Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells

In the present study, we show that human self-MHC-reactive (autoreactive) T cell clones are functionally distinct from Ag-specific T cell clones. Self-MHC-reactive T cells exhibited helper function for B cell Ig production when cultured with non-T cells alone, and they exhibit suppressor function when cultured with PWM- or rCD40 ligand (rCD40L)-activated non-T cells, whereas tetanus toxoid (TT)-specific clones exhibited only helper function in the presence of TT with or without PWM or rCD40L. Addition of neutralizing Abs to the cultures showed that the suppression was mediated by TGF-beta but not by IL-10 or IFN-gamma. The self-MHC-reactive clones also inhibited proliferation of primary CD4+ T cells and TT-specific T cell clones, but in this case the inhibition was mediated by both IL-10 and TGF-beta. In further studies, the interactions between self-MHC-reactive T cell clones and non-T cells that led to suppressor cytokine production have been explored. We found that prestimulation of non-T cells for 8 h with PWM or for 48 h for rCD40L results in non-T cells capable of inducing self-MHC-reactive T cell to produce high levels of TGF-beta and IL-10. In addition, these prestimulation times coincided with peak induction of HLA-DR and costimulatory B7 molecule (especially CD86) expression on B cells. Finally, addition of CTLA-4/Fc or blocking F(ab')2 anti-CTLA-4 mAb, plus optimally stimulated non-T cells, to cultures of self-MHC-reactive clones inhibited the induction of TGF-beta but not IL-10 or IFN-gamma production. In summary, these studies show that activated self-MHC-reactive T cells have the cytokine phenotype of Th3 or T regulatory cell 1 and thus may be important regulatory cells that mediate oral and peripheral tolerance and prevent the development of autoimmunity.     

HLA-DR antigens render resting T cells sensitive to interleukin-2 and induce production of the growth factor in the autologous mixed lymphocyte reaction

Monoclonal anti-HLA-DR (anti-Ia) antibodies inhibited autologous mixed lymphocyte reactions (AMLR) when added from the initiation of the cultures, but not 72 hr later. The suppressive principle was removed by the stimulator non-T cells, but not by the responding T cells. Antibody-treated non-T cells lost their ability to activate T cells, whereas antibody-treated T cells could still respond to untreated non-T cells. The anti-DR antibodies prevented T cells from acquiring responsiveness to Interleukin-2 (IL-2). However, T cells previously activated by AMLR responded to IL-2 even in the presence of the anti-DR antibodies. OKT4⁺ lymphocytes synthesized IL-2 in the AMLR while OKT8⁺ cells did not. Anti-DR antibodies caused OKT4⁺ cells to become unresponsive to Interleukin-1 stimulation and inhibited the production of IL-2. Interleukin-1 (IL-1) promoted the synthesis of IL-2 in non-anti-DR-treated AMLR cultures. Since resting T cells are unresponsive to IL-2 and resting OKT4⁺ lymphocytes are unable to produce IL-2 even in the presence of IL-1, it is concluded that HLA-DR antigens render resting T cells sensitive to IL-2 and enable OKT4⁺ lymphocytes to respond to IL-1 and subsequently, to produce Interleukin-2.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.     

Stage-related, cell-mediated cytotoxicity with effector cell analysis and computation of cytotoxic activity of T cells and non-T cells.

Cell-mediated cytotoxicity (CMC) by lymphocytes from patients with oral squamous cell carcinoma, as well as from nonmalignant control donors, was tested by a microcytotoxicity assay against a cultured cell line derived from an oral squamous cell carcinoma. In terms of the degree of CMC, stage-related cytotoxicity was observed. A further study of the effector cell analysis revealed that the cytotoxic effects of lymphocytes from both patients and control donors were largely attributable to non-T cells. However, the effectors were also stage related, and in early stage patients, T-cell-mediated cytotoxicity was super-imposed on non-T-cell-mediated cytotoxicity. This conclusion was further supported by the evidence of elevated cytotoxic activity of T cells in early stage patients, which was computed from simultaneous equations proposed in the present paper for computing the cytotoxic activity of T cells (CTA_T) and non-T cells (CTA_non-T).     

Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity,which is suppressed by regulatory T cells in breast cancer

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8⁺ T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8⁺ T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8⁺ T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8⁺ T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8⁺ T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8⁺ T cells but could significantly enhance IL-2-mediated promotion of CD8⁺ T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8⁺ T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4⁺CD25⁺ regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8⁺ T cells, an effect suppressible by CD4⁺CD25⁺ Treg cells.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Cytotoxic immune cells with specificity for defined soluble antigens. II. Chasing the killing cells

Cells from mice immune against soluble antigens were tested for their in vitro cytotoxicity against chicken red blood cells (CRBC) coated with these antigens. In parallel, cells from mice immune against allogeneic P815 mastocytoma cells were tested for their in -vitro cytotoxicity against P815 cells. Before the cytotoxicity test, the immune cell populations were fractionated, using four different types of techniques, to check the impact of the removal of given subpopulations of cells on cytotoxic activity.Fractionation on anti-immunoglobulin coated columns did not affect the anti P815 cytotoxicity, but markedly decreased the cytotoxicity against antigen-coated CRBC. The same results were obtained by incubation on a plastic surface and/or an “ironplus-magnet” technique. Preincubation of the cytotoxic cell populations with homologous anti-θ or heterologous anti-T antiserum, plus complement, abolished both types of cytotoxicity. However, preincubation with anti-θ or anti-T antiserum alone, without complement, also blocked the cytotoxicity against antigen-coated CRBC, but not the anti P815 cytotoxicity. In vivo injection of heterologous anti-lymphocyte gammaglobulin completely abolished the anti P815 cytotoxicity, but not the cytotoxicity against antigen-coated CRBC.These results confirm that T cells (thymus-processed lymphocytes) can kill autonomously in the anti P815 system. They also suggest that, in the cytotoxicity against antigen-coated CRBC, as effector cells, (1) non-T cells are involved, (2) T cells might not be involved.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Presentation of Class I antigens on T cells can induce the generation of antigen-specific CTL

Cytotoxic T lymphocyte (CTL) lines generally require a source of specific antigen to continue to proliferate in vitro. We previously showed that populations of mononuclear cells grown in medium containing interleukin 2 (IL 2) and composed largely of activated T cells were able to present Class I alloantigen to CTL lines. On the basis of these findings we were interested to know whether T cells themselves were able to present antigen or whether this was a function of the small number of contaminating non-T cells. To answer this question, populations of activated or resting mononuclear cells were rigorously depleted of non-T cells before use as antigen-presenting cells. We observed that populations composed of greater than 99% T cells were able to support the differentiation of antigen-specific CTL. These results were confirmed by using cells from an established T cell line. The proliferation of the same lines, however, was less than that of lines grown in the presence of antigen-presenting cells containing some non-T cells. These results suggest that although T cells can present Class I alloantigens to CTL, they may be less effective in triggering cell division than populations containing a source of non-T cells.     

A close relationship between cytotoxic T lymphocytes generated in the mixed lymphocyte culture and insulin receptor-bearing lymphocytes: enrichment by density gradient centrifugation.

In contrast to liver, fat, muscle, the fibroblast, and the monocyte, the lymphocyte does not bear insulin receptors unless it is activated by antigen or mitogen. Antigen stimulation by skin graft or in the mixed lymphocyte culture (MLC) generates a population of T lymphocytes, the effector function of which can be augmented by insulin. In the same pool of cells are found T lymphocytes with newly emergent insulin receptors capable of supporting this augmentation. This study demonstrates the close relationship between the augmentable effector T cell and the insulin receptor-bearing cell and strongly suggests that these cells are identical. Splenic lymphocytes from unidirectional murine MLCs were separated into light and heavy fractions by discrete density gradient eentrifugation daily and assayed for cellular-mediated cytotoxicity and for insulin receptors. Receptor-bearing and cytotoxic lymphocytes waxed and waned together primarily in the light fraction. Receptor-positive cells preceded effectors by 24 hr and the two characteristics were highly correlated over time (r ≥ 0.95). T-Cell depletion by specific antisera or by immunoabsorbent column chromatography demonstrated that most, but not all, receptor-bearing cells were T cells and that virtually all effectors were also receptor positive. When the insulin receptor was functionally removed from the lymphocyte membrane by trypsin proteolysis, effector function ceased. The return of cytotoxicity was accompanied by return of the lymphocyte insulin receptor. Receptor-bearing cells were predominantly of the Ly 2⁺3⁺ pedigree but Ly 1⁺ cells were also induced to bear the insulin receptor along with a few non-T cells. These data show that the emergence of a lymphocyte insulin receptor is not just a fortuitous marker event of cellular activation but provides a structure capable of supporting lymphocyte effector function. The appearance of Ly 1⁺ receptor-bearing cells suggests the alloactivation of T helpers and their participation in a T-T cooperative event.     

CD4+ T cells modulate expansion and survival but not functional properties of effector and memory CD8+ T cells induced by malaria sporozoites

CD4⁺ helper T cells are critical orchestrators of immune responses to infection and vaccination. During primary responses, naïve CD8⁺ T cells may need “CD4 help” for optimal development of memory populations. The immunological factors attributed to CD4 help depend on the context of immunization and vary depending on the priming system. In response to immunization with radiation-attenuated Plasmodium yoelii sporozoites, CD8⁺ T cells in BALB/c mice fail to generate large numbers of effector cells without help from CD4⁺ T cells – a defect not observed in most systems. Given this unique early dependence on CD4 help, we evaluated the effects of CD4⁺ cells on the development of functional properties of CD8⁺ T cells and on their ability to abolish infection. First, we determined that this effect was not mediated by CD4⁺ non-T cells and did not involve CD1d-restricted NKT cells. We found that CD8⁺ T cells induced by sporozoites without CD4 help formed memory populations severely reduced in magnitude that could not limit parasite development in the liver. The inability of these “helpless” memory T cells to protect is not a result of defects in effector function, as their capacity to produce cytokines and undergo cytotoxic degranulation was indistinguishable from control memory T cells. These data indicate that CD4⁺ T help may not be necessary to develop the functional attributes of CD8⁺ T cells; however they are crucial to ensure the survival of effector and memory cells induced in primary responses.     

Activation of immunoglobulin allotype-specific T cells by B cells

To investigate the role of Ia and immunoglobulin (Ig) molecules of B cells in alloantigen-specific and nominal antigen-specific T-cell activations, the ability of B cells to stimulate Ig allotype-specific T cells was examined. T15-primed B10.BR T cells responded to MOPC 315 (IgA myeloma protein derived from BALB/c) as well as T15 but not to MOPC31c (IgG, myeloma protein). These T cells were stimulated by papain-digested Fc fragment of T15. Thus, T15-primed B10.BR T cells were shown to be specific for Ig allotype of T15, that is, Igh-2a. T15-specific B10.BR T cells were selected by 10-day cultures with T15 in vitro. They responded to BALB.K spleen cells without addition of soluble T15 antigen to the assay culture. Stimulator cells in this mixed lymphocyte reaction (MLR)-like response between T15-specific B10.BR T cells and BALB.K spleen cells were Thy-1⁻, Ia⁺ cells and these responses were blocked by anti-Ia^κ antibodies. Furthermore, Sephadex G-10-passed BALB.K B cells stimulated the proliferation of T15-specific B10.BR T cells, while they failed to stimulate allogeneic BALB/c spleen cells. The stimulating ability of B cells in this MLR-like response of T15-specific B10.BR T cells was shown to be genetically restricted, namely, both H-2 and non-H-2 genes are involved in the manifestation of the stimulating ability. This system will provide a useful model for studying the role of B-cell surface Ig and Ia molecules in the activation of antigen-specific T cells and alloreactive T cells.     

T Cells Contribute to Stroke-Induced Lymphopenia in Rats

Stroke-induced immunodepression (SIID) results when T cell and non-T immune cells, such as B cells, NK cells and monocytes, are reduced in the peripheral blood and spleen after stroke. We investigated the hypothesis that T cells are required for the reductions in non-T cell subsets observed in SIID, and further examined a potential correlation between lymphopenia and High-mobility group protein B1 (HMGB1) release, a protein that regulates inflammation and immunodepression. Our results showed that focal ischemia resulted in similar cortical infarct sizes in both wild type (WT) Sprague Dawley (SD) rats and nude rats with a SD genetic background, which excludes the possibility of different infarct sizes affecting SIID. In addition, the numbers of CD68-positive macrophages in the ischemic brain did not differ between WT and nude rats. Numbers of total peripheral blood mononuclear cells (PBMCs) or splenocytes and lymphocyte subsets, including T cells, CD4⁺ or CD8⁺ T cells, B cells and monocytes in the blood and spleen, were decreased after stroke in WT rats. In nude rats, however, the total number of PBMCs and absolute numbers of NK cells, B cells and monocytes were increased in the peripheral blood after stroke; nude rats are athymic therefore they have few T cells present. Adoptive transfer of WT splenocytes into nude rats before stroke resulted in lymphopenia after stroke similar to WT rats. Moreover, in vitro T cell proliferation stimulated by Concanavalin A was significantly inhibited in WT rats as well as in nude rats receiving WT splenocyte adoptive transfer, suggesting that T cell function is indeed inhibited after stroke. Lastly, we demonstrated that stroke-induced lymphopenia is associated with a reduction in HMGB1 release in the peripheral blood. In conclusion, T cells are required for stroke-induced reductions in non-T immune cells and they are the most crucial lymphocytes for SIID.     

Establishment of human T cell clones exhibiting natural killer-like activity

We have succeeded in establishing a method to reproducibly immortalize human T cells by oncogene(s) transfection (Alam, 1997). This study was based on our previous discoveries that these immortalized T cell lines contained T cells which showed cytotoxicity against K562 cells in MHC-nonrestricted manner. Then we attempted to obtain human T cell clones exhibiting natural killer-like activity. Here, we tried to establish clones from these immortalized T cell lines by limiting dilution after stimulation with K562 cells, and then obtained 16 T cell clones. Two clones among them maintained their stability and showed vigorous growth phenotype. Thus we selected these two clones for further analysis. One is derived from the T cell line transfected with oncogenes ras and fos, the other is from the T cell line transfected with myc and fos. Both clones were demonstrated to be CD4⁺ T cells, indicating that CD4⁺ T cells were preferably expanded from T cell lines immortalized by oncogene transfection. These two clones showed cytotoxicity against K562 cells, indicating that these two T cell clones still retain a natural killer-like activity of killing target cells of K562 cells in a MHC-nonrestricted manner. The natural killer-like activity of the T cell clones was shown to be stable for more than 2 yr when cultured in the presence of IL-2, indicating that introduction of two oncogenes such as ras/fos or myc/fos resulted in the acquisition of infinite replicative life-span but not in transformational alteration of cellular function. This revised version was published online in August 2006 with corrections to the Cover Date.     

Surface markers on the T cells that regulate cytotoxic T-cell responses

Regulatory T cells can be obtained from primary mixed lymphocyte cultures of CBA spleen cells responding to BALB/c stimulators. At day 3 of culture, T cells are generated which can either help or suppress the generation of cytotoxic T cells in a second primary MLC culture. The regulatory activity observed depends on the conditions employed in the assay system allowing independent assay of different functional cell types which coexist in the cultures. Both the helper activity and the suppressor activity are mediated by differentiated antigen-specific T cells whose function is radioresistant. The Ly phenotype of these regulatory cells was tested. At day 3 of the first-step culture, the phenotype of the helper cells is Ly 1.1⁺ Ly 2.1^–, whereas the inhibitory cells are Ly 1.1⁺ Ly 2.1⁺. At day 5 of MLC culture, suppressor activity and helper activity are also observed. However, at this point, a suppressor cell which is Ly 1.1^– Ly 2.1⁺ represents the major inhibitory activity. It is not clear whether this change in suppressor cell phenotype as a function of time in culture represents one differentiation pathway or cells derived from two different precursor cells. The Ly phenotype of helper or cytotoxic T cells did not change as a function of time in culture. In day 5 first-step cells, the cytotoxic cells were typed as Ly 1.1⁺ 2.1⁺, whereas the inhibitory cells present in aliquots of the same treated cell population expressed the Ly 1.1^– Ly 2.1⁺ phenotype. Taken together, these observations show that the antigen-specific suppressor cells and helper cells which regulate the generation of cytotoxicity, and the cytotoxic cells themselves represent physically distinct subclasses of T cells.     

Analysis of the role of xenogeneic antigens in the proliferation of human T cells stimulated with autologous non-T cells and phytohemagglutinin-activated T cells

Since conflicting results have been reported about the role of xenoantigens in the proliferation of T cells stimulated with autologous non-T cells, the effect of the exposure of cells to xenogeneic proteins during the isolation procedure and/or the culture period on autologous mixed lymphocyte reactions (AMLR) with non-T cells and phytohemagglutinin-activated T cells as stimulators was investigated. T and non-T cells were isolated by rosetting with 2-aminoethylisothiuronium bromide-treated sheep red blood cells (AET-SRBC), by nylon-wool nitration, and by positive or negative selection with anti-class II HLA antigens and anti-T-cell monoclonal antibodies. Isolation and cultures were performed in presence of fetal calf serum (FCS) or of autologous serum. In both types of AMLR, proliferation of responding cells did not require exposure to xenoantigens. However xenoantigens enhanced the proliferation of cells from some, although not all, the donors tested. There were differences in the degree of proliferation of the cells from the donors tested, but without correlation with the two types of AMLR. These results suggest that both types of AMLR reflect a self-recognition event and not a response to xenoantigens. However the potential interference of xenoantigens, as well as the individual variability, should be taken into account when interpreting the significance of abnormalities of AMLR in immunopathologic processes.     

Growth and characterization of T cell colonies from human thymus

A semisolid microculture system was used to study T cell colonies grown from human thymocytes. Colony growth was absolutely dependent upon media conditioned by peripheral blood leukocytes (PBL) in the presence of phytohemagglutinin. Plating efficiency was further enhanced by the addition of a non-T, adherent, radiation-resistant (7500 rad) PBL subpopulation, but was not enhanced by culture supernatants of these cells. The T colony precursor cell in the thymus occurred with a frequency of 8.0 X 10(-3) and had a surface receptor for the OKT3 monoclonal antibody. Thymocyte colony cells were functionally distinct from PBL and the major thymocyte population. The colony cells proliferated in response to T cell mitogens, but only in the presence of exogenous growth factors. The cells stimulated normal PBL in mixed leukocyte culture (MLC), but did not respond to alloantigens in MLC or in assays of spontaneous cytotoxicity. This culture system should prove helpful in the study of human thymocyte differentiation.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

Regulatory T cells induced by B cells: a novel subpopulation of regulatory T cells

Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4⁺Foxp3⁺ regulatory T cells, several subsets of Foxp3^- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4⁺CD25^- T cells into CD25⁺Foxp3^- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3⁺ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.