In susceptible mice,Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1−

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-γ (IFN-γ) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-γ dependent. Treatment of resistant C57BL/6 mice with anti-IFN-γ allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in Vβ8, 7, 2^? CD4⁺ cells in BALB/c mice and NK1.1^? CD4⁺ cells in anti-IFN-γ treated C57BL/6 mice. These results show that the NK1.1⁺ CD4⁺ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.     

NK1.1+ CD4+ CD8- thymocytes with specific lymphokine secretion

CD4+8^? or CD4^?8⁺ thymocytes have been regarded as direct progenitors of peripheral T cells. However, recently, we have found a novel NK1.1⁺ subpopulation with skewed T cell antigen receptor (TcR) Vβ family among heat-stable antigen negative (HSA^?) CD4⁺8^? thymocytes. In the present study, we show that these NK1.1⁺ CD4+8^? thymocytes, which represent a different lineage from the major NK1.1^? CD4⁺8^? thymocytes or CD4⁺ lymph node T cells, vigorously secrete interleukin (IL)-4 and interfron (IFN)-γ upon stimulation with immobilized anti-TcR-αβ antibody. On the other hand, neither NK1.1^? CD4+8^?thymocytes nor CD4⁺ lymph node T cells produced substantial amounts of these lymphokines. A similar pattern of lymphokine secretion was observed with the NK1.1⁺ CD4⁺ T cells obtained from bone marrow. The present findings elucidate the recent observations that HSA^? CD4⁺8^? thymocytes secrete a variety of lymphokines including IFN-γ, IL-4, IL-5 and IL-10 before the CD4⁺8^? thymocytes are exported from thymus. Our evidence indicates that NK1.1⁺ CD4⁺8^? thymocytes are totally responsible for the specific lymphokine secretions observed in the HSA- CD4⁺8^? thymocytes.     

CD38+ CD45RBlow CD4+ T cells: a population of T cells with immune regulatory activities in vitro

An antibody reactive with CD38 revealed both phenotypic and functional heterogeneity amongst CD45RB^low cells. Functional analysis of the CD38⁺ and CD38⁻ fractions showed that the latter contained T cells which responded to recall antigens and produced high levels of cytokine in response to polyclonal stimulation. In contrast, the CD38⁺ population failed to proliferate or to produce detectable levels of cytokines. Despite appearing unresponsive, the CD38⁺ population significantly inhibited anti-CD3-induced proliferation and cytokine secretion by the reciprocal CD38⁻ population. Immune suppression required stimulation through the TCR and was dependent on a physical interaction between regulatory and responding CD4⁺ populations. It did not involve killing of the responding T cells or secretion of IL-10 or TGF-β. Despite some similarities there is no direct correlation between the in vitro suppression characteristic of the CD38⁺ CD45RB^low subset and in vivo suppression which has been shown to be mediated by unseparated CD45RB^low CD4⁺ T cells. However, these results demonstrate that two functionally distinct subsets of T cells reside within the antigen-exposed or CD45RB^low CD4⁺ T cell population and are thus generated in vivo: (1) conventional memory T cells which proliferate and secrete cytokines in response to activation and (2) a population of regulatory T cells which inhibit T cell activation in vitro. Antibodies reactive with CD38 may provide a useful tool with which to study the role of these T cell subsets in the induction and regulation of the immune response.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.     

Mouse γδ TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I independent,and are developmentally related to αβ TCR+NK1.1+ thymocytes

Mouse T cells co-expressing an αβ T cell receptor (TCR) and the NK1.1 antigen have been shown to be major interleukin (IL)-4-producing cells and could therefore regulate cell-mediated immune responses. We have identified a related subset of thymocytes co-expressing a γδ TCR and NK1.1 which also produce IL-4. Unlike αβ⁺NK1.1⁺ thymocytes, the selection of γδ⁺NK1.1⁺ thymocytes is not dependent upon β2-microglobulin (β2m)-associated class I molecule expression because these cells are present in β2m-deficient mice. This suggests that γδ⁺NK1.1⁺ T cells may regulate immune responses to a different variety of antigens. However, the development of αβ⁺NK1.1⁺ and αβ⁺NK1.1⁺ thymocytes appears to be related. Analysis of different mutant mice lacking αβ⁺NK1.1⁺ thymocytes revealed a specific increase in γδ⁺NK1.1⁺ thymocyte production when the block in αβ⁺NK1.1⁺ thymocyte differentiation occurs after β TCR rearrangement.     

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor β (IL-2Rβ CD122) and the polymorphic antigen NK1.1. These “NK-like” T cells are distinguished by their highly skewed Vα and Vβ repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1⁺ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2Rβ⁺ T cells with a skewed Vβ repertoire characteristic of “NK-like” T cells. Furthermore lack of NK1.1 expression on IL-2Rβ⁺ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2Rβ⁺ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that “NK-like” T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.     

Expansion of CD56+ NK T and γδ T cells from cord blood of human neonates

A particular T cell population expressing NK cell markers, CD56 and CD57, exists in humans. Many CD56⁺ T and CD57⁺ T cells (i.e. NK T cells) exist in the liver and increase in number in the blood with ageing. They may be a human counterpart of extrathymic T cells, similar to NK1.1⁺ CD3^int cells seen in mice. We investigate here the existence of such NK T cells in human cord blood and the in vitro expansion of these cells by the stimulation of human recombinant IL-2 (rIL-2). There were very small populations (< 1.0%) of CD56⁺ T cells, CD57⁺ T cells, and γδ T cells in cord blood. However, all of these populations increased in number after birth and with ageing. When lymphocytes in cord blood were cultured with rIL-2 (100 U/ml) for 14 days, CD56⁺ T cells expanded up to 25% of T cells. CD57⁺ T cells were never expanded by these in vitro cultures. The expansion of γδ T cells (mainly Vγ9^? non-adult type) also occurred in the in vitro culture. A considerable proportion of CD56⁺ T cells was found to use Vα24 (i.e. equivalent to invariant Vα14 chain used by murine NK T cells) for TCR αβ. These results suggest that neonatal blood contains only a few NK T cells but CD56⁺ NK T cells and γδ T cells are able to expand in vitro.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

NK 1.1+ T cell: a two-faced lymphocyte in immune modulation of the IL-4/IFN-gamma paradigm

T lymphocytes expressing NK1.1 marker (NK1.1⁺) have been suggested as being important in peripheral immune modulation. Alteration of the balance between Th1 proinflammatory and Th2 anti-inflammatory cytokine-producing cells can ameliorate immune-mediated disorders. The aim of the study was to determine the role of NK1.1⁺ lymphocytes in the pathogenesis of tolerance and proinflammatory states and to determine their role in altering the Th1/Th2 balance in experimental colitis. Colitis was induced in C57/B6 mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS colitis colonic wall. Standard clinical, macroscopic, and microscopic scores were used for colitis assessment. Liver-associated lymphocytes and splenocytes were harvested 14 days following tolerance induction. Depletion of NK 1.1+ lymphocytes was performed 36 hr before lymphocyte harvesting. Lymphocytes were cultured for 12 hr with Con A and colitis extracted proteins. To evaluate the role of NK1.1⁺ lymphocytes in keeping a balance between immunogenic and tolerogenic subsets of cells, intracellular staining and flow cytometry assays were performed in tolerized and nontolerized mice. IL-4, IL-12, and IFN- levels were measured by ELISA. Administration of mouse-derived colitis-extracted proteins ameliorated experimental colitis. Tolerized mice exhibited significant improvement in all macroscopic and microscopic parameters for colitis. Depletion of NK1.1 following tolerance induction significantly decreased the CD4⁺IL-4+/CD4⁺IFN-⁺ ratio in tolerized mice. However, depletion of NK1.1 lymphocytes in nontolerized mice increased the CD4⁺IL-4⁺/CD4⁺IFN-⁺ ratio, compared with nondepleted nontolerized mice. Induction of tolerance led to an increase in IL4 and a decrease in IFN- levels. In the experimental colitis model NK1.1+ lymphocytes play a dual role: In the presence of peripheral tolerance they may be accountable for keeping the high CD4⁺IL-4⁺/CD4⁺IFN-⁺ ratio and disease alleviation. However, in nontolerized conditions they may induce a proinflammatory shift.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

NK1.1+ T cells from IL-7-deficient mice have a normal distribution and selection but exhibit impaired cytokine production

Particular subsets of T cells expressing the NK1.1 antigen havebeen proposed to play an immune regulatory role by their fastand strong production of cytokines, in particular IL-4. We soughtto determine factors driving the functional differentiationof NK1.1⁺ T cells. Since NK1.1⁺ T cells are exquisitely sensitiveto IL-7 stimulation, we analyzed the development, selectionand IL-4 production of NK1.1⁺ T cells in IL-7 deficient mice(IL-7–/–mice). Besides a sharp reduction of allT cell subsets, NK1.1⁺ T cells develop at normal relative frequenciesin IL-7–/–;mice. They also undergo a normal selectionprocess, as revealed by the biased V_ß TCR repertoireidentical to the one in IL-7+/+ mice. However, NK1.1₊ T cellsfrom IL-7+/+ mice were found to be impaired in IL-4 and IFN-production in in vitro and in vivo models. In addition, IL-7was able to restore IL-4 production by NK1.1⁺ thymocytes fromIL-7–/– mice. Finally, IL-7 but not IL-4 was ableto maintain and increase IL-4 production by NK1.1⁺ thymocytesfrom normal mice. These data suggest that the functional maturationof NK1.1⁺ T cells requires a cytokine-driven differentiationprocess, in which IL-7 plays a major role.     

Marked anti-tumor effects of CD8+CD62L+ T cells from melanoma-bearing mice

CD8⁺CD62L⁺ T cells have been shown to play pivotal roles in anti-viral immunity, chronic myeloid leukemia and renal cell carcinoma. Recently, CD8⁺CD62L⁺ T cells from naïve mice (nCD8⁺CD62L⁺ T cells) have shown superior anti-tumor properties in melanoma-bearing mice. Considering that antigen-specific memory T cells have shown to possess more potent immunity than non-specific memory T cells, we hypothesized that CD8⁺CD62L⁺ T cells from tumor-bearing individuals (mCD8⁺CD62L⁺ T cells) might have superior anti-tumor effect than nCD8⁺CD62L⁺ T cells. Therefore, we investigated phenotypes, functions and the in vivo distribution of mCD8⁺CD62L⁺ T cells in tumor-bearing mice. We found that, while keeping the features of central memory T cells, the frequency of mCD8⁺CD62L⁺ T cell in the spleen of tumor-bearing mice was significantly higher than that the one of nCD8⁺CD62L⁺ T cell in naive mice. Moreover, we demonstrated that mCD8⁺CD62L⁺ T cells had higher proliferation rate and IFN-γ production than nCD8⁺CD62L⁺ T cells, in vitro. We performed adoptive transfer of mCD8⁺CD62L⁺ T cells into melanoma-bearing mice and tracked them in spleen, lymph nodes and in melanoma tissues. Our results show that mCD8⁺CD62L⁺ T cells had stronger in vivo anti-tumoral activity than nCD8⁺CD62L⁺ T cells. This study highlights the therapeutic potential of mCD8⁺CD62L⁺ T cells in the immunotherapy of melanoma and possibly other tumors.     

Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse

An immunoregulatory role has recently been attributed to the discrete subset of major histocompatibility complex class I-restricted NK1⁺ mature heat-stable antigen⁻ (HSA⁻) thymocytes expressing an unusual Vβ8-biased T cell receptor repertoire. NK1⁺ T cells are the main interleukin (IL)-4 producers upon priming. We have studied the size and the function of this subset in the nonobese diabetic (NOD) mouse, a model of spontaneous T cell-mediated autoimmune insulin-dependent diabetes. This study was complicated by the absence in this strain of the NK1.1 allele, the only one for which an antibody is available. To circumvent this difficulty, the cells, hereafter designated the NK1⁺-like T subset, were characterized by the use of monoclonal antibodies which showed the Vβ8 bias in the CD44⁺ Ly-49⁺ MEL-14⁻ 3G11⁻ thymocyte subset of non-autoimmune strains and of its absence in class I-deficient (β2-microglobulin^−/−) mice. A clear deficit in the number of NK1⁺-like cells was evidenced at 3 weeks of age in NOD mice. It was still present at 8 weeks of age in the double-negative CD4⁻CD8⁻ population. The functional anomaly was even more striking: NOD mouse NK1⁺-like thymocytes virtually lacked the ability to produce IL-4 at 3 weeks and still showed a very reduced capacity at 8 weeks. NK1⁺ T cell deficiency was also suggested in the periphery by the reduction of Ly-49A⁺ cells in the spleen of 3- and 8-week-old NOD mice and the absence of short-term production of IL-4 in vitro by NOD mouse spleen cells 90 min after the administration of anti-CD3 antibody, a response attributed to NK1⁺ T cells. Taken together, these data demonstrate a very early defect in NK1⁺-like T cells which could be involved in the genesis of autoimmunity in NOD mice through a deficiency in Th2 cell function.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4.

In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti-CD3 and anti-CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non-specific mechanism. C57BL/6 mice were inoculated with anti-natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T-cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin-4 (IL-4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T-cell deficient [beta 2-microglobulin beta 2m-deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when beta 2m-deficient mice were reconstituted with NK T cells. NK-specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL-4.     

TCR-mediated target cell lysis by CD4+NK1+ liver T lymphocytes

In the liver, an unusual T lymphocyte population exists with the intriguing phenotype CD4+NK1+ TCR alpha beta int. Thus far, functions of these lymphocytes remained elusive. Recently, however, CD4+NK1+ liver T lymphocytes have been shown to produce cytokines. Here we show that sorted CD4+NK1+ liver lymphocytes from naive mice lyse target cells after TCR alpha beta or CD3, but not TCR gamma delta, engagement. Liver lymphocytes from beta 2-microglobulin-deficient gene disruption mutant mice failed to express such cytolytic activities and in vivo treatment with anti-NK1.1 mAb or anti-CD4 mAb, but not anti-CD8 mAb, markedly reduced target cell lysis. In vivo administration or rIL-12 impaired TCR alpha beta-mediated target cell lysis by liver lymphocytes. A similar down-regulation of cytolytic activities was observed with liver lymphocytes from mice infected with Listeria monocytogenes or Mycobacterium bovis BCG, which are potent IL-12 inducers. We anticipate (i) that cytolytic CD4+NK1+ T lymphocytes contribute to immunosurveillance of inflammatory processes in the liver and (ii) that they are influenced by IL-12.      

CD4+CD8− thymocytes that express L-selectin protect rats from diabetes upon adoptive transfer

Previous studies have shown that insulin-dependent diabetes can be induced in normal PVG.RT1^u rats by a protocol of adult thymectomy and irradiation. The injection of CD4⁺ T cells from non-irradiated syngeneic donors prevents the onset of disease in approximately 50 % of pre-diabetic recipients but all rats are protected if a particular subset of CD4⁺ cells is transferred. These protective cells express TCRαβ and have a memory phenotype, being CD45RC^low RT6⁺. Further studies have demonstrated that the transfer of CD4⁺CD8⁻ thymocytes, like that of unfractionated CD4⁺ peripheral T cells, also protects approximately half of recipients from diabetes suggesting that, as with the peripheral T cells, a functional heterogeneity may exist amongst CD4⁺CD8⁻ thymocytes. In this study, we show that L-selectin is expressed by 50–60 % of all CD4⁺CD8⁻ thymocytes from 6-week-old rats. Adoptive transfer of these populations into thymectomized and irradiated rats revealed that the protection from diabetes observed by CD4⁺CD8⁻ thymocytes was mediated almost entirely by the L-selectin⁺ subset. Cells with this phenotype were also able to mediate both humoral and cell mediated responses, providing primed B cells with help for secondary antibody responses and mediating local graft-versus-host reactions. L-selectin⁻ CD4⁺CD8⁻ thymocytes failed to mediate these responses. These data indicate that CD4⁺CD8⁻ thymocytes must mature to the stage of L-selectin expression, before they can mediate normal T cell function. The implications of these results are discussed with respect to the possible role of murine NK1.1⁺ thymocytes in the control of autoimmunity.     

Characterization of human CD25+ CD4+ T cells in thymus,cord and adult blood

CD4⁺ CD25⁺ regulatory T cells prevent organ‐specific autoimmune diseases in various animal models. We analysed human lymphoid tissues to identify similar CD25⁺ regulatory T cells. Adult peripheral blood contained two populations of CD4⁺ T cells that expressed CD25 at different densities. The larger population (≈ 40%) expressed intermediate levels of CD25 (CD25⁺) and displayed a memory T‐cell phenotype (CD45RA^?/RO⁺, CD45RB^low, CD95⁺, CD62L^low, CD38^low). The smaller population of cells (≈ 2%) expressed very high levels of CD25 (CD25⁺⁺). In addition to the activation/memory T‐cell antigens mentioned above they also expressed intracellular CD152 (CTLA‐4) as well as enhanced levels of cell‐surface CD122, similar to the murine CD4⁺ CD25⁺ regulatory counterpart. To exclude that the CD25⁺⁺ cells had not been recently primed by external antigen we analysed cord blood and thymus. CD25⁺⁺, CD152⁺ and CD122⁺⁺ cells were present in paediatric thymus (10% of CD4⁺ CD8^? thymocytes) expressing signs of recent selection (CD69⁺) and in cord blood (5% of CD4⁺ cells) where they showed a naive phenotype. In addition, cord blood contained a small population of CD25⁺ cells (≈ 2% of CD4 T cells) that were CD152^? and CD122^low and displayed signs of activation. Together with published data that CD25⁺ CD25⁺⁺ cells from the thymus and peripheral blood are regulatory, our results suggest that regulatory CD25⁺ T cells leave the thymus in a naïve state and become activated in the periphery.     

Co-stimulatory pathways controlling activation and peripheral tolerance of human CD4+CD28− T cells

Co-stimulation mediated by the CD28 molecule is considered critical in the activation of CD4⁺ T cells. In patients with rheumatoid arthritis and infrequently in normal individuals, CD4⁺ T cells lacking CD28 expression are expanded and contain clonogenic populations. To analyze whether these cells are independent of co-stimulatory requirements or whether they use co-stimulatory signals distinct from the CD28 pathway, we have compared CD4⁺ CD28⁺ and CD4⁺ CD28^?T cell clones isolated from rheumatoid arthritis patients. Accessory cells supported the induction of CD25 expression as well as of proliferative responses after anti-CD3 cross-linking and prevented the induction of anergy in CD4⁺ CD28^? T cell clones. In contrast to CD4⁺CD28⁺ T cells, the presence of accessory cells did not enhance the secretion of interleukin (IL)-2, interferon-γ, or IL-4. The co-stimulatory signals did not involve CD28/CTLA-4–CD80/CD86 receptor-ligand interactions. The proliferative response of CD4⁺CD28^? T cells could not be blocked by anti-CD2, anti-CD18, and anti-CD58 antibodies, suggesting that these receptor-ligand interactions cannot provide CD28^? independent co-stimulation. Our data suggest that CD4⁺CD28^? T cells require co-stimulatory signals for optimal induction of cell growth and CD25 expression as well as for the prevention of anergy. The co-stimulatory receptor-ligand interaction is independent of the CD28 pathway and may be involved in the oligoclonal expansion of the CD4⁺ CD28^? T cell subset in rheumatoid arthritis.