)-transformed B cells is mediated by CD2/lymphocyte function-associated antigen (LFA)-3 and LFA-1/intracellular adhesion molecule (ICAM)-1. Although some anti-CD4 antibodies block the antigen-independent adhesion of CD4+ T lymphocytes, the CD4-HLA class II interaction does not appear to significantly contribute to the forces of cell adhesion since CD4+ T cells equally bind HLA class II+ and HLA class II- mutant B cells. In addition, conjugates formed between CD4+ T cells and HLA class II- B cells remain stable for at least 1 h while CD4+T/HLA class II+ B cell conjugate percentages promptly drop off. Down-regulation of CD4 or spontaneous low expression of CD4 also results in a persistance of conjugates formed with B cells. The role of the CD4-HLA class II interaction has been further studied by investigating the inhibitory effect of synthetic 12-mer peptides analogous to HLA class II and containing the Arg-Phe-Asp-Ser sequence conserved in the beta 1 domain. These peptides were previously found to inhibit HLA class II-restricted T cell responses, this sequence being thought to be involved in CD4-HLA class II interaction. These peptides block conjugate formation of CD4+ resting T cells or clones but not of CD8+ T cells, by interacting with the T cells as shown by preincubation experiments. Down-regulation of CD4 or spontaneous low expression results in the loss of the inhibitory activity. The peptide-mediated inhibition is neutralized by a soluble dimeric CD4 molecule. Alteration within the Arg-Phe-Asp-Ser sequence results in a significant loss of inhibition. It is thus proposed that the CD4-HLA class II interaction negatively regulates antigen-independent adhesion of T cells, this interaction involving the highly conserved Arg-Phe-Asp-Ser sequence in the HLA class II beta 1 sequence as a CD4-binding site.     

HLA class II molecules on monocytes regulate T cell proliferation through physical interaction in the CD3 activation pathway

HLA class II molecules are involved in the OKT3-induced T cell activation, since monoclonal antibodies (mAb) to monomorphic determinants of class II antigens are able to inhibit T cell proliferation. This effect involves several of the events leading to T cell activation and proliferation, i.e. interleukin (IL) 1, IL 6 and IL2 secretion and IL2 receptor expression. The main target of the inhibition is represented by monocytes, and the interference of anti-class II mAb in the direct interaction of monocytes with T cells is likely to play a relevant role in the inhibition mechanism. To test this hypothesis, we investigated in the present study the effect of anti-class II mAb on the proliferation of T cells stimulated with mAb OKT3 in the presence of paraformaldehyde-treated monocytes. We show that the inhibition of proliferation is specific and dose dependent, and seems to involve particular class II epitopes. Addition of fixed monocytes to inhibited T cell cultures restores proliferation to a moderate extent, only if monocytes are added within the first 12 h of culture, suggesting that class II antigens or spatially related molecules deliver signals concurrently with the mitogenic stimulus. The blocking capability of anti-class II mAb was not restricted to the CD4+ or the CD8+ T cell subsets, suggesting that, under inhibitory conditions, these mAb affect other structures on the T cell surface, relevant to the monocyte-T cell interaction.     

Liver-derived T cell clones in autoimmune chronic active hepatitis: accessory cell function of hepatocytes expressing class II major histocompatibility complex molecules

Thirty T cell clones were generated from T cell blasts, infiltrating the liver of autoimmune chronic active hepatitis (CAH) patients, stimulated with autologous hepatocytes expressing class II major histocompatibility complex (MHC) molecules and interleukin 2 (IL2). Sixteen clones were CD4+ and 14 were CD8+; all were CD25+ and WT31+, revealing that all cell lines expressed the alpha/beta chains of T cell receptor. Five CD4+ and 4 CD8+ T clones proliferated in response to hepatocytes expressing both class I and class II antigens. The hepatocyte recognition was MHC restricted because only class II MHC-matched hepatocytes were able to stimulate the CD4+ T clones, while only class I-matched hepatocytes stimulated CD8+ T clones, and because MoAbs to monomorphic determinants of class II antigens or to class I antigens appeared to block the response of the CD4+ and CD8+ T clones, respectively. These findings, together with the observation that autologous irradiated peripheral blood mononuclear cells (iPBMC) were unable to stimulate the clones, indicate that the response of these clones was directed to a liver membrane antigen in association with class II or class I MHC molecules on the surface of the hepatocytes. All the CD8+ T clones and 5 CD4+ T clones expressed high cytotoxic activity in a lectin-dependent cell-mediated cytotoxicity assay; 10 CD8+ and 3 CD4+ T clones also showed natural killer (NK)-like function. The cytolytic machinery was also present in those clones (both CD8 and CD4) recognizing the HLA-matched hepatocytes. All liver-derived T clones were able to produce high amounts of interferon (IFN)-gamma, as well as being capable of secreting IL2, following PHA stimulation.     

The response of gamma delta T cells to Plasmodium falciparum is dependent on activated CD4+ T cells and the recognition of MHC class I molecules.

Peripheral blood gamma delta T cells from non-exposed individuals respond to antigens of the malaria parasite, Plasmodium falciparum, in vitro. This response, largely caused by T cells bearing the V gamma 9+ chain of the T-cell receptor, is stimulated by components of the parasite expressed on the schizont stage and released at schizont rupture. The response of V gamma 9+ T cells to parasite components is inhibited by antibodies to major histocompatibility complex (MHC) class I and class II. However, the inhibition by anti-MHC class II antibodies can be overcome by the addition of interleukin-2 (IL-2) to the cultures, suggesting that gamma delta T cells themselves do not recognize MHC class II molecules but require an MHC class II-dependent response taking place in the culture. In contrast, the inhibition by anti-class I antibodies cannot be reversed by addition of IL-2. Since an accompanying CD4+ T-cell response occurred in peripheral blood mononuclear cells cultured with P falciparum antigens, it was considered that these cells provide the cytokines necessary for the subsequent activation and expansion of V gamma 9+ T cells recognizing components of the parasite and MHC class I molecules. This was confirmed by reconstituting the response of enriched gamma delta T cells to P falciparum schizont extract by addition of purified CD4+ T cells.