Parallel expansion of human virus-specific FoxP3- effector memory and de novo-generated FoxP3+ regulatory CD8+ T cells upon antigen recognition in vitro

Although FoxP3 has been shown to be the most specific marker for regulatory CD4(+) T cells, its significance in the CD8(+) T cell population is not well understood. In this study, we show that the in vitro stimulation of human PBMC with hepatitis C virus or Flu virus-specific peptides gives rise to two distinct Ag-specific T cell populations: FoxP3(-) and FoxP3(+)CD8(+) T cells. The FoxP3(+) virus-specific CD8(+) T cells share phenotypical markers of regulatory T cells, such as CTLA-4 and glucocorticoid-induced TNFR family-related gene, and do produce moderate amounts of IFN-gamma but not IL-2 or IL-10. IL-2 and IL-10 are critical cytokines, however, because the expansion of virus-specific FoxP3(+)CD8(+) T cells is blocked by IL-2- or IL-10-neutralizing mAbs. The virus-specific FoxP3(+)CD8(+) T cells have a reduced proliferative capacity, indicating anergy, and display a cell-cell contact-dependent suppressive activity. Taken together, our results indicate that stimulation with a defined viral Ag leads to the expansion of two different cell populations: FoxP3(-) memory/effector as well as FoxP3(+) regulatory virus-specific CD8(+) T cells.     

Induction of FoxP3+CD4+CD25+ regulatory T cells by a bone marrow population distinct from plasmacytoid-DC

Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3ε⁺ and FC-CD3ε⁻ subpopulations in the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells. Here, we demonstrate that the induction of FoxP3⁺CD4⁺CD25⁺ regulatory T cells in coculture is mediated by not only the FC-CD3ε⁻ subset but also the FC-CD3ε⁺ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3⁺CD4⁺CD25⁺ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.     

Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age

The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age.     

Spatial coordination of CD8 and TCR molecules controls antigen recognition by CD8+ T-cells

The interactions between the TCR and peptides bound to class I MHC encoded molecules (pMHC) and a mechanism for CD8 cooperation in this process are reviewed. Observation of two TCR/CD8 populations with different lateral diffusion rate constants as well as two distinct association phases of class I MHC tetramers ((pMHC)4) with T-cells suggest that the most efficient pMHC-T-cell association route corresponds to a fast tetramer binding to a colocalized CD8/TCR population, which apparently resides within membrane rafts. Thus, ligand-cell association starts by pMHC binding to the CD8. This rather fast step promotes pMHC association with CD8-proximal TCRs and thereby enhances the overall association process. The model suggests that this raft-associated CD8-TCR subpopulation is responsible for evoking T-cell activation.     

Osteoclast bone resorption is enhanced in the presence of osteoblasts

Bone resorption activity by osteoclasts has been evaluated in a co-culture system in which osteoclasts have been plated in the presence of osteoblasts. The system prevents cell-cell contact but permits diffusion of molecules through the pores of a millipore membrane that separates the two compartments in which the two cell types have been plated. Results demonstrated that osteoblasts exert a stimulatory effect over osteoclast bone resorption due to soluble molecules capable of passing through the membrane pores. The effect is specific since periosteal cells, which do not express osteoblastic characteristics, fail to induce changes in the osteoclast activity. PTH does not affect osteoblast-mediated enhancement of bone resorption, indicating that the stimulatory effect that the hormone exert in vivo occurs via a different cellular system.     

Mycobacterial trehalose-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+ T-cells

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4+ and CD8+ T-cell subsets. One of the key features of the tubercle bacillus is its cell envelope, characterized by extremely abundant and specific lipids. The cell-surface glycolipid 2,3-di-O-acyl-trehalose (DAT) has been consistently found in M. tuberculosis strains. In this study, analysis of proliferation, activation markers and cytokine release was performed in human peripheral blood mononuclear cells (PBMC) activated in the presence and absence of DAT. We present evidence that mycobacterial DAT is able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets. We show that the effect is associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69, and down-modulation of IL-2, IL-12, TNF-alpha and IL-10 cytokines. Data indicating that fine acyl chain structural variations in the trehalose-containing lipid may be involved in the degree of immune modulation are also presented.     

Herpesvirus ateles immortalizes in vitro a CD3+CD4+CD8+ marmoset lymphocyte with NK function

Herpesvirus ateles, nonpathogenic in its natural host, the spider monkey, induces a fatal lymphoproliferative syndrome in a variety of New World primate species. Whereas the closely related New World primate virus Herpesvirus saimiri immortalizes in vitro common marmoset lymphocytes that express a TCR and phenotypically are CD4-CD8+NKH1+, we now show that H. ateles-immortalized marmoset lymphocytes are CD3+ and CD4+. Furthermore, these CD4+ lymphocytes coexpress CD8 and NKH1. The NK function of cloned H. ateles-immortalized lymphocyte populations is proportional to the extent to which they express the CD8 antigen. These studies illustrate an interesting example of restricted viral tropism and may indicate a potentially useful means of generating phenotypically stable, functionally competent, cloned lymphocyte populations for study.     

Small intestine CD11c+ CD8+ T cells suppress CD4+ T cell-induced immune colitis

The large (LI) and small intestine (SI) differ in patterns of susceptibility to chronic mucosal inflammation. In this study, we evaluated whether this might, in part, reflect differences in resident mucosal CD11c(+) T cells. These cells comprised 39-48% (SI) and 12-17% (LI) of the intraepithelial compartment, most of which were T-cell receptor-αβ(+). In the SI, the majority of these cells were CD103(+) CD8(+) NK1.1(-), whereas the opposite phenotype prevailed in the LI. In transfer models of CD4(+) T cell-induced colitis, small numbers (2.5 × 10(5)) of SI CD11c(+) CD8(+) T cells suppressed proinflammatory cytokine-producing CD4(+) T cells in mesenteric lymph nodes and mucosa-associated lymphoid compartments (SI and LI) and protected mice from chronic inflammation. On a per-cell basis, the regulatory function of SI CD11c(+) T cells in CD4(+) T cell colitis was potent compared with other reported regulatory CD4(+) or CD8(+) T cells. In contrast, neither LI CD11c(+) T cells nor SI CD11c(-) T cells were effective in such immunoregulation. SI CD11c(+) CD8(+) T cells were similarly effective in suppressing CD4(+)CD45RB(hi) T cell colitis, as evidenced by inhibition of intracellular proinflammatory cytokine expression and histological inflammation. These findings indicate that SI CD11c(+) CD8(+) T cells are a distinct intestinal T cell population that plays an immunoregulatory role in control of proinflammatory CD4(+) T cells and maintenance of intestinal mucosal homeostasis.     

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.     

Prognostic Impact of FoxP3+ Regulatory T Cells in Relation to CD8+ T Lymphocyte Density in Human Colon Carcinomas

Background

T-lymphocyte infiltration into colon carcinomas can influence clinical outcome, and interactions among T cell subsets may be more informative than either subset alone. Our objective was to examine the prognostic impact of tumor-infiltrating FoxP3⁺ regulatory T cells (Tregs) in relation to cytotoxic CD8⁺ T lymphocytes in patients with colon carcinomas characterized by DNA mismatch repair (MMR) status who participated in adjuvant chemotherapy trials.

Methods

FoxP3⁺ and CD8⁺ densities in tumor epithelial and stromal compartments were analyzed by immunohistochemistry and quantified in resected, stage II and III colonic carcinomas (N = 216). Immune marker density was dichotomized at the median and categorized as high vs low. MMR status was classified as MMR deficient (dMMR) or proficient (pMMR). Cox models were adjusted for age, stage, and tumor grade.

Results

The density of FoxP3+ infiltration was similar in tumor stroma and epithelia, whereas CD8+ was higher in stroma. The prognostic impact of FoxP3+ and CD8+ T cell infiltration was stronger in stroma vs epithelia, and the density of each marker in stroma was independently associated with improved overall survival (OS). However, the impact of FoxP3+ on survival was dependent upon CD8+ density (P interaction  = .040). Among CD8+_low tumors, FoxP3+_high cases had significantly improved OS compared to FoxP3+_low cases after adjustment for covariates (hazard ratio 0.43; 95% confidence interval 0.19 to 0.95; P = .030). In contrast, FoxP3+ was not prognostic among CD8+_high tumors. FoxP3+ remained prognostic in CD8+_low tumors after further adjustment for MMR or BRAF ^V600E mutation status. Additionally, these immune markers identified a pMMR subgroup with a similarly favorable OS as for dMMR tumors.

Conclusions

The prognostic impact of FoxP3+ and CD8+ T cell density are inter-dependent, whereby FoxP3+ exerts a favorable influence on survival only in colon cancers with low CD8+ infiltration.     

FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeficiency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load

The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25⁺ FoxP3⁺ regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4⁺ regulatory T cells have been studied in the most detail, but CD8⁺ CD25⁺ FoxP3⁺ T cells also have regulatory properties. We monitored the dynamics of CD25⁺ FoxP3⁺ T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4⁺ CD25⁺ FoxP3⁺ T cells paralleled that of memory CD4⁺ T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25⁺ FoxP3⁺ T cells was observed in peripheral lymph nodes. A small number of CD4⁺ CD25⁺ FoxP3⁺ T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8⁺ CD25⁺ FoxP3⁺ T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4⁺ T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8⁺ CD25⁺ FoxP3⁺ T cells being indicative of a poor prognosis.     

Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction, we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.     

Low doses of the novel caspase-inhibitor GS-9450 leads to lower caspase-3 and -8 expression on peripheral CD4+ and CD8+ T-cells

Chronic hepatitis C virus (HCV) infection is characterized by increased rates of apoptotic hepatocytes and activated caspases have been shown in HCV-infected patients. GS-9450, a novel caspase-inhibitor has demonstrated hepatoprotective activity in fibrosis/apoptosis animal models. This study evaluated the effects of GS-9450 on peripheral T-cell apoptosis in chronic HCV-infected patients. As sub study of the GS-US-227-0102, a double-blind, placebo-controlled phase 2a trial evaluating the safety and tolerability of GS-9450, apoptosis of peripheral CD4+ and CD8+ T-cells was measured using activated caspase-3, activated caspase-8 and CD95 (Fas). Blood samples were drawn at baseline, day 14 after therapy and at 5 weeks off-treatment follow-up in the first cohort of 10 mg. In contrast to the placebo-treated patients, GS-9450 caused a median of 46% decrease in ALT-values from baseline to day 14 in all treated patients (median of 118–64 U/l) rising again to a median of 140 U/l (19%) at 5 weeks off-treatment follow-up. In GS9450-treated patients, during treatment and follow-up, percentages of activated caspase-3+ and caspase-8 expression tended to decrease, in contrast to placebo-treated patients. Interestingly, compared to healthy controls, higher percentages of caspase-3 and caspase-8 positive CD4+ and CD8+ T-cells were demonstrated in HCV-infected patients at baseline. Decreased ALT-values were observed in all HCV-infected patients during treatment with low dose of the caspase-inhibitor GS-9450 accompanied by a lower expression of caspase-3 and -8 on peripheral T-cells. Furthermore, at baseline percentages of activated caspase-3, activated caspase-8 and CD95+ T-cells were higher in chronic HCV-infected patients compared to healthy controls.     

In vitro and in vivo analysis of bone marrow-derived CD3+, CD4-, CD8-, NK1.1+ cell lines

The development of methods of avoiding graft-versus-host disease (GVHD) while retaining the alloengraftment-promoting and anti-leukemic effects of allogeneic T cells is a major goal of research in bone marrow transplantation (BMT). We have recently obtained evidence suggesting that natural suppressor (NS) cells derived from T cell-depleted (TCD) syngeneic marrow can protect against GVHD while permitting alloengraftment. We have now attempted to enrich and then propagate NS cells in vitro, with the goal of obtaining an enhanced anti-GVHD effect by adoptive transfer in vivo. Two long-term cell lines were generated culturing BMC depleted of Mac1-positive cells and of Mac1-positive plus Thy1-positive cells in high concentrations of IL-2. Both cell lines showed anti-GVHD effects when administered along with a GVHD-producing inoculum, while permitting complete allogeneic reconstitution. A clone derived from Mac1-depleted BMC protected completely against a more chronic pattern of GVHD. These cell lines demonstrated suppressive activity in vitro, cytolytic activity against a broad range of natural killer (NK)-sensitive and NK-resistant targets, and a novel cell surface phenotype, with characteristics of both alpha beta-TcR-bearing T cells and of NK cells. In some respects, these cells resemble LAK cells and differ from fresh NS cells, and from the cloned NS cells derived from spleens of total lymphoid irradiation (TLI)-treated mice and neonatal mice. To our knowledge, this is the first detailed phenotypic analysis of cell lines with in vivo anti-GVHD activity. If applicability can be demonstrated in large animal models, the ability to use bone marrow as a source of such protective cell lines might also have potential utility in clinical BMT.     

fat-1 transgene expression prevents cell culture-induced loss of membrane n-3 fatty acids in activated CD4+ T-cells

In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4⁺ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4⁺ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4⁺ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4⁺ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4⁺ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.     

HIV-specific IL-10-positive CD8+ T cells suppress cytolysis and IL-2 production by CD8+ T cells

IL-10 producing T cells inhibit Ag-specific CD8+ T cell responses and may play a role in the immune dysregulation observed in HIV infection. We have previously observed the presence of HIV-specific IL-10-positive CD8+ T cells in advanced HIV disease. In this study, we examined the suppressive function of the Gag-specific IL-10-positive CD8+ T cells. Removal of these IL-10-positive CD8+ T cells resulted in increased cytolysis and IL-2, but not IFN-gamma, production by both HIV- and human CMV-specific CD8+ T cells. In addition, these IL-10-positive CD8+ T cells mediated suppression through direct cell-cell contact, and had a distinct immunophenotypic profile compared with other regulatory T cells. We describe a new suppressor CD8+ T cell population in advanced HIV infection that may contribute to the immune dysfunction observed in HIV infection.     

Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.     

Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.