CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

CD4+CD8- thymocytes from neonatal mice induce IgM production in SCID mice.

Defective recombination of both the TCR and Ig genes results in the absence of mature lymphocytes in mice with the scid mutation. We have shown previously that the transfer of neonatal, but not adult, thymocytes results in high levels of Ig production in 100% of C.B-17-scid (SCID) mice, in contrast to the 10 to 25% of SCID mice spontaneously producing low levels of oligoclonal Ig. In this report we demonstrate that neonatal CD4+8- thymocytes were able to induce this response; the CD4+8+ and CD4-8+ subpopulations were totally inactive and CD4-8- T cells had only limited activity several weeks after transfer. The stimulation of IgM production in SCID mice was detectable by 1 wk posttransfer of CD4+8- thymocytes or splenic T cells, and could be achieved with as few as 300 cells. The ability of neonatal CD4+8- thymocytes to induce Ig diminished gradually to insignificant levels at 3 wk postbirth; this loss of function was not associated with differential survival of neonatal T cells. Neonatal CD4+8- thymocytes from C.B-17 and other H-2d strains rescued Ig production, whereas cells from H-2b, H-2a, and H-2k strains were much less effective. These results suggest that a CD4+8- subpopulation found in both neonatal thymus and peripheral lymphoid tissues is able to induce the expansion or differentiation of the small numbers of functional B lymphocytes in SCID mice, and that the inducing T cell disappears shortly after birth, perhaps during the acquisition of self-tolerance.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.     

Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor-inducer lymphocytes and CD8+ suppressor/cytotoxic lymphocytes

In vivo exposure of human epidermis to UV abrogates the function of T6+DR+ Langerhans cells and induces the appearance of Ag-presenting T6-DR+ OKM5+ cells in the epidermis. Since UV exposure of murine skin results in Ts lymphocyte activation, we investigated the capacity of human epidermal cells (EC) harvested 3 days after in vivo UV exposure to activate regulatory and effector autologous T lymphocyte subsets. T lymphocytes were separated into CD8+ suppressor/cytotoxic lymphocytes and CD4+ helper/inducer lymphocytes by C lysis and panning. The CD4+ subset was further divided by using the 2H4 mAB to obtain CD4+2H4+ lymphocytes (inducers of TS lymphocytes) and CD4+2H4- lymphocytes (inducers of B cell Ig production and inducers of cytotoxic T cells). Unirradiated suction blister-derived EC from control skin (C-EC) and from skin exposed in vivo to UV (UV-EC) were cultured with purified autologous T lymphocyte subsets in the absence of added Ag. The resultant T lymphocyte proliferation was detected by [3H]thymidine uptake. UV-EC were highly effective in the stimulation of CD4+ lymphocytes, whereas C-EC were poor stimulators. The stimulator effect of UV-EC was abrogated after depletion of DR+ UV-EC. When CD4+ lymphocytes were fractionated, UV-EC consistently demonstrated enhanced ability to stimulate suppressor-inducer CD4+2H4+ lymphocytes relative to C-EC. Although less responsive than CD4+2H4+ lymphocytes, CD4+2H4- lymphocytes also demonstrated greater proliferation to UV-EC than to C-EC. Neither UV-EC nor C-EC were able to activate CD8+ lymphocytes devoid of CD4+ lymphocytes. However, after addition of rIL-2 at concentrations that allow binding only to the high affinity IL-2R on T lymphocytes, UV-EC induced vigorous proliferation of CD8+ lymphocytes, whereas C-EC induced only background levels of proliferation. C lysis of leukocytes resident within UV-EC resulted in 66 to 70% reduction of CD8+ lymphocyte proliferation. In conclusion, UV-EC may activate CD8+ lymphocytes by at least two pathways: (1) UV-EC activation of CD4+2H4+ lymphocytes may induce differentiation/proliferation of CD8+ suppressor cells and (2) UV-EC activation of CD4+ cells may induce IL-2 production, that, in combination with UV-induced epidermal leukocytes, stimulates CD8+ cells.     

Tumor-selective cytolysis is executed exclusively by CD45R+ CTL whereas allo-specific cytotoxicity can be executed also by CD45R- CTL

Naive and memory CD4+ T helper cells can be distinguished on the basis of expression of the CD45R molecule. Whether this dichotomy applies also to CD8+ T cells has not yet been established. In the present investigation the cytolytic activity of peritoneal CD8+CD45R+ and CD8+CD45R- T cells from tumor- and allo-immunized rats has been studied. More than 90% of the CD8+ peripheral blood T lymphocytes expressed the CD45R molecule, whereas in the peritoneal cavity about 60% of the CD8+ T cells displayed the CD45R+ phenotype. Analysis of cytotoxicity of sorted peritoneal cells of W439 tumor-immunized donors demonstrated selective cytolytic activity of the CD5+CD4-CD8+CD45R+ subpopulation to W439 lymphoma target cells but no effect of CD5+CD4-CD8+CD45R- lymphocytes. None of these lymphocyte populations exhibited cytolytic activity to the NK-sensitive cell line YAC-1, whereas the CD5-CD45R+ population showed strong cytotoxicity to YAC-1 cells. In allo-immunized rats both CD5+CD4- CD8+CD45R+ and CD5+CD4-CD8+CD45R- peritoneal cells exhibited strong allo-specific cytolytic activity, but no activity to YAC-1 cells. Both CD5+CD4-CD8+CD45R+ and CD5+CD4-CD8+CD45R- cells from tumor-immunized rats proliferated in response to Con A and rIL-2. This is the first study demonstrating that tumor-selective cytolytic CD8+ T cells express the CD45R molecule and that allo-specific cytolytic CD8+ T cells are found in both the CD45R+ and CD45R- populations.     

Predominant involvement of CD8+CD28- lymphocytes in human immunodeficiency virus-specific cytotoxic activity.

Distinct functional CD8+ T-cell populations have been observed during human immunodeficiency virus (HIV) infection. One of these functions is the inhibition of viral replication by a noncytotoxic mechanism, which was shown to be mediated by the CD8+CD28+ subpopulation. On the other hand, CD8+ T cells exert an HIV-specific cytotoxic activity. The present study shows that CD8+CD28- lymphocytes display this HIV-specific cytotoxic activity, which is detectable immediately after the cells are purified from peripheral blood. The CD28- population is also able to proliferate and to retain its cytotoxic activity after in vitro restimulation with autologous blast cells. Finally, HIV-specific cytotoxic T cells can be obtained in vitro from the CD8+CD28+ population.     

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.     

Alteration of circadian rhythmicity of CD3+CD4+ lymphocyte subpopulation in healthy aging

The CD4+ T helper/inducer and the CD8+ T suppressor/cytotoxic are major lymphocyte subsets that play a key role in cell-mediated immunity. Aging-related changes of immune function have been demonstrated. The purpose of this study is to analyze the dynamics of variation of these specific lymphocyte subsets in the elderly. In our study cortisol and melatonin serum levels were measured and lymphocyte subpopulation analyses were performed on blood samples collected every four hours for 24 hours from fifteen healthy young middle-aged subjects (age range 36-55 years) and fifteen healthy elderly male subjects (age range 67-79 years). A clear circadian rhythm was validated for the time-qualified changes of CD3+ and CD4+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in young middle-aged subjects and for the time-qualified changes of CD3+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in elderly subjects. No clear circadian rhythm was validated for the time-qualified changes of CD4+ cells in elderly subjects. No statistically significant correlation among lymphocyte subsets was found in elderly subjects. In elderly subjects CD3+ lymphocyte percentage was higher in the photoperiod and in the scotoperiod and cortisol serum level were higher in the scotoperiod in respect to young middle-aged subjects. In the elderly there is an alteration of circadian rhythmicity of T helper/inducer lymphocytes and this phenomenon might contribute to the aging-related changes of immune responses.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.     

Radiosensitivity of CD4 or CD8 positive human T-lymphocytes by an in vitro colony formation assay

The recent development of an in vitro lymphocyte colony assay makes it possible to examine variations in the radiosensitivity of humans using peripheral blood lymphocytes (PBL) instead of the skin fibroblast assay. Our recent study (M. Hakoda et al., Mutat. Res. 197, 161-169, 1988) showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4+ and CD8+ cells in PBL differs among individuals, we suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4+ and CD8+ cells were different from each other. In the present study, CD4+ (helper/inducer T) and CD8+ (suppressor/cytotoxic T) lymphocytes were isolated from PBL and their dose-survival curves were determined. The results showed that the D10 (dose required to reduce the surviving fraction to 10%) was similar for these two types of cells [3.13 +/- 0.10 Gy (mean +/- SD) for CD4+, 3.34 +/- 0.50 Gy for CD8+ and 3.14 +/- 0.17 Gy for the unsorted cells], supporting the use of the whole PBL population for the screening of individuals with altered radiosensitivity.     

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

CD45 isoforms associated with distinct functions of CD4 cells derived from unusual healthy donors lacking CD45RA- T lymphocytes.

We now report two healthy individuals whose T lymphocytes were over 95% positive for CD45RA antigen expression. However, these donors normally expressed both the CD29 high (CD29+) and CD45RO high (CD45RO+) antigens on approximately 40 and 50% of their CD4 cells, respectively. Despite the strong CD45RA expression on the surface of almost all CD4 cells, the CD29 marker allowed T cells from these donors to be divided phenotypically into subsets having distinct in vitro function. CD4+CD29+ cells from these donors responded maximally to recall antigens such as TT and provided strong helper function for B cell Ig synthesis. In contrast, CD4+CD29- cells responded poorly to recall antigens and had poor helper function for B cell Ig synthesis, but had strong suppressor activity. Thus, CD29 antigen expression was still predictive of the in vitro functional activity as previously described for normal donors. Furthermore, biochemical analysis of the distribution of individual CD45 isoforms on the surface of these subsets of CD4 cells revealed distinct differences. The CD4+CD29 high (CD4+CD29+) subset of cells primarily expressed the 180-, 190-, and 205-kDa CD45 isoforms, while the CD4+CD29 low (CD4+CD29-) cells primarily expressed the 190-, 205-, and 220-kDa CD45 isoforms. These results suggest that despite the superficial phenotypic similarity of CD4 cells in these donors, distinctions in the distribution of both CD29 and the 180- and 220-kDa CD45 isoforms exist and might play a role in the different functions of freshly isolated CD4 lymphocytes.     

CD4 positive Leu-8 negative helper-inducer T cells predominate in the human intestinal lamina propria

The regulatory function of peripheral blood CD4 T cells correlates with the presence or absence of the membrane glycoprotein recognized by anti-Leu-8 antibody; CD4,Leu8- T cells help Ig synthesis and CD4,Leu-8+ T cells suppress Ig synthesis. In contrast to CD4 T cells from the peripheral blood and organized gut-associated lymphoid tissues, intestinal lamina propria CD4 T cells were found to have diminished expression of the Leu-8 Ag. Therefore, studies were performed to determine whether the decreased expression of the Leu-8 Ag on lamina propria CD4 T cells correlates with a difference in the ability of peripheral blood and lamina propria CD4 T cells to regulate PWM-stimulated Ig synthesis. At high T cell to non-T cell ratios, the helper function of lamina propria CD4 T cells was significantly higher than that of peripheral blood CD4 T cells. When CD4 T cells were incubated with anti-Leu-8 antibody, the suppressor function of peripheral blood CD4 T cells was increased, but lamina propria CD4 T cells did not suppress Ig synthesis. No difference was found between the helper function of CD4,Leu-8- T cells and the suppressor function of CD4, Leu-8+ T cells isolated from either the peripheral blood or the lamina propria. Thus, the difference in the regulatory function of CD4 T cells from the peripheral blood and the lamina propria is due to the quantitative difference in CD4,Leu-8+ T cells in these sites. Consequently, the intestinal lamina propria is a site enriched in CD4,Leu-8- T cells which predominantly mediate help for Ig synthesis.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

Rapid memory CD8+ T-lymphocyte induction through priming with recombinant Mycobacterium smegmatis

The most promising vaccine strategies for the induction of cytotoxic-T-lymphocyte responses have been heterologous prime/boost regimens employing a plasmid DNA prime and a live recombinant-vector boost. The priming immunogen in these regimens must elicit antigen-specific memory CD8+ T lymphocytes that will expand following the boosting immunization. Because plasmid DNA immunogens are expensive and their immunogenicity has proven disappointing in human clinical trials, we have been exploring novel priming immunogens that might be used in heterologous immunization regimens. Here we show that priming with a prototype recombinant Mycobacterium smegmatis strain expressing human immunodeficiency virus type 1 (HIV-1) gp120-elicited CD4+ T lymphocytes with a functional profile of helper cells as well as a CD8+ T-lymphocyte population. These CD8+ T lymphocytes rapidly differentiated to memory cells, defined on the basis of their cytokine profile and expression of CD62L and CD27. Moreover, these recombinant-mycobacterium-induced T lymphocytes rapidly expanded following boosting with a recombinant adenovirus expressing HIV-1 Env to gp120-specific CD8+ T lymphocytes. This work demonstrates a remarkable skewing of recombinant-mycobacterium-induced T lymphocytes to durable antigen-specific memory CD8+ T cells and suggests that such immunogens might be used as priming vectors in prime/boost vaccination regimens for the induction of cellular immune responses.     

Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.     

Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity.

This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals. Elimination of CD25-expressing T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them. The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4-8- cytotoxic cells akin to NK cells. Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4-8- cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation. In this culture, self-reactive CD25-4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2. The IL-2 thus produced appeared to be responsible for the generation of CD4-8- NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4-8- cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4-8- cells with CD25-4+ T cells from IL-2-deficient mice did not. Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones. This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.     

Cutting edge: activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin generates a population of CD4+ CD25+ cells with characteristics of regulatory T cells

Activation of the aryl hydrocarbon receptor (AhR) by its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to immune suppression in mice. Although the underlying mechanisms responsible for AhR-mediated immune suppression are not known, previous studies have shown that activation of the AhR must occur within the first 3 days of an immune response and that CD4+ T cells are primary targets. Using the B6-into-B6D2F1 model of an acute graft-vs-host response, we show that activation of AhR in donor T cells leads to the generation of a subpopulation of CD4+ T cells that expresses high levels of CD25, along with CD62L(low), CTLA-4, and glucocorticoid-induced TNFR. These donor-derived CD4+ CD25+ cells also display functional characteristics of regulatory T cells in vitro. These findings suggest a novel role for AhR in the induction of regulatory T cells and provide a new perspective on the mechanisms that underlie the profound immune suppression induced by exposure to TCDD.     

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.