Immune CD4+ T cells specific for Theileria parva-infected lymphocytes recognize a 24-kilodalton protein.

Theileria parva is a protozoan parasite that infects and transforms bovine lymphocytes. Here we report the partial purification of a T. parva-specific protein from infected lymphocytes that is recognized by CD4+ parasite-specific T-cell clones derived from immune cattle. T. parva-infected lymphocytes were homogenized in Dulbecco's phosphate-buffered saline in the presence of protease inhibitors. The antigen was purified from a postmicrosomal supernatant by using a combination of DEAE-cellulose chromatography and hydroxylapatite column chromatography. After labelling with 125I, the antigen preparation was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to contain 8 to 10 proteins. This preparation was subjected to chromatography in phosphate-buffered saline on HPLC TSK-250/125 columns coupled in tandem. A radiolabelled protein of M(r) 24,000 correlated with antigenic activity.     

Cytotoxic CD4+ T cells specific for Francisella tularensis.

The specific cell-mediated cytotoxicity of tularaemia-immune human T lymphocytes were studied in vitro. Peripheral blood mononuclear cells (PBMC) of six tularaemia-vaccinated healthy subjects were stimulated with F. tularensis LVS whole cell antigen for 6 days and used as effector cells in a conventional 4-h 51Cr release cytotoxicity assay. The target cells were phagocyting autologous monocytes, which were pulsed with F. tularensis or PPD antigen. The specific lysis of the F. tularensis-pulsed cells (42.6% +/- 11.7) was significantly higher (P less than 0.05) than that of the PPD-pulsed ones (22.2% +/- 8.3) or unpulsed control cells (15.9% +/- 5.2). The cytotoxicity was associated with CD4+ F. tularensis-specific T cell clones (TLC), which killed 36.3% +/- 12.3 of the F. tularensis-pulsed targets but only 6.9% +/- 6.5 of the unpulsed control targets. Their lysing was inhibited by monoclonal anti-HLA-DR and anti-HLA-DQ antibodies, but not by CD15 (monocyte/macrophage) antibody. The functional role of CD4+ lymphocytes in tularaemia immunity is discussed.     

Interactions of CD4+ and CD8+ human T lymphocytes from malaria-unprimed donors with Plasmodium falciparum schizont stage.

During Plasmodium falciparum malaria, a wide spectrum of parasite-encoded blood-stage proteins is presented to the immune system of the host. To explore their multiple interactions with T cells from donors who have had no previous exposure to the parasite, whole schizont extract was used in vitro. Both CD4+ and CD8+ lymphocytes from all individuals tested were stimulated to proliferate. The responses were dependent on the presence of accessory cells and were only partially replaced by recombinant interleukin-1. Responses were inhibited by monoclonal antibodies to CD3, the alpha beta-chain T-cell receptor, or CD4 molecules but not to CD2. P. falciparum schizont extract-specific T-cell clones were generated and maintained by using sole stimulation by P. falciparum extract with autologous accessory cells or recombinant interleukin-2. Monoclonal antibodies to CD3 (or the alpha beta-chain T-cell receptor) blocked cloned T-cell responses to the schizont extract, and although the responses of the majority of the CD4+ or CD8+ T-cell clones were restricted by autologous accessory cells and inhibited by monoclonal antibodies to either CD4 or CD8, other clones responded to P. falciparum in the absence of accessory cells and were not regulated by the same monoclonal antibodies. The last category of clones consisted of autoreactive T cells. These data suggest that at the first contact with P. falciparum, requirements are met for significant T-cell stimulation.     

Cytotoxic human CD4(+) T cells

The induction of adaptive immune responses critically depends on helper signals provided by CD4(+) T cells. These signals not only license antigen presenting cells (APC) to activate na?ve CD8(+) T cells leading to the formation of vast numbers of cytotoxic T lymphocytes but also support the differentiation of B cells into immunoglobulin-secreting plasma cells. Next to these helper functions, a subpopulation of CD4(+) T cells can also directly function as effector cells by executing cytotoxicity in a peptide-specific and MHC class II-restricted manner. Cytotoxic CD4(+) T cells may function in combating pathogens but additionally their presence has been associated with autoimmune disease and vascular damage. On the contrary, the induction of cytotoxic CD4(+) T cells may be a future target for vaccine strategies.     

Natural amino acid polymorphisms of the circumsporozoite protein of Plasmodium falciparum abrogate specific human CD4+ T cell responsiveness

Sequence polymorphism has been reported for virtually all malaria antigens and, in the case of the circumsporozoite (CS) protein, this variation is in the form of point mutations concentrated primarily in several regions recognized by T cells. The factors responsible for the variation are unknown. We studied the T cell responses to all known variants in malaria-exposed Thais. Memory CD4⁺ T cells responded to variants of a polymorphic immunodominant region (denoted Th2R), and CD4⁺ T cell clones specific for one Thai Th2R variant were generated. There was minimal cross-reactivity to any of the naturally occurring variants, including the other Thai variant, and competition studies performed with the clones using analog peptides demonstrated that all the substitutions of the polymorphic residues modulate either the binding of the peptide to major histocompatibility complex (MHC) molecules or the recognition by the T cell receptor of the peptide-MHC complex. Our data suggest that CD4⁺ T cells may be able to select parasites expressing variant sequences and have implications for development of a CS-based vaccine.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Evidence for CD4+ T cell responses common to Plasmodium falciparum and recall antigens

In individual donors which have never been exposed to malaria parasites, the CD4+ T cell precursor frequencies for tetanus toxoid (TT) and Plasmodium falciparum responses are similar (range 1:850- 1:4800). Limiting dilution cultures set up in response to P. falciparum trophozoites can be re-stimulated with the same stage of the parasite or TT and respond with similar frequencies. A substantial overlap in the responses to different agents was confirmed in suicide selection experiments where cells responding to malaria parasite, TT or influenza virus antigens were deleted using the cell cycle inhibitor cytosine arabinoside (Ara-C). The responses of the remaining cells to P. falciparum were almost completely abrogated and only weak responses were observed to different recall antigens (0.2-21% of untreated control). Little or no effect was observed on the responses to superantigen or mitogen. Furthermore, in contrast to superantigen, the observed responses to TT and Plasmodium were polyclonal, the blastoid cells generated reacting with a range of anti-TCR Vbeta antibodies with little preferential usage.      

Cytotoxic T cell reactivity and HLA-B35 binding of the variant Plasmodium falciparum circumsporozoite protein CD8+ CTL epitope in naturally exposed Kenyan adults

In this study, we have investigated the extent of natural polymorphism in the CD8⁺ cytotoxic T lymphocyte (CTL) determinant (amino acids 368–390) of circumsporozoite (CS) protein of Plasmodium falciparum field isolates from a holoendemic region of Kenya, and determined how this variation affects the CTL reactivities in clinically immune adults and binding specificities to human histocompatibility leukocyte antigen (HLA)-B35. Among the eight variant sequences that were found in this region, four were new and not seen in parasites from other geographical regions. When synthetic peptides corresponding to the eight variants were used to test the presence of CTL response in different donors, a different spectrum of CTL reactivity to these variants was noticed. While CTL from some donors recognized the P1 sequence (the most prevalent type of sequence) but not P8 (another major variant), other donors showed a reverse pattern of reactivity. Although none of the donors was able to recognize all the variants, CTL responses to all the eight variant sequences were found in this population. An octamer peptide with P1 sequence KPKDELDY in this polymorphic determinant was known to bind HLA-B35. When we tested the effect of natural variation in this octamer sequence on HLA-B35 binding, it became evident that SP13 with D → N substitution retained its binding specificity to HLA-B35. On the other hand, the SP12 octamer sequence which had two substitutions did not bind HLA-B35. The most interesting finding was the observation that a D → G substitution at position 374 rescued the binding ability of SP14, which otherwise could not bind to this HLA molecule due to E → Q amino acid substitution at position 372. To our knowledge, this is the first demonstration showing that a natural polymorphism can rescue the binding specificity to an HLA-class I molecule that was lost due to another natural amino acid substitution. Altogether, these results demonstrate that natural polymorphism in the CS protein affects both the CTL reactivity and the ability to bind to HLA-B35.     

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.     

Regulated secretion from CD4+ T cells

The regulated secretion of cellular proteins is central to the correct function of many cell types, including immune cells. Lymphocyte control of the storage, transport and exocytosis of immunomodulatory molecules is a highly specialised task triggered by T cell receptor engagement. The regulated secretory pathway in CD8+ T and NK cells has been the focus of much research, and recent advances have provided insight into the molecular mechanisms governing secretory organelle biogenesis, trafficking and killing. By contrast, regulated secretory pathways in CD4+ T cells have not been studied extensively. Aside from their physiological function in normal T cells, components of CD4+ T cell secretory pathways might be implicated in the assembly of HIV-1. Here, we review findings that shed light on CD4+ T cell secretion in health and disease.     

Human CD8+ T cell clone regulates autologous CD4+ myelin basic protein specific T cells.

Normal human CD8+ T cell clones were co-isolated from the same culture wells as CD4+ T effector cell clones specific for myelin basic protein (MBP). Microcultures from which the CD8+ clones were isolated initially proliferated weakly to whole MBP and to an MBP peptide spanning residues 90-170. This pattern of response was similar to strongly proliferating wells that yielded CD4+ T cell clones specific for the 90-170 peptide. After repeated stimulation, however, no response to MBP or MBP 90-170 was detected, even though the number of cells increased after stimulation. Phenotyping and TCR analyses revealed the presence of two CD8+, CD4-, IL-2R+ T cell isolates that expressed a single V beta gene (V beta 17) that differed from the CD4+ isolates that uniformly expressed V beta 14. One of these CD8+ clones (C9) inhibited the antigen-driven proliferation of an autologous MBP 90-170 reactive clone but not an autologous clone specific for Herpes simplex virus (HSV), without affecting MHC non-restricted mitogen responses of the same clones. Moreover, C9 did not inhibit heterologous CD4+ T cell clones specific for MBP 1-38 or 90-170. A culture supernatant of the CD8+ clone showed the same pattern but lower levels of inhibition. C9 had mild cytolytic activity when incubated at high ratios with an autologous MBP-specific CD4+ clone. Lysis was blocked completely by anti-MHC class I antibodies, but not by anti-MHC II antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin 10-mediated immunosuppression by a variant CD4 T cell epitope of Plasmodium falciparum.

The immunodominant CD4 T cell epitope region, Th2R, of the circumsporozoite protein of Plasmodium falciparum is highly polymorphic. Such variation might be utilized by the parasite to escape from or interfere with CD4 T cell effector functions. Here, we show that costimulation with naturally occurring altered peptide ligands (APL) can induce a rapid change from IFNgamma production to the immunosuppressive mediator interleukin 10 (IL-10). This mechanism may contribute to the low levels of T cell responses observed to this pathogen in malaria-endemic areas.     

Specific CD8+ T cells recognize human herpesvirus 6B

The importance of human herpesvirus 6 (HHV‐6) species as human pathogens is increasingly appreciated. However, we do not understand how infection is controlled in healthy virus carriers, and why control fails in patients with disease. Other persistent viruses are under continuous surveillance by antigen‐specific T cells, and specific T‐cell repertoires have been well characterized for some of them. In contrast, knowledge on HHV‐6‐specific T‐cell responses is limited, and missing for CD8⁺ T cells. Here we identify CD8⁺ T‐cell responses to HHV‐6B, the most widespread HHV‐6 species, in healthy virus carriers. HHV‐6B‐specific CD8⁺ T‐cell lines and clones recognized HLA‐A2‐restricted peptides from the viral structural proteins U54 and U11, and displayed various antigen‐specific antiviral effector functions. These CD8⁺ T cells specifically recognized HHV‐6B‐infected primary CD4⁺ T cells in an HLA‐restricted manner, produced antiviral cytokines, and killed infected cells, whereas HHV‐6A‐infected cells were not recognized. Thus, HHV‐6B‐specific CD8⁺ T cells are likely to contribute to control of infection, overcoming the immunomodulatory effects exerted by the virus. Potentially, HHV‐6‐associated disease could be addressed by active or passive immunotherapy that reconstitutes virus‐specific CD8⁺ T‐cell responses.     

CD4+CD25+-regulatory T cells from mouse to man

Immunological tolerance is one of the fundamental concepts of the immune system. During the past decade, CD4+CD25+-regulatory T cells have emerged as key players in the development of tolerance to autoantigens as well as to foreign antigens. Still many questions remain illusive regarding the basic properties of CD4+CD25+-regulatory T cells. This review aims to recapitulate some of the current understandings about the phenotype, function and clinical relevance of murine and human CD4+CD25+-regulatory T cells.     

Hodgkin's reed-sternberg cell line (KM-H2) promotes a bidirectional differentiation of CD4+CD25+Foxp3+ T cells and CD4+ cytotoxic T lymphocytes from CD4+ naive T cells

A recent report revealed that a large population of Hodgkin's lymphoma-infiltrating lymphocytes (HLILs) consisted of regulatory T cells. In this study, we cocultured CD4+ naive T cells with KM-H2, which was established as a Hodgkin's Reed-Sternberg cell line, to clarify their ability to induce CD25+ Forkhead box P3+ (Foxp3+) T cells. The characteristic analyses of T cells cocultured with KM-H2 revealed the presence of CD4+CD25+ T cells. They expressed CTLA-4, glucocorticoid-induced TNFR family-related gene, and Foxp3 and could produce large amounts of IL-10. Conversely, KM-H2 also generated CD4+ CTLs, which expressed Granzyme B and T cell intracellular antigen-1 in addition to Foxp3+ T cells. They exhibit a strong cytotoxic effect against the parental KM-H2. In conclusion, KM-H2 promotes a bidirectional differentiation of CD4+ naive T cells toward Foxp3+ T cells and CD4+ CTLs. In addition to KM-H2, several cell lines that exhibit the APC function were able to generate Foxp3+ T cells and CD4+ CTLs. Conversely, the APC nonfunctioning cell lines examined did not induce both types of cells. Our findings suggest that the APC function of tumor cells is essential for the differentiation of CD4+ naive T cells into CD25+Foxp3+ T cells and CD4+ CTLs and at least partly explains the predominance of CD25+Foxp3+ T cells in HLILs and their contribution to a better prognosis. Therefore, in APC-functioning tumors, including classical Hodgkin lymphomas, which generate Foxp3+ T cells and CD4+ CTLs, these T cell repertories play a beneficial role synergistically in disease stability.     

CTLA-4 x Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells

CTLA-4 x Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4 x Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4+CD25- T cells into Foxp3+ regulatory T (T(reg)) cells, as well as to expand their numbers. The CD4+CD25+Foxp3+ T(reg) generated by CTLA-4 x Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4 x Ig increases the percentage of CD4+CD25(hi)Foxp3+ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4+CD25- T cells into T(reg) cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4 x Ig-driven T(reg) induction may be predicated upon active CTLA-4 x Ig to B7-2 signaling within APC, which elicits from them T(reg)-inducing potential. These findings extend CTLA-4 x Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.     

CD4+CD25+ T cells as immunoregulatory T cells in vitro

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4(+)25(+) T cells. Following TCR ligation, DO.11.10 CD4(+)25(+) T cells suppress the activation of OT-1 CD8(+)25(-) T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4(+)25(+) T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4(+)25(+) T cell regulation. CTLA-4 blockade failed to abrogate suppression by CD4(+)25(+) T cells in mixing experiments. Although CD4(+)25(+) T cells failed to respond following cross-linking of TCR, they could be induced to proliferate following the addition of exogenous IL-2, allowing the generation of a T cell line from CD4(+)25(+) T cells. After the first in vitro restimulation, CD4(+)25(+) T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.     

Human CD4+CD25+ regulatory T cells

In this report, we review studies of human CD4+CD25+ regulatory T cells (T-reg). Although lagging a few years behind the discovery of these cells in the mouse, the equivalent population of CD4+CD25+ regulatory T cells has also been isolated from human peripheral blood, thymus, lymph nodes and cord blood. In general, the characteristics of this T cell subset are strikingly similar between mouse and man. In the recent explosion of research reports on human CD4+CD25+ cells, although the majority of the characteristics ascribed to these cells appear to be consistent, contrasting results have been found primarily in regards to potential involvement of TGFbeta and production of IL-10. One explanation for this variability may reside in the fact that markedly different techniques are used to isolate human CD4+CD25+ T-reg cells and thus may result in the comparison of T-reg populations that differ in cellular composition and/or activation state. Another potential explanation for differences in human T-reg function may rest on the extreme variability of the culture conditions and TCR stimuli that have been used to test the functional properties of these cells in vitro. The strength of the TCR signal provided to the culture greatly affects the functional outcome of the co-culture and can result in the difference between suppression and full activation. Surprisingly, it appears that stronger stimulation has a greater and more rapid effect on the T-resp cell than on the T-reg cell as it causes T-resp cells to quickly become resistant to suppression. Thus, the details of in vitro culture conditions may at least partially account for disparate findings in regard to the functional characterization of human CD4+CD25+ cells. Here we review the evidence regarding the identification of human CD4+CD25+ regulatory T cells and their possible mechanism(s) of function.