TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.     

Analysis of tumor-infiltrating lymphocytes in cutaneous squamous cell carcinoma

OBJECTIVE: To characterize tumor-infiltrating lymphocytes (TILs) within lesions of cutaneous squamous cell carcinoma (SCC) and related disorders. DESIGN: Case series with 1- and 2-color immunohistologic, molecular biological analysis of T-cell clonality and in vitro cytotoxicity assays. SETTING: Academic medical center. PATIENTS: Twenty-one patients, including 6 with actinic keratoses, 4 with SCC in situ, and 11 with invasive SCC. RESULTS: CD8+ TILs were present within lesions of cutaneous SCC and AK. These cells constituted a variable minority of the total T-cell infiltrate, and many expressed a phenotype consistent with major histocompatibility complex-restricted cytotoxic T lymphocytes: CD3+, TIA1+, CD16-, CD56-, CD57-. They also expressed HLA-DR, suggesting their activation in vivo. Virtually all T cells were T-cell receptor (TCR)-beta + delta, indicating that they expressed the TCR-alpha beta protein heterodimer. Molecular biological analysis of TCR-gamma gene rearrangements by the polymerase chain reaction and denaturing gradient gel electrophoresis technique indicated that the TILs were polyclonal. Functional studies suggested that TILs derived from SCC lesions were cytotoxic for autologous tumor cell targets. CONCLUSION: Tumor-infiltrating lymphocytes within cutaneous SCC lesions contain a subpopulation of polyclonal, major histocompatibility complex-restricted cytotoxic T lymphocytes expressing the TCR-alpha beta heterodimer.     

Bone marrow-derived dendritic epidermal T cells express T cell receptor-alpha beta/CD3 and CD8. Evidence for their extrathymic maturation

The majority of murine Thy-1+ dendritic epidermal T cells (DETC) express virtually identical TCR encoded by V gamma 5 and V delta 1 genes and are derived from early fetal thymic precursors. However, not consistent with this notion is an early finding that DETC arise continuously from bone marrow (BM) precursors by a thymus-independent mechanism. To address this issue, donor-type DETC were characterized in lethally irradiated mice that were reconstituted by Thy-1-disparate BM cells with or without a thymus. The BM-derived DETC, unlike their normal TCR-gamma delta counterparts, were found to express the TCR-alpha beta/CD3 complex and CD8, and their migration to the epidermis dermis occurred independently of the thymus. The numbers of the BM-derived DETC increased with time and reached a plateau 6 mo after BM transfer, at which time the TCR-alpha beta/CD3 complex was expressed on a small fraction of the DETC in athymic BM chimeras. Although no further increase in the number was observed at later times, at 1 yr after transfer most of the BM-derived DETC came to express the TCR-alpha beta/CD3 complex in the absence of thymic influence. By contrast, most of BM-derived T cells in other lymphoid organs from athymic BM chimeras still failed to express the TCR-alpha beta/CD3 complex even at 1 yr after transfer. These results suggest that extrathymic differentiation of BM-derived DETC could occur with the epidermal microenvironment.     

SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model

In contrast to conventional T cells, natural killer (NK) 1.1+ T cell receptor (TCR)-alpha/beta+ (NK1+T) cells, NK cells, and intestinal intraepithelial lymphocytes (IELs) bearing CD8-alpha/alpha chains constitutively express the interleukin (IL)-2 receptor (R)beta/15Rbeta chain. Recent studies have indicated that IL-2Rbeta/15Rbeta chain is required for the development of these lymphocyte subsets, outlining the importance of IL-15. In this study, we investigated the development of these lymphocyte subsets in interferon regulatory factor 1-deficient (IRF-1-/-) mice. Surprisingly, all of these lymphocyte subsets were severely reduced in IRF-1-/- mice. Within CD8-alpha/alpha+ intestinal IEL subset, TCR-gamma/delta+ cells and TCR-alpha/beta+ cells were equally affected by IRF gene disruption. In contrast to intestinal TCR-gamma/delta+ cells, thymic TCR-gamma/delta+ cells developed normally in IRF-1-/- mice. Northern blot analysis further revealed that the induction of IL-15 messenger RNA was impaired in IRF-1-/- bone marrow cells, and the recovery of these lymphocyte subsets was observed when IRF-1-/- cells were cultured with IL-15 in vitro. These data indicate that IRF-1 regulates IL-15 gene expression, which may control the development of NK1+T cells, NK cells, and CD8-alpha/alpha+ IELs.     

Study of activation of murine T cells with bacterial superantigens. In vitro induction of enhanced responses in CD4+ T cells and of anergy in CD8+ T cells

Primary and secondary responses of murine CD4+ T cells and CD8+ T cells upon stimulation with staphylococcal enterotoxin E (SEE) bearing superantigenic properties were examined. Both isolated C57BL/6 splenic CD4+ T cells and CD8+ T cells proliferated and produced IL-2 and IFN-gamma upon stimulation with SEE in substantial levels. The amounts of IL-2 were greater in CD4+ T cells and those of IFN-gamma were somewhat greater in CD8+ T cells. SEE-induced CD4+ T lymphoblasts, larger parts of which bore the V beta 11 element in their TCR, proliferated, produced IL-2 and IFN-gamma, and showed toxin-dependent cytotoxicity in substantial levels upon restimulation with SEE. By contrast, SEE-induced CD8+ T lymphoblasts, the larger part of which bore the V beta 11 element, did not show the first two of the three responses at all upon restimulation with SEE, whereas these cells showed greater cytotoxicity. The CD8+ T lymphoblasts did not suppress the reactivity of the CD4+ T lymphoblasts. Both SEE-induced CD4+ T lymphoblasts and CD8+ T lymphoblasts proliferated and produced IL-2 and IFN-gamma in comparable levels upon stimulation with rIL-2 or mAb to CD3 or V beta 11.     

Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.     

Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.     

In situ non-radioactive detection of nuclear factors in paraffin sections by Southwestern histochemistry

CD8+ T cells often differentiate into highly cytotoxic cells, secreting a Th1-like or type 1 cytokine pattern characterized by the production of IFN-gamma. However, cytotoxic, and in some reports, noncytotoxic, type 2 cells that secrete IL-4, IL-5, or IL-10 instead of IFN-gamma, can be generated when CD8+ T cells are primed in the presence of IL-4. Here, we show that IL-4 can also generate typical CD8 type 1 responses. Indeed, while presence of TGF-beta biases the development of CD8 T cells that, then, produce little cytolytic activity and IFN-gamma, addition of IL-4 results in the recovery of cytotoxicity and IFN-gamma production. The cooperative effects of TGF-beta and IL-4 imply dual functions, not only for IL-4, but also for TGF-beta. Indeed, depending on the presence or absence of IL-4, TGF-beta either inhibits or induces the generation of type 1 CD8+ T cells. Physiologically, the ratio of local IL-4/TGF-beta concentration may therefore be a critical element in determining the outcome of T cell responses to pathogen and autoantigens. It allows CD8 T cells to switch from an immunotolerant state in the presence of only TGF-beta or IL-4, to an immunocompetent proinflammatory type 1 state in the absence or presence of both cytokines.     

Low dose TGF-beta attenuates IL-12 responsiveness in murine Th cells

Expression of IL-12Rs is one important checkpoint for Th1 development. BALB/c DO11.10 CD4+ T cells stimulated by Ag in neutral conditions lose expression of the IL-12R beta 2 subunit and become unresponsive to IL-12. In contrast, B10.D2 or F1 (BALB/c x B10.D2) DO11.10 CD4+ T cells maintain IL-12R beta 2 expression when stimulated similarly. Here we show that the loss of IL-12 responsiveness by BALB/c T cells involves the action of endogenous TGF-beta. BALB/c T cells stimulated in the presence of anti-TGF-beta specifically maintain IL-12 responsiveness, express IL-12R beta 2 mRNA, and can stimulate nitric oxide production in peritoneal exudate cells. Low concentrations of TGF-beta added exogenously during primary activation of B10.D2 or F1 T cells significantly inhibit their development of IL-12 responsiveness. These effects of anti-TGF-beta are dependent on endogenous IFN-gamma and are inhibited by exogenously added IL-4. Thus, at least one effect of TGF-beta on Th1/Th2 development may be the attenuation of IL-12R beta 2 expression.     

Increased in vitro induced CD4+ and CD8+ T cell IFN-gamma and CD4+ T cell IL-10 production in stable relapsing multiple sclerosis

Multiple sclerosis (MS) is presumed to be a T-cell mediated chronic inflammatory disease of the central nervous system. Investigators previously demonstrated increased IFN-gamma (pro-inflammatory) and IL-10 (counterregulatory anti-inflammatory) in MS. The balance of pro-inflammatory and counterregulatory anti-inflammatory cytokines may be important in the stabilization of disease activity. Purified CD4+ and CD8+ T cells from patients with clinically definite, stable relapsing MS (RRMS) were stimulated by anti-CD3 mAb or Con A for 48 hours and cytokine supernatants analysed for production of IL-2, IL-6, IFN-gamma, TNF-alpha (potential pro-inflammatory) and IL-4, IL-10, and TGF-beta (potential counterregulatory anti-inflammatory). Con A activated CD4+ and CD8+ T cell proinflammatory cytokine IL-2 secretion, CD4+ T cell IL-6 secretion, CD4+ and CD8+ T cell TNF-alpha secretion and CD8+ T cell IFN-gamma secretion was decreased significantly in RRMS subjects compared to controls. CD3 activated CD4+ and CD8+ T cell IL-6 secretion and CD4+ T cell TNF-alpha secretion was significantly decreased in MS subjects compared to controls. In contrast, there was increased CD3-induced IFN-gamma in both CD4+ and CD8+ T cells and counterregulatory anti-inflammatory CD3-induced IL-10 secretion in CD4+ T cells in RRMS compared to controls. These data suggest that an equilibrium of a pro-inflammatory (IFN-gamma) and a counterregulatory anti-inflammatory (IL-10) cytokine may define stable clinically definite early RRMS.     

CD4+ type 1 and CD8+ type 2 T cell subsets in human leishmaniasis have distinct T cell receptor repertoires

The mechanism of protective immunity and immunologic resistance against intracellular pathogens is believed to involve the activation of Ag-specific T cells. The T cells involved in protection/resistance to Leishmania can be studied using localized American cutaneous leishmaniasis (LCL) as a model, because the disease is often self-healing. Our study was undertaken to identify specific T cell populations that had accumulated in LCL lesions on the basis of TCR V beta gene usage. RNA was derived from skin lesions and blood of eight LCL patients, as well as from purified CD4+ and CD8+ subsets from the lesions and blood of three patients. After synthesis of cDNA, V beta gene usage was assessed by polymerase chain reaction. In all eight patients, several V beta gene families were overrepresented in lesions compared to blood. More importantly, the TCR V beta repertoires of both lesional CD4+ and CD8+ subsets were skewed compared to the repertoire of the respective subsets in the blood of the same donor. The overrepresented V beta s in the CD4+ and CD8+ subsets from lesions were in most instances disparate, particularly with the V beta 6 TCR skewed in the lesional CD8+ subset. Not only were the TCR repertoires of the overrepresented V beta in the lesional CD4+ and CD8+ subsets generally distinct, but the cytokine mRNA expressed by these subsets were also discrete. Strikingly, the CD4+ subset was characterized by IFN-gamma mRNA expression and the CD8+ subset by IL-4 and IL-10 mRNA expression. These data indicate that the pathogenesis of human leishmaniasis may be explained by the balance of CD4+ type 1 and CD8+ type 2 T cells, which probably recognize distinct sets of Ag.     

CD8+ myelin peptide-specific T cells can chemoattract CD4+ myelin peptide-specific T cells: importance of IFN-inducible protein 10

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+ CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+ CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1alpha and -1beta, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+ CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.     

The role of B7-1 and LFA-3 in costimulation of CD8+ T cells

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.     

Stimulation of basal and L-DOPA-induced motor activity by glutamate antagonists in animal models of Parkinson''s disease

Peripheral T-cell antigen receptor V beta (TCRV beta) repertoire is influenced by clonal deletion both in the thymus and periphery. Developing thymocytes expressing certain TCRV beta are deleted by endogenous superantigens presented on major histocompatibility complex (MHC) molecules in the thymus. Likewise, mature T cells bearing particular TCRV beta chains can be clonally deleted by superantigens in the periphery. The efficiency with which T cells expressing particular V beta subunits are deleted differs depending upon which coreceptor is expressed. Indeed, while deletion of V beta 11+ splenic T cells in CBA/J (Mls-1, a I-E, + MTV 9+) mice is quite efficient for CD4+ spleen T cells, it is much less efficient for CD8+ splenic T cells. If the difference in the efficiency of deletion is due solely to the coreceptor expressed, then a transgene encoding CD4 should increase the efficiency with which CD8+ cells are deleted. To address this question, we have produced CD4 transgenic (TG) mice that express physiologic levels of CD4 on all thymocytes and peripheral CD8 T cells. CD4 molecules expressed on CD8+ splenic T cells were associated with P56lck tyrosine kinase, and were functional as evidenced by their ability to facilitate class II alloreactivity. Furthermore, we found that ectopic expression of TG CD4 molecules on CD8+ cells was able to affect the efficiency of deletion in response to superantigen stimulation. In particular, deletion of TCRV beta 11+ T cells was much less efficient for CD8+ than for CD4+ T-cell subpopulations in (CBA/J x B6) F1 mice. However, expression of the CD4 transgene on CD8+ splenic T cells from these mice increased the efficiency of deletion in the CD8+ V beta 11 T cells. Interestingly, this effect was not observed in a mature CD8+ thymocyte subpopulation. The results in this report demonstrate that CD4 molecules are involved in peripheral deletion of TCRV beta 11+ T cells in (CBA/J x B6) F1 mice, and that the TCRV beta repertoire can be altered by ectopic expression of CD4 on all T-lineage cells.     

Generation of CD8+ and CD4+ T-cell response to dendritic cells genetically engineered to express the MART-1/Melan-A gene

Both CD8+ and CD4+ T cells have demonstrated roles in antitumor immune response in many animal tumor systems. In many human tumor systems, although abundant literature exists on the evidence of tumor antigen-specific CD8+ CTL response, only limited information is available on tumor antigen-specific CD4+ T-cell response. Using the MART-1/Melan-A (MART-1) antigen system as a prototype human tumor-associated antigen (TAA)- and dendritic cell (DC)-based MART-1 antigen presentation system (i.e., DCs transduced with an adenoviral vector-based construct carrying the MART-1 gene), we explored, in vitro, the feasibility of generating both CD8+ and CD4+ T-cell responses in the same individual. Here, we show that autologous DCs from both HLA-A2-positive melanoma patients and normal healthy individuals that are transduced with an adenoviral vector containing the MART-1 antigen are capable of inducing both MART-1-specific CD8+ and CD4+ T cells in in vitro coculture. After several rounds of stimulation, both the CD4+ and CD8+ T cells synthesized IFN-gamma when they were specifically stimulated. The CD8+ T cells generated in such cocultures also recognized the MART-1(27-35) peptide, AAGIGILTV, in 4-h cytotoxicity assays. These observations, therefore, suggest that Th1-type responses can be generated, in vitro, by stimulation with DCs that are genetically modified to express a TAA. Although the outcome of this type of genetically engineered DC-based stimulation may vary from system to system, this type of in vitro antigen presentation may be very useful in more comprehensive analyses of CD4+ T-cell response to defined TAAs, and such genetically engineered autologous DCs might be better candidates to serve as surrogate cancer vaccines.     

Administration of IL-12 induces a CD3+ CD4- CD8- B220+ lymphoid population capable of eliciting cytolysis against Fas-positive tumor cells

The present study investigates the effect of IL-12 administration on the generation of lymphoid cells that exhibit cytotoxicity against tumor cells expressing Fas Ag. Systemic injection of rIL-12 into BALB/c or (B6C3)F1 mice bearing syngeneic CSA1M or OV-HM tumor induced complete tumor regression. CSA1M tumor cells expressed Fas Ag, and exposure of these cells to IFN-gamma enhanced Fas expression. In contrast, Fas Ag was hardly detected on OV-HM cells even after IFN-gamma exposure. Only CSA1M cells were lysed by anti-Fas mAb or cells expressing Fas ligand (FasL), indicating that Fas on CSA1M cells is functional in mediating cell death. An increase in the frequency of lymphoid cells characterized as CD3+ CD4- CD8- B220+ was observed in spleens from both CSA1M and OV-HM tumor-bearing mice after IL-12 treatment. A splenic population enriched in cells with these unique phenotypes exhibited considerable degrees of cytotoxicity against Fas+ CSA1M, but not against Fas- OV-HM tumor cells. The lysis of CSA1M cells was almost completely blocked by addition of Fas-Fc, a fusion protein between the extracellular domain of mouse Fas and the Cgamma1 domain of human Ig. Regressing CSA1M and OV-HM tumor masses after IL-12 treatment exhibited a massive lymphoid cell infiltration and expressed significant levels of FasL mRNA, suggesting the infiltration of FasL-expressing cells to tumor sites. These results indicate that IL-12 induces the expansion of lymphoid cells that exhibit FasL-mediated cytolytic activity and accumulate into regressing tumor masses.     

Defective interactions between TCR chains and CD3 heterodimers prevent membrane expression of TCR-alpha beta in human T cells

The human TCR complex is composed of two clonotypic polypeptide chains, TCR-alpha and TCR-beta (or TCR-gamma and TCR-delta) associated with CD3 gamma-, delta-, and epsilon-chains and zeta 2 homodimers. All six polypeptide chains are indispensable for TCR membrane expression and signaling function. In the present paper is described the analysis of a new TCR membrane-negative Jurkat T cell variant: E6.R3. The defect in this variant bears on the interaction between TCR and CD3 chains. E6.R3 cells have deleted three nucleotides in the TCR-alpha transmembrane (TM) region, which consequently lacks a leucine. This defect causes 1) lack of association between TCR alpha-chains and CD delta epsilon heterodimers; 2) lack of formation of disulphide-linked, fully glycosylated TCR-alpha beta heterodimers; and 3) lack of interaction between TCR-alpha beta/CD3 complexes and zeta-chains. Despite these defective interactions, TCR alpha-chains appear to become fully glycosylated, i.e., they are not retained in the endoplasmic reticulum but are further processed in the Golgi apparatus without such interactions. The defect may be due to the observation that in the E6.R3 TCR alpha- chains TM region, the two charged amino acids are situated on the same side of the alpha-helix; these two amino acids are exposed on opposite faces of the TM alpha-helix in normal TCR alpha-chains, possibly allowing TCR alpha-chains to interact with both CD3 delta- and CD3 epsilon-chains. Further possible consequences of the leucine deletion in the E6.R3 TCR-alpha TM region are discussed.     

Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.     

IL-4 protects adult C57BL/6 mice from prolonged Cryptosporidium parvum infection: analysis of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in gut-associated lymphoid tissue during resolution of infection

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+alpha beta+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-gamma mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-gamma mAb present in treated mouse sera suggested that IFN-gamma may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in Peyer's patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+alpha beta+IFN-gamma+ lymphocytes, and eventual resolution of infection was associated with CD4+alpha beta+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+alpha beta+IL-4+ lymphocytes, but not CD4+alpha beta+IFN-gamma+ lymphocytes. The relevance of CD4+alpha beta+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4(-/-)). Adult IL-4(-/-) mice excreted oocysts in feces approximately 23 days longer than IL-4(+/+) mice. Further, anti-IFN-gamma mAb treatment increased the severity and the duration of infection in IL-4(-/-) mice compared with those in IL-4(+/+) mice. Together, the data demonstrated that IFN-gamma was important in the control of severity of infection, and either IFN-gamma or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-gamma was required for the final clearance of infection from the intestinal tract of adult mice.     

The proliferative response of HIV+ T-cells (CD4+ and CD8+) are severely suppressed via CD28 coactivation

Several studies have suggested that regulation of expression of the costimulatory molecule CD28 on the T-cell surface may play an important role in AIDS pathogenesis. In a study of T-cells from HIV+ donors, we find that activation with anti-CD3 plus anti-CD28 results in a mitogenic response which was approximately 86% suppressed for both CD4+ and CD8+ T-cells when compared to normal control cells. With PMA costimulation (instead of anti-CD28), the anti-CD3 response was suppressed much less, by 64 and 61%, respectively. With Con A as opposed to CD3 stimulation, the degree of suppression was less with either coactivator but still more severe with CD28 than with PMA coactivation. It has been reported that the CD28 subset of CD8+ T-cells is diminished in HIV+ individuals and could account for these results. It is possible as well that the CD28 costimulatory pathway in the CD4+ T-cells particularly is altered due to intervention by the HIV. While our data do not differentiate between these two possibilities, it show that the immune status is compromised in the HIV+ individual not only in terms of number of CD4+ T-cells, but in their activation response as well.