The majority of CD4+8- thymocytes are functionally immature.

The thymus is the major site of T cell development and repertoire selection. During these processes, T cells segregate into two subsets that express either CD4 or CD8 accessory molecules, the phenotype of peripheral T cells. Analysis of CD4+8- thymocytes revealed that the majority of these cells express the heat-stable Ag (HSA) but not the nonclassical class I Ag, Qa-2. This HSA+, Qa-2- phenotype is similar to that of the less mature, CD4+8+ thymocytes. The remaining CD4+8- thymocytes possess the HSA-, Qa-2+ phenotype of peripheral T cells. To determine whether the Qa-2-, CD4+8- thymic subset is fully mature, we have analyzed the functional status of these CD4+8- subpopulations. The results indicate that only those thymocytes which express Qa-2 are fully responsive to anti-TCR stimulation in a manner analogous to peripheral T cells. The Qa-2- subset is nonresponsive to stimulation by anti-TCR antibodies that have been immobilized to plastic, even in the presence of lymphokines or syngeneic APC. This subset is, however, capable of proliferating to allogeneic cells or to anti-TCR on the surface of syngeneic APC, although not to the levels achieved by Qa-2+ thymocytes. Thus, the Qa-2- subset appears to require additional interactions which are not necessary for peripheral T cells or Qa-2+ thymocytes. Relevant to this issue, the Qa-2+ thymocyte subset does not appear until relatively late in development, and does not reach adult frequencies until several weeks after birth. These results would suggest that there is a progression from HSA+, Qa-2- to HSA-, Qa-2+ which parallels the maturation of functional responsiveness. These findings are important to understanding T cell selection since thymocytes with such a decreased responsiveness may have a differential capacity for tolerance induction. The results presented suggest that the bulk of CD4+8- thymocytes are not fully mature and that Qa-2 may serve as a marker for T cells with a more complete functional competence.     

Fas ligand costimulates the in vivo proliferation of CD8+ T cells

Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8(+) T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL(+) CD8(+) T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL(-)counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8(+) T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8(+) T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo.     

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.     

Immature CD4- CD8+ murine thymocytes

Mature thymocytes are usually defined and separated from other less mature thymocytes on the basis of their mutually exclusive expression of either CD4 or CD8. However, such murine "single positives" include a subpopulation of immature cells with properties resembling CD4- CD8- thymocytes or CD4+ CD8+ cortical blasts. Most of these immature single positives are CD4- CD8+, some expressing relatively low levels of CD8. They are large, dividing cortisone-sensitive cells found in the outer cortex. They express high levels of the heat-stable antigen (recognized by the monoclonals M1/69, B2A2, and J11d) but they are MEL-14-. The absence of detectable surface CD3, the absence of alpha-chain messenger RNA, and the predominance of the truncated form of the beta-chain messenger RNA all indicate that they do not express the T-cell antigen-receptor complex. Strategies for eliminating such immature cells from preparations of mature thymocytes are given, and their developmental significance is discussed.     

CD8+ T lymphocytes are not required for murine resistance to human filarial parasites.

Mice are resistant to the establishment of infection with the nematode parasite Brugia malayi, an etiologic agent of human lymphatic filariasis. We have recently shown that T and B lymphocyte-deficient C.B.-17 scid/scid mice are permissive for infection with this parasite, whereas coisogenic C.B.-17+/+ mice are resistant. This observation suggests that T and B lymphocytes that comprise the antigen-specific immune system orchestrate murine resistance to B. malayi. In order to define the component of the antigen-specific immune response that is responsible for this resistance, we have tested the susceptibility of beta 2M-/- mice to infection with B. malayi L3 larvae. These mice are homozygous for insertional disruption of their B2m genes, which encode beta 2-microglobulin, the small subunit of the major histocompatibility (MHC) antigens. They do not express beta 2-microglobulin and, as a consequence, fail to express the class I major histocompatibility antigens, and they do not develop the CD8+ class I MHC-restricted cytotoxic T cell subset. We find that these mice are completely resistant to B. malayi, indicating that the CD8+ T lymphocyte subset is not an obligate requirement for murine resistance to human filarial parasites.     

Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii.

C57BL/6 mice chronically infected with an avirulent strain (ME-49) of Toxoplasma gondii were used to study the mechanisms by which T lymphocytes and IFN-gamma prevent reactivation of latent infection. Infected animals were treated with mAb, either anti-CD8, anti-CD4, anti-CD4 plus anti-CD8, anti-IFN-gamma, or anti-CD4 plus anti-IFN-gamma and the mice followed for survival, histopathology, cyst numbers, and spleen cell cytokine responses. In agreement with previously published findings, treatment with anti-IFN-gamma antibodies fully reactivated the asymptomatic infection, inducing massive necrotic areas in the brain with the appearance of free tachyzoites and death of all animals within 2 wk. Mice treated with the combination of anti-CD4 plus anti-CD8 antibodies showed augmented pathology and mortality nearly identical to the anti-IFN-gamma- treated animals. In contrast, treatment with anti-CD4 or anti-CD8 mAb alone failed to result in significantly enhanced brain pathology or mortality. In additional experiments, full reactivation of infection was observed in mice treated with anti-CD4 plus anti-IFN-gamma indicating that CD4+ lymphocytes are not required for the pathology resulting from IFN-gamma neutralization. Cytokine measurements on parasite Ag-stimulated spleen cells from mAb-treated mice indicated that both CD4+ and CD8+ cells produce IFN-gamma whereas only CD4+ cells contribute to parasite Ag-induced IL-2 synthesis. Together, these results suggest that CD4+ and CD8+ lymphocytes act additively or synergistically to prevent reactivation of chronic T. gondii infection probably through the production of IFN-gamma.     

The FOXP3+ subset of human CD4+CD8+ thymocytes is immature and subject to intrathymic selection

FOXP3, believed to be the regulatory T (Treg)-cell determining factor, is already expressed at the CD4+CD8+ thymocyte stage, but there is disagreement whether these cells are the precursors of mature CD4+CD8(-) Treg cells. Here, we provide a quantitative analysis of FOXP3 expression in the human thymus. We show that a subset of CD4+CD8+ cells already expressed as much FOXP3 as the FOXP3+ CD4+CD8(-) cells, and like mature Treg cells were CD127 low. In contrast to earlier data, CD8+CD4(-) thymocytes expressed significantly lower levels of FOXP3 than either the CD4+CD8+ or CD4+CD8(-) subsets. The CD4+CD8+ double-positive cells also expressed recombination-activating gene-2, suggesting that they were still immature. Although the FOXP3+ double-positive cells are thus putatively the precursors of the mature CD4+CD8(-)FOXP3+ subset, their frequency did not predict the frequency of more mature Treg cells, and analysis of T-cell antigen receptor repertoire showed clear differences between the two subsets. Although these data do not rule out an independent CD4+CD8+ Treg cell subset, they are consistent with a model of human Treg cell development in which the upregulation of FOXP3 is an early event, but the first FOXP3+ population is still immature and subject to further selection. The upregulation of FOXP3 may thus not be the final determining factor in the commitment of human thymocytes to the Treg cell lineage.     

Cutting edge: blockade of the CD28/B7 costimulatory pathway inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells.

The effect of blocking the CD28/B7 costimulatory pathway on intestinal allograft rejection was examined in mice. Murine CTLA4Ig failed to prevent the rejection of allografts transplanted into wild-type or CD4 knockout (KO) mice but did inhibit allograft rejection by CD8 KO recipients. This effect was associated with decreased intragraft mRNA for IFN-gamma and TNF-alpha and increased mRNA for IL-4 and IL-5. This altered pattern of cytokine production was not observed in allografts from murine CTLA4Ig-treated CD4 KO mice. These data demonstrate that blockade of the CD28/B7 pathway has different effects on intestinal allograft rejection mediated by CD4+ and CD8+ T cells and suggest that these T cell subsets have different costimulatory requirements in vivo. The results also suggest that the inhibition of CD4+ T cell-mediated allograft rejection by CTLA4Ig may be related to down-regulation of Th1 cytokines and/or up-regulation of Th2 cytokines.     

Subpopulations of mature murine thymocytes: properties of CD4-CD8+ and CD4+CD8- thymocytes lacking the heat-stable antigen

The heat-stable antigen (HSA), recognized by the monoclonal antibodies M1/69, B2A2, and J11d, is low or absent on the surface of most murine peripheral T cells but present on all but 3% of thymocytes. The CD4-CD8+ and CD4+CD8- or "single positive" thymic populations may be divided into further subgroups based on surface HSA expression. One group, CD4-CD8+ and expressing very high levels of HSA (HSA++), is an immature, T cell antigen receptor (TcR) negative, outer cortical blast cell. However, a further subdivision of CD4-CD8+ and CD4+CD8- single positives may be made, into those negative to low for HSA (HSA-) and those expressing moderate amounts of HSA (HSA+). The proportion of HSA- single positives is low in the thymus of young mice, whereas the proportion of HSA+ single positives is similar to that of the adult. Both the HSA- and the HSA+ subsets of single positive thymocytes from adult mice are CD3+ and express the normal peripheral T cell incidence of V beta 8 determinants on the TcR. On stimulation with concanavalin A in limit-dilution culture both HSA- and HSA+ subsets of single positive thymocytes give a high frequency of proliferating clones, and the clones from both HSA- and HSA+ subsets of CD4-CD8+ thymocytes are cytotoxic. Thus both HSA- and HSA+ single positive thymocytes are functionally mature. The HSA- subsets of single positive thymocytes differ from the HSA+ subsets in being slightly larger in size, in expressing higher levels of MEL-14, in binding more peanut agglutinin, and in including a proportion of cells expressing high levels of the Pgp-1 glycoprotein. It is suggested that HSA- CD4-CD8+ and HSA- CD4+CD8- thymocytes are more mature than their HSA+ counterparts, and might represent a previously activated or "memory" thymic subpopulation.     

CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.     

IL-2 is not required for the initiation of CD8 T cell cycling but sustains expansion

Based primarily on in vitro data, IL-2 is believed to be the key cytokine for initiation of the cell cycle of activated T cells. However, the role of IL-2 remains unresolved for T cell responses in vivo. We examined whether the absence of IL-2-mediated signaling in CD8 T cells affected initiation of proliferation. Our results conclusively demonstrated that initial division of Ag-specific CD8 T cells following priming was IL-2 independent, regardless of the context in which Ag was presented. In contrast, the latter stage of the proliferative phase was IL-2-dependent, particularly in nonlymphoid tissues. Thus, activated CD8 T cells initially undergo IL-2-independent proliferation, but reach a critical juncture where the requirement for IL-2 as a growth factor gains prominence.     

Human T-cell leukemia virus type I-induced proliferation of human immature CD2+CD3- thymocytes.

The mitogenic activity of human T-cell leukemia virus type I (HTLV-I) is triggering the proliferation of human resting T lymphocytes through the induction of the interleukin-2 (IL-2)/IL-2 receptor autocrine loop. This HTLV-I-induced proliferation was found to be mainly mediated by the CD2 T-cell antigen, which is first expressed on double-negative lymphoid precursors after colonization of the thymus. Thus, immature thymocytes express the CD2 antigen before that of the CD3-TCR complex. We therefore investigated the responsiveness of these CD2+CD3- immature thymocytes and compared it with that of unseparated thymocytes, containing a majority of the CD2+CD3+ mature thymocytes, and that of the CD2-CD3- prothymocytes. Both immature and unseparated thymocytes were incorporating [3H]thymidine in response to the virus, provided that they were cultivated in the presence of submitogenic doses of phytohemagglutinin. In contrast, the prothymocytes did not proliferate. Downmodulation of the CD2 molecule by incubating unseparated and immature thymocytes with a single anti-CD2 monoclonal antibody inhibited the proliferative response to HTLV-I. These results clearly underline that the expression of the CD2 molecule is exclusively required in mediating the proliferative response to the synergistic effect of phytohemagglutinin and HTLV-I. Immature thymocytes treated with a pair of anti-CD2 monoclonal antibodies were shown to proliferate in response to HTLV-I, even in the absence of exogenous IL-2. We further verified that the proliferation of human thymocytes is consecutive to the expression of IL-2 receptors and the synthesis of IL-2. These observations provide evidence that the mitogenic stimulus delivered by HTLV-I is more efficient than that provided by other conventional mitogenic stimuli, which are unable to trigger the synthesis of endogenous IL-2. Collectively, these results show that the mitogenic activity of HTLV-I is able to trigger the proliferation of cells which are at an early stage of T-cell development. They might therefore represent target cells in which HTLV-I infection could favor the initiation of the multistep lymphoproliferative process leading to adult T-cell leukemia.     

CTLA-4 is not required for induction of CD8(+) T cell anergy in vivo.

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.     

CD36 is differentially expressed by CD8+ splenic dendritic cells but is not required for cross-presentation in vivo

Cross-presentation allows the processing of Ags from donor cells into the MHC class I presentation pathway of dendritic cells (DCs). This is important for the generation of cytotoxic T cell immunity and for induction of self tolerance. Apoptotic cells are reported to be efficient targets for cross-presentation, and in vitro studies using human DCs have implicated CD36 in their capture. In support of a role for CD36 in cross-presentation, we show that this molecule is differentially expressed by CD8(+) splenic DCs, which previously have been identified as responsible for cross-presentation in the mouse. Three different cross-presentation models were examined for their dependence on CD36. These included cross-priming to OVA-coated spleen cells and cross-tolerance to OVA transgenically expressed in the pancreatic islet beta cells under constitutive conditions or during beta cell destruction. In these models, CD36 knockout DCs were equivalent to wild-type DCs in their capacity to cross-present either foreign or self Ags, indicating that CD36 is not essential for cross-presentation of cellular Ags in vivo.     

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.     

Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.     

CTLA-4 engagement acts as a brake on CD4+ T cell proliferation and cytokine production but is not required for tuning T cell reactivity in adaptive tolerance

Adaptive tolerance is the physiologic down-regulation of T cell responsiveness in the face of persistent antigenic stimulation. In this study, we examined the role of CTLA-4 in this process using CTLA-4-deficient and wild-type TCR transgenic, Rag2(-/-), CD4(+) T cells transferred into a T cell-deficient, Ag-expressing host. Surprisingly, we found that the tuning process of adoptively transferred T cells could be induced and the hyporesponsive state maintained in the absence of CTLA-4. Furthermore, movement to a deeper state of anergy following restimulation in vivo in a second Ag-bearing host was also unaffected. In contrast, CTLA-4 profoundly inhibited late T cell expansion in vivo following both primary and secondary transfers, and curtailed IL-2 and IFN-gamma production. Removal of this braking function in CTLA-4-deficient mice following Ag stimulation may explain their lymphoproliferative dysregulation.     

IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.