Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Peripheral CD8+CD25+ T lymphocytes from MHC class II-deficient mice exhibit regulatory activity

We characterized CD8(+) T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4(+)CD25(+) T cells, CD8(+)CD25(+) T cells appear late in the periphery during ontogeny. Peripheral CD8(+)CD25(+) T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4(+)CD25(+) T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25(-) T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4(+)CD25(+) T cells. However, unlike CD4(+)CD25(+) T cells CD8(+)CD25(+) T cells from MHC class II-deficient mice strongly proliferate and produce IFN-gamma in vitro in response to stimulation in the absence of exogenous IL-2.     

Defective clonogenic potential of CD8+ T lymphocytes in patients with AIDS. Expansion in vivo of a nonclonogenic CD3+CD8+DR+CD25- T cell population

This study examines the potential mechanism(s) responsible for the defective clonability of CD8+ T lymphocytes in patients with AIDS. By the combined use of one- and two-color fluorescence cytofluorometry we have shown an increase in the number of circulating DR+ cells due to the expression of DR on a relatively large proportion of T lymphocytes (one-third of CD3+ cells), the majority of them belonging to the CD8+ subset. In addition, the majority of CD8+DR+ cells in AIDS patients did not express CD25 Ag (the receptor for IL-2), a surface marker generally expressed on normal activated T lymphocytes. Sorted CD8+DR+ and CD8+DR- cell populations were analyzed comparatively for their ability to proliferate in response to different stimuli, including anti-CD3, anti-CD2, alone or in combination with anti-CD28 mAb and mitogens such as PHA, alone or in combination with PMA. We have demonstrated that CD8+DR+ cells were severely defective in their proliferative response to triggering via these major pathways of T cell activation even when an exogenous source of IL-2 or IL-4 was added to the microcultures 24 h after initiating the cultures. In contrast, CD8+DR- cells showed a significant proliferation in response to the different stimuli and the proliferative response was strongly enhanced by the addition of IL-2 or IL-4. At the end of the stimulation period CD8+DR+ and CD8+DR- proliferating populations were analyzed for CD25 Ag expression. Only 1 to 10% of CD8+DR+ cells expressed CD25 antigen compared with 40 to 50% of CD8+DR- cells. The proliferative defect of CD8+DR+ cells was further confirmed in experiments performed at the clonal level. The analysis of the frequency of proliferating T lymphocyte-precursors in both CD8+DR+ and CD8+DR- subsets showed that the defective clonogenic potential of CD8+ cells in AIDS patients could be in large part ascribed to CD8+DR+ cells. Five percent of CD8+DR+ cells showed a clonogenic potential compared to the 25% of CD8+DR- cells. Finally, we analyzed the surface expression of VLA-2 Ag, a marker of a chronic state of T cell activation, on circulating T lymphocytes. We have shown that a large proportion of CD3+DR+CD25- cells (50 to 80% in the different patients with AIDS analyzed) expressed VLA-2 Ag.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential in vitro activation of CD8-CD4+ and CD4-CD8+ T lymphocytes by combinations of anti-CD2 and anti-CD3 antibodies

Purified peripheral blood T lymphocytes and the CD8-CD4+ and CD4-CD8+ T cell subsets, exhaustively depleted of APC have been studied for their capacity to respond to mAb directed against the CD3-Ti Ag-specific TCR complex and against the CD2 SRBCR. It is demonstrated that high affinity IL-2R can be readily induced by either anti-CD3 and/or anti-CD2 stimulation. However, IL-2 production can be observed only in the CD4+CD8- T cell subset. These results clearly contrast data obtained with purified CD4-CD8+ T cells, which are able to proliferate when the CD3-Ti complex is activated in the presence of APC. The data presented in the present study demonstrate that a simplified model for T cell activation and clonal expansion of the two major T cell subsets involve only the CD3-Ti complex and the CD2 Ag. Under conditions where the activation signals for the T cells are restricted only to the activation of CD3-Ti and CD2, the CD4+CD8- T cells respond with IL-2 production and expression of high affinity IL-2R, whereas the CD4-CD8+ T cell subset depends on exogenous IL-2 provided by the CD4+CD8- cells. These data do not, however, exclude an involvement of other cell-surface signals for regulation and control of T cell activation and T cell effector functions.     

T lymphocytes that express CD4 and the alpha beta-T cell receptor but lack Thy-1. Preferential localization in Peyer's patches

Thy-1- T cells expressing CD4 and the alpha beta-TCR have been identified in murine lymphoid tissues. These cells are particularly prevalent in Peyer's patches (PP), representing 17 +/- 3% of PP CD4 T cells, whereas they are much less prevalent in spleen, lymph nodes, lamina propria, or peritoneum. Phenotypic studies of fresh-isolated PP T cells demonstrate that all PP CD4 T cells (both Thy-1- and Thy-1+) express CD3, alpha beta-TCR, and CD5 (Lyt-1), whereas none coexpress CD8 (Lyt-2). Thy-1- and Thy-1+ CD4 T cell lines generated from PP also coexpress CD3 and alpha beta-TCR, but are heterogeneous in expression of CD5 and again do not coexpress CD8. Further studies revealed that Thy-1- CD4+ T cells were not present in nude mice. Short term stimulation of Thy-1+ CD4+ PP T cells with anti-CD3 resulted in loss of Thy-1 in a substantial fraction of these cells. Functional studies of Thy-1- and Thy-1+ CD4+ PP T cells indicate that fresh-isolated Thy-1- CD4+ cells do not proliferate in response to insoluble anti-CD3 but do proliferate when stimulated with soluble anti-CD3 in the presence of feeder cells. In contrast, Thy-1+ CD4+ cells proliferate well to both stimuli. However, Thy-1- CD4+ PP T cells adapted to in vitro culture exhibit vigorous proliferative responses when stimulated with either form of anti-CD3. Evaluation of lymphokine secretion by fresh-isolated Thy-1- and Thy-1+ CD4+ PP T cells revealed that both make substantial amounts of IL-2; however, Thy-1- T cells made less IL-4 than their Thy-1+ counterparts. Neither population made IL-5 or IFN-gamma. Similarly, Thy-1- and Thy-1+ CD4 T cell lines made similar amounts of IL-2; again Thy-1- T cells made less IL-4; and in this case Thy-1- T cells made IL-5 albeit significantly less than the Thy-1+ cells. Finally, immunohistochemical studies suggested that many of the CD4+ T cells in PP germinal centers were Thy-1-, indicating that Thy-1- and Thy-1+ CD4 T cells differ in their distribution within the PP. These studies thus define a phenotypically and functionally distinct T cell population which is most prevalent in murine Peyer's patches.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.     

In vivo functional clonal deletion of recipient CD4+ T helper precursor cells that can recognize class II MHC on injected donor lymphoid cells.

Intravenous injection of semiallogeneic (C57BL/6XDBA/2)F1 lymphocytes into adult C57BL/6 recipient mice not only, as previously reported, reduces the recipients' cytotoxic T lymphocyte response in a subsequent in vitro mixed lymphocyte reaction against the injected cell type, but also reduces Th cell function in the same MLR. Thus lymphoid cells derived from the injected mice were greatly reduced in their ability to proliferate and to produce IL-2 in response to (C57BL/6XDBA/2)F1 stimulator cells in vitro, whereas third party responses were unaffected. This appears to be due to a reduction in the precursor frequency of IL-2-producing T lymphocytes specific for the injected cells as measured by limiting dilution analysis. Similar donor-specific reduction in the frequency of precursors of IL-2-producing cells was seen after i.v. injection of A.TL lymphocytes into A.TH recipients (differing at class II determinants I-A and I-E, but identical at K and D). Here there also appeared to be a functional clonal deletion of precursors of IL-2-producing Th cells, shown directly to be class II MHC reactive and CD4+. There is strong evidence that the reduction of class I-specific cytotoxic responses in the injected mice is a manifestation of donor cells that function as veto cells, i.e., that function as deletional APC that inactivate class I-reactive CTL precursors that recognize them. Our data in this study show that class II-specific Th responses are similarly reduced in the injected mice and suggest that CD4+ class II-reactive precursors of Th cells may be functionally inactivated in vivo by donor cells via a veto-like mechanism.     

Murine and human IL-7 activate STAT5 and induce proliferation of normal human pro-B cells

The role of IL-7 in lymphoid development and T cell homeostasis has been extensively documented. However, the role of IL-7 in human B cell development remains unclear. We used a xenogeneic human cord blood stem cell/murine stromal cell culture to study the development of CD19+ B-lineage cells expressing the IL-7R. CD34+ cord blood stem cells were cultured on the MS-5 murine stromal cell line supplemented with human G-CSF and stem cell factor. Following an initial expansion of myeloid/monocytoid cells within the initial 2 wk, CD19+/pre-BCR- pro-B cells emerged, of which 25-50% expressed the IL-7R. FACS-purified CD19+/IL-7R+ cells were larger and, when replated on MS-5, underwent a dose-dependent proliferative response to exogenous human IL-7 (0.01-10.0 ng/ml). Furthermore, STAT5 phosphorylation was induced by the same concentrations of human IL-7. CD19+/IL-7R- cells were smaller and did not proliferate on MS-5 after stimulation with IL-7. In a search for cytokines that promote human B cell development in the cord blood stem cell/MS-5 culture, we made the unexpected finding that murine IL-7 plays a role. Murine IL-7 was detected in MS-5 supernatants by ELISA, recombinant murine IL-7 induced STAT5 phosphorylation in CD19+/IL-7R+ pro-B cells and human B-lineage acute lymphoblastic leukemias, and neutralizing anti-murine IL-7 inhibited development of CD19+ cells in the cord blood stem cell/MS-5 culture. Our results support a model wherein IL-7 transduces a replicative signal to normal human B-lineage cells that is complemented by additional stromal cell-derived signals essential for normal human B cell development.     

Frequency analysis of class I MHC-reactive Lyt-2+ and class II MHC-reactive L3T4+ IL 2-secreting T lymphocytes

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T responder cells were not the numerical superposition of the two individual T cell subsets (Lyt-2+ + L3T4+); the latter finding is likely to reflect regulatory influences of Lyt-2+ T cells on IL 2-secreting L3T4+ T cells.     

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas

Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63+/-8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32+/-6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Human thymocytes do not respond to interleukin-2 after removal of mature "bright" CD5 positive cells

Interleukin-2 receptors (IL-2R) are expressed on minor populations of immature and mature human thymocytes. These studies were designed to determine if immature T cells could respond to the mitogen phytohemagglutinin (PHA-P) plus IL-2 in vitro by increasing the expression of IL-2R and by proliferation. Using monoclonal antibodies to CD5 and magnetic immunobeads we were able to remove all mature, "bright" CD5+ cells from nylon wool-purified thymocytes and to obtain less mature cells which consisted almost completely of cells with the CD4+CD8+ phenotype. These immature cells were mostly "dim" CD5+ and less than 5% CD5- and a small percentage expressed the IL-2R. After culture in serum-free medium with PHA-P, these cells showed only a slight increase in the percentage of IL-2R+ cells and the addition of IL-2 did not increase the percentage of IL-2R+ cells and no proliferation was observed. Unseparated, nylon wool-purified thymocytes contained 14% bright CD5+ cells. These bright CD5+ cells had a mature phenotype of CD4+CD8- (52%) and CD4-CD8+ (27%) cells. A small percentage of these cells were IL-2R+. These bright CD5+IL-2R+ cells were predominantly mature CD4+CD8- cells as measured by three-color flow cytometry. After culture with PHA-P and IL-2, the percentage of IL-2R+ cells increased and they were now found not only on CD4+CD8- but also on CD4-CD8+ and on CD4+CD8+ cells. IL-2 plus PHA-P increased proliferation of these cells as compared to those cultured in medium with PHA-P without IL-2. Thus, we show that human immature thymocytes in contrast to mature thymocytes are not responsive to IL-2 as measured by a lack of IL-2R expression and proliferation. These data indicate that mature thymocytes can express a functional high affinity receptor for IL-2 and suggest that immature thymocytes may not possess a (functional) p75 chain of the IL-2R.     

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.     

Gamma-irradiated peripheral blood mononuclear cells can express LAK activity

Peripheral blood mononuclear cells (PBMC) irradiated with high dose gamma-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such gamma-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, gamma-irradiated LAK cells are significantly increased by the addition of the mitogen, phytohemagglutinin (PHA). Purification of the gamma-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3+ cells also expressed moderate LAK activity. The ability of gamma-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 + PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidine uptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such gamma-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.     

Monocyte tissue factor induction by activation of beta 2-glycoprotein-I-specific T lymphocytes is associated with thrombosis and fetal loss in patients with antiphospholipid antibodies

Antiphospholipid (aPL) syndrome (APS) is characterized by thromboembolic events, thrombocytopenia, or recurrent miscarriage associated with aPL Abs with specificity for beta2-glycoprotein-I (beta2GPI). We recently reported that at least 44% of patients with the APS possess circulating type 1 (Th1) CD4+ T cells that proliferate and secrete IFN-gamma when stimulated with beta2GPI in vitro. In this study, we show that stimulation of PBMCs from 20 APS patients with beta2GPI induced substantial monocyte tissue factor (TF) (80 +/- 11 TF stimulation index (TF-SI)), whereas no induction was observed using PBMCs from 13 patients with aPL Abs without APS (6 +/- 1 TF-SI) or 7 normal and 7 autoimmune controls (5 +/- 1 and 3 +/- 1 TF-SI, respectively) (p < 0.0001). TF induction on monocytes by beta2GPI was dose dependent and required CD4+ T lymphocytes and class II MHC molecules. Because monocyte TF induction by beta2GPI was observed in all patients with APS, but not in any patient with aPL Abs without APS, this response is a potentially useful predictor for APS in patients with aPL Abs, as well as providing mechanistic insight into thrombosis and fetal loss in these patients.     

Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.     

Defective activation of protein kinase C and Ras-ERK pathways limits IL-2 production and proliferation by CD4+CD25+ regulatory T cells

Naturally occurring CD4+CD25+ regulatory T cells (Tregs), which play an important role in the maintenance of self-tolerance, proliferate poorly and fail to produce IL-2 following stimulation in vitro with peptide-pulsed or anti-CD3-treated APCs. When TCR proximal and distal signaling events were examined in Tregs, we observed impairments in the amplitude and duration of tyrosine phosphorylation when compared with the response of CD4+CD25- T cells. Defects were also seen in the activity of phospholipase C-gamma and in signals downstream of this enzyme including calcium mobilization, NFAT, NF-kappaB, and Ras-ERK-AP-1 activation. Enhanced stimulation of diacylglycerol-dependent pathways by inhibition of diacylglycerol metabolism could overcome the "anergic state" and support the ability of Tregs to up-regulate CD69, produce IL-2, and proliferate. Our results demonstrate that Tregs maintain their hyporesponsive state by suppressing the induction and propagation of TCR-initiated signals to control the accumulation of second messengers necessary for IL-2 production and proliferation.     

CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.     

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.