In vivo behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes.

The selectins and the beta 2-integrins (CD11/CD18) mediate distinct adhesive interactions between neutrophils and endothelial cells. Selectins are believed to initiate binding by mediating neutrophil rolling, whereas beta 2-integrins are required for subsequent activation-induced firm sticking and emigration. In vitro evidence suggests that two endothelial cell selectins, P- and E-selectin, can mediate rolling by binding to the carbohydrate ligand sialyl-Lewisx (sLex) on neutrophil surface glycoconjugates. To test the relative contribution of selectins and beta 2-integrins in vivo we used intravital microscopy to study the behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes. Neutrophils from a patient suffering from CD18 deficiency showed normal rolling behavior but were incapable of sticking or emigrating upon chemotactic stimulation. Neutrophils from a second patient with a newly described adhesion deficiency had normal CD18 but did not express sLex. These neutrophils rolled poorly and also failed to stick in venules under shear force. Under static conditions, however, chemoattractant-induced sticking and emigration could be observed. This demonstrates that both selectin-carbohydrate-mediated initiation of adhesion and subsequent activation-induced beta 2-integrin engagement are essential for the normal function of human neutrophils in vivo.     

T helper cell 1-type CD4+ T cells, but not B cells, mediate colitis in interleukin 10-deficient mice

Mice rendered deficient in the production of interleukin 10 (IL-10-/-) develop a chronic inflammatory bowel disease (IBD) that predominates in the colon and shares histopathological features with human IBD. Our aim was to identify which cell type(s) can mediate colitis in IL-10-/- mice. We detected an influx of immunoglobulin-positive cells into the colon and the presence of colon-reactive antibodies in the serum of IL- 10-/- mice. To assess a pathogenic role for B cells, we generated a B cell-deficient (B-/-) strain of IL-10-/- mice. B-/-IL-10-/- mice acquired a severe colitis analogous to that IL-10-/- mice, implying that B cells were not the primary mediator of IBD in this model. A series of cell transfer experiments was performed to assess a pathogenic role for T cells. When IL-10-/- T cell-enriched lamina propria lymphocytes (LPL) or intraepithelial lymphocytes (IEL) were transferred into immunodeficient recombinase-activating gene (RAG)-2-/- recipients, a mild to severe colitis developed, depending on the cell number transferred. Lymphocytes recovered from the colon of transplanted RAG-2-/- mice with colitis were predominantly alpha beta TCR+CD4+, including a large proportion of CD4+CD8 alpha + cells. These cells were also CD45RB-/low and CD44+, indicative of an activated/memory population. Individual populations of CD4+CD8 alpha-, CD4+CD8 alpha + and CD4-CD8 alpha + T cells were then isolated from the lamina propria compartment of IL-10-/- mice and transferred into RAG-2- /- recipients. Only IL-10-/- CD4-expressing LPL, including both the CD4+CD8 alpha- and CD4+CD8 alpha + populations, induced colitis in recipient mice. Interferon-gamma, but little to no IL-4, was produced by CD4+CD8 alpha- and CD4+CD8 alpha + LPL recovered from the inflamed colons of RAG-2-/- recipients implicating alpha T helper cell 1 (TH1)- mediated response. We thus conclude that colitis in IL-10-/- mice is predominantly mediated by TH1-type alpha beta TCR+ T cells expressing CD4 alone, or in combination with the CD8 alpha molecule.     

Expression of the alpha1beta1 integrin, VLA-1, marks a distinct subset of human CD4+ memory T cells

The alpha1beta1 integrin, very late antigen-1 (VLA-1), is a collagen receptor expressed in many CD4+ T cells localizing to inflamed tissues. Here we show that the expression of VLA-1 is a stable marker of a distinct subset of CD4+ memory T cells. Thus, in human peripheral blood lymphocytes (PBLs), approximately 1-4% of the CD4+ T cells express VLA-1, and following T cell receptor activation ex vivo, the percentage of VLA-1+ cells increases within the CD45RO+ population. Importantly, the activated VLA-1+ and VLA-1- cells can be isolated and maintained in culture as phenotypically stable subsets. Functionally, CD4+ memory T cells, operationally defined as the cells that divide rapidly following stimulation with a recall antigen, are highly enriched for VLA-1+ cells. Moreover, depletion of the small fraction of VLA-1+ cells present in CD4+ PBLs prior to stimulation significantly abrogated the proliferative response to recall antigens. Notably, the VLA-1+ cells in fresh CD4+ PBLs are composed of resting CD45RO+/RA-, CCR7-, CD62L+, CD25-, and VLA-4hi cells. Interestingly, this VLA-1+ subset is enriched for Th1-type cells, and Th1-polarizing conditions during T cell activation favor the emergence of VLA-1+ cells. Thus, VLA-1 expression is a stable marker of a unique subset of human memory CD4+ T cells that predominantly differentiates into Th1 cells.     

Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.     

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.     

The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo

A role for the alpha 4-integrin (alpha 4 beta 1 or alpha 4 beta 7), has been implicated in the recruitment of peripheral blood mononuclear cells (PBMCs) to sites of inflammation. However, the adhesive interactions (i.e., tethering, rolling, and adhesion) mediated by the alpha 4-integrin have not been characterized in vivo. The objective of this study was to establish a model wherein postcapillary venules were chronically inflamed, and then use intravital microscopy to identify the adhesive interactions mediated by the alpha 4-integrin in vivo. Between 4 and 20 d after immunization with Mycobacterium butyricum, animals developed a systemic vasculitis characterized by large increases in the numbers of rolling and adhering leukocytes within mesenteric venules. The selectins could only account for approximately 50% of the leukocyte rolling whereas the remaining cells rolled exclusively via the alpha 4-integrin. Anti-alpha 4 therapy also eliminated the increase in leukocyte adhesion observed in this model, whereas selectin therapies and an anti-CD18 (beta 2-integrin) monoclonal antibody (mAb) did not reduce adhesion. A serum against polymorphonuclear leukocytes (PMNs) was used to confirm that a significant proportion of rolling cells, and most of the adhering cells were PBMCs. Sequential treatment with anti-PMN serum and the anti-alpha 4 mAb demonstrated that alpha 4-dependent rolling was distinct from PMN rolling populations. Initial leukocyte tethering via the alpha 4- integrin could not be demonstrated in this model, whereas L-selectin did support leukocyte tethering. These data suggest that the alpha 4- integrin can mediate both rolling and adhesion in the multistep recruitment of PMBCs in vivo, and these interactions occur independently of the selectins and beta 2-integrins.     

CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin- mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1- mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR- CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin- mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1- dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Induction of interleukin 2 receptor beta chain expression by self- recognition in the thymus

1-2% of adult mouse thymocytes express the T cell receptor alpha/beta (TCR-alpha/beta) together with the interleukin (IL) 2R beta (p70), but not the alpha (p 55) chain. We show that the previously described alpha/beta-TCR +CD4-8- and the partially overlapping Ly6C+ thymocytes are contained within this subset. Most IL-2R beta+ alpha/beta-TCR+ cells have a mature and activated (heat stable antigen [HSA]-, thymic shared antigen 1 [TSA-1]-, CD44high, CD69+) phenotype. Overrepresentation of V beta 8.2 in both CD4-8- and CD4 and/or CD8+ IL- 2R beta+ thymocytes suggests that IL-2R beta expression is induced by a TCR-mediated activation event. In mice transgenic for an H-2Kb-specific TCR, IL-2R beta+ cells were abundant under conditions of mainstream negative selection, i.e., in the presence of Kb, but absent under conditions of mainstream positive selection or in a nonselecting environment. Together, these results show that in addition to clonal deletion, self-recognition by immature thymocytes leads to phenotypic maturation of a small subset of thymocytes expressing IL-2R beta. IL-2- deficient mice contain normal numbers of IL-2R beta+ alpha/beta-TCR+ thymocytes, indicating that like mainstream T cell development, this minor pathway of positive selection does not depend on IL-2. However, in the absence of IL-2, the CD4/CD8 subset composition of IL-2R beta+ thymocytes is skewed towards CD4-8+, mostly at the expense of CD4-8-. A possible relevance of this finding for the development of the immune pathology of IL-2-deficient mice is discussed.     

Precursors of CD3+CD4+CD8+ cells in the human thymus are defined by expression of CD34. Delineation of early events in human thymic development

Studies of the most immature T cell progenitors in the human thymus have been hampered by the lack of markers and assays that define these cells. In this report we used a novel human fetal thymic organ culture system to determine the potential of T cell precursors isolated from human postnatal thymus, to differentiate into CD3+ thymocytes, and to investigate early stages of human T cell development. It was found that thymocytes that lack the markers CD3, CD4, and CD8 (triple negative [TN]) can differentiate in an allogeneic organotypic thymic culture. The capacity of TN thymocytes to differentiate was exclusively confined to the CD34+ population. CD34- TN thymocytes failed to differentiate in this system. In contrast, cloned lines of CD3- thymocytes could only be established from CD34- TN thymocytes. Five subsets of CD3- thymocytes were found with the following phenotype: CD1-TN, CD1+TN, CD1+CD4+CD8-, CD1+CD4+CD8 alpha+ beta-, and CD1+CD4+CD8 alpha beta+. These subpopulations expressed decreasing levels of CD34. The CD1-CD3- population expressed the highest levels of CD34 supporting the notion that this population is the most immature T cell precursor in the thymus, whereas the CD1+CD4+CD8 alpha+ beta+ which did not express CD34 seems to be the most mature of these CD3- populations. This notion is supported by the observations that CD34+ cells isolated from fetal liver, which differentiated into T cells in a FTOC, developed into CD3+ cells via CD1- and CD4+CD8- intermediates. Based on these data, we present a model of early stages in human intrathymic development.     

Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level

The development of CD4+ T helper (Th) type 1 and 2 cells is essential for the eradication of pathogens, but can also be responsible for various pathological disorders. Therefore, modulation of Th cell differentiation may have clinical utility in the treatment of human disease. Here, we show that interleukin (IL) 12 and IL-4 directly induce human neonatal CD4- T cells, activated via CD3 and CD28, to differentiate into Th1 and Th2 subsets. In contrast, IL-13, which shares many biological activities with IL-4, failed to induce T cell differentiation, consistent with the observation that human T cells do not express IL-13 receptors. Both the IL-12-induced Th1 subset and the IL-4-induced Th2 subset produce large quantities of IL-10, confirming that human IL-10 is not a typical human Th2 cytokine. Interestingly, IL- 4-driven Th2 cell differentiation was completely prevented by an IL-4 mutant protein (IL-4.Y124D), indicating that this molecule acts as a strong IL-4 receptor antagonist. Analysis of single T cells producing interferon gamma or IL-4 revealed that induction of Th1 cell differentiation occurred rapidly and required only 4 d of priming of the neonatal CD4+ T cells in the presence of IL-12. The IL-12-induced Th1 cell phenotype was stable and was not significantly affected when repeatedly stimulated in the presence of recombinant IL-4. In contrast, the differentiation of Th2 cells occurred slowly and required not only 6 d of priming, but also additional restimulation of the primed CD4+ T cells in the presence of IL-4. Moreover, IL-4-induced Th2 cell phenotypes were not stable and could rapidly be reverted into a population predominantly containing Th0 and Th1 cells, after a single restimulation in the presence of IL-12. The observed differences in stability of IL-12- and IL-4-induced human Th1 and Th2 subsets, respectively, may have implications for cytokine-based therapies of chronic disease.     

A role for interleukin 4 in the differentiation of mature T cell receptor gamma/delta + cells from human intrathymic T cell precursors

We have analyzed the effect of human recombinant interleukin 4 (rIL-4) on the growth and differentiation of human intrathymic pre-T cells (CD7+2+1-3-4-8-). We describe that this population of T cell precursors proliferates in response to rIL-4 (in the absence of mitogens or other stimulatory signals) in a dose-dependent way. The IL-4-induced proliferation is independent of the IL-2 pathway, as it cannot be inhibited with an anti-IL-2 receptor alpha chain antibody. In our culture conditions, rIL-4 also promotes the differentiation of pre-T cells into phenotypically mature T cells. Although both CD3/T cell receptor (TCR)-alpha/beta + and CD3-gamma/delta + T cells were obtained, the preferential differentiation into TCR-gamma/delta + cells was a consistent finding. These results suggest that, in addition to IL-2, IL-4 plays a critical role in promoting growth and differentiation of intrathymic T cell precursors at early stages of T cell development.     

Thalidomide Costimulates Primary Human T Lymphocytes,Preferentially Inducing Proliferation,Cytokine Production,and Cytotoxic Responses in the CD8+ Subset

The efficacy of thalidomide (α-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor α. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2–mediated T cell proliferation and interferon γ production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.     

T lymphocyte differentiation in vitro from adult human prethymic CD34+ bone marrow cells

Pluripotent lymphohematopoietic stem cells are probably confined to bone marrow cells expressing CD34 surface molecules. To investigate the capacity of adult human CD34+ bone marrow cells to differentiate along the T lymphoid lineage, we plated purified CD34+ cells from healthy adults in liquid culture on adherent thymic stromal cells prepared from HLA- or blood group-mismatched postnatal thymic tissue. We show that purified CD34+CD3-CD4-CD8- bone marrow cells contained progenitors with the ability to differentiate into CD4+ and CD8+ T lymphocytes expressing surface (s)CD3 and T cell receptor alpha/beta in vitro. These progenitors were found in the CD34+CD2+sCD3-CD4-CD8-, CD34+CD7+sCD3-CD4-CD8-, and CD34+CD2+CD7+sCD3-CD4-CD8-, as well as in the CD34+CD2-sCD3-CD4-CD8-, CD34+CD7-sCD3-CD4-CD8-, and CD34+CD2-CD7- sCD3-CD4-CD8- subsets, indicating that T lymphocyte progenitors sensitive to signals mediated by thymic stroma in vitro are not restricted to CD34+ cells already coexpressing early T lymphocyte- associated markers. Finally, we show that T lymphopoiesis was enhanced by c-kit ligand.     

Beta 2-microglobulin-dependent T cells are dispensable for allergen- induced T helper 2 responses

CD4+ and CD8+ alpha/beta+ T cells of the T helper cell (Th)2 phenotype produce the cytokines IL-4, IL-5, and IL-13 that promote IgE production and eosinophilic inflammation. IL-4 may play an important role in mediating the differentiation of antigenically naive alpha/beta+ T cells into Th2 cells. Murine NK1.1+ (CD4+ or CD4-CD8-) alpha/beta+ T cells comprise a beta 2-microglobulin (beta 2m)-dependent cell population that rapidly produces IL-4 after cell activation in vitro and in vivo and has been proposed as a source of IL-4 for Th2 cell differentiation. alpha/beta+ CD8+ T cells, most of which require beta 2m for their development, have also been proposed as positive regulators of allergen-induced Th2 responses. We tested whether beta 2m- dependent T cells were essential for Th2 cell-mediated allergic reactions by treating wild-type, beta 2m-deficient (beta 2m -/-), and IL-4-deficient (IL-4 -/-) mice of the C57BL/6 genetic background with ovalbumin (OVA), using a protocol that induces robust allergic pulmonary disease in wild-type mice. OVA-treated beta 2m -/- mice had circulating levels of total and OVA-specific IgE, pulmonary eosinophilia, and expression of IL-4, IL-5, and IL-13 mRNA in bronchial lymph node tissue similar to that of OVA-treated wild-type mice. In contrast, these responses in OVA-treated IL-4 -/- mice were all either undetectable or markedly reduced compared with wild-type mice, confirming that IL-4 was required in this allergic model. These results indicate that the NK1.1+ alpha/beta+ T cell population, as well as other beta 2m-dependent populations, such as most peripheral alpha/beta+ CD8+ T cells, are dispensable for the Th2 pulmonary response to protein allergens.     

Interleukin 2 abrogates the nonresponsive state of T cells expressing a forbidden T cell receptor repertoire and induces autoimmune disease in neonatally thymectomized mice

Under physiological conditions, the vast majority of T cells differentiate in the thymus, an organ that provides an optimal microenvironment for T cell maturation and shapes the T cell repertoire via positive and negative selection processes. In the present report, we demonstrate that neonatal thymectomy of CBA/H mice results in a diminution of T cells in peripheral lymphoid organs (spleen, lymph nodes), but is followed by a marked transient (12 wk) increase in Thy-1+ CD3+ cells in the peritoneal cavity. These cells exhibit predominantly a double-negative (CD4-CD8-) phenotype among which products of the T cell receptor (TCR) V beta 11 gene family (i.e., an I-E-reactive TCR normally deleted in I-E-bearing CBA/H mice) are selectively overexpressed. This observation suggests that, under athymic conditions, T cell differentiation and/or accumulation may occur in the peritoneal cavity. Intraperitoneal inoculation of an interleukin 2 (IL-2) vaccinia virus construct that releases high titers of human IL-2 in vivo induces conversion of these double-negative T cells to either CD4+ CD8- or CD4- CD8+ single positives, and allows in vitro stimulation of TCR V beta 11-bearing cells with a clonotypic anti-V beta antibody. Since IL-2 induces autoimmune manifestations (DNA autoantibodies, rheumatoid factors, and interstitial nephritis) in thymectomized CBA/H mice, but not in sham-treated littermates, this lymphokine is likely to enhance the autoaggressive function of T cells that bear forbidden, potentially autoreactive TCR gene products and that are normally deleted in the thymus.     

A novel subset of CD2-, CD3/T cell receptor alpha/beta+ human peripheral blood T cells. Phenotypic and functional characterization of interleukin 2-dependent CD2-CD3+ T cell clones

It is generally believed that CD2 (T11, sheep erythrocyte receptor) is expressed on all human T cells. In the present study we have identified and characterized a minor subset of CD2- CD3/TCR alpha/beta+ T cells in the peripheral blood of healthy individuals. CD2-CD3+ T cells were enriched in PBMC depleted of plastic-adherent macrophages, E-rosetting (i.e., CD2+) T cells and surface Ig+ B cells. CD2-CD3+ T cells accounted for 0.1-0.8% of PBMC in six individuals. IL-2-dependent long-term clones of CD2-CD3+ T cells neither reacted with a panel of anti-CD2 mAbs nor expressed detectable levels of CD2 mRNA by Northern blot analysis. These clones, however, expressed a full-length TCR C beta mRNA and reacted with mAbs against TCR-alpha/beta, CD3, and CD4, and thus were mature T cells. CD2-CD3/TCR+ T cell clones could be triggered into proliferation, IL-2 production, and cytotoxic effector activity by anti-CD3 and anti-TCR mAbs. We conclude that (a) a minor subset of CD2-, CD3/TCR-alpha/beta+ T cells is present in normal peripheral blood; and (b) expression of CD2 at the level of protein and/or mRNA is not required for T cell signaling via the CD3/TCR molecular complex.     

Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.     

Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection

We analyzed at clonal level the functional profile of circulating or skin-infiltrating T lymphocytes from two individuals infected with the human immunodeficiency virus type 1 (HIV-1), suffering from a Job's- like syndrome (eczematous dermatitis, recurrent skin and sinopulmonary infections, and hypergammaglobulinemia E) and showing virtually no circulating CD4+ T cells. Most of the CD3+ T cell clones generated from both patients were CD4- CD8+ TCR alpha beta +. The others were CD4- CD8- TCR alpha beta + which exhibited reduced mRNA expression for the CD8 molecule or no mRNA expression for either CD4 or CD8 molecules. The great majority of both CD4- CD8+ and CD4- CD8- did not produce interferon (IFN) gamma and exhibited reduced cytolytic activity. Rather, most of them produced large amounts of both interleukin (IL) 4 and IL-5 and provided B cell helper function for IgE synthesis. These data suggest that a switch of cytolytic CD8+ T cells showing a Th1-like cytokine secretion profile to cells that make Th2-type cytokines, exhibit reduced cytolytic potential, and provide B cell helper function can occur in the course of HIV-1 infection. These cells may contribute to the reduced defense against viral infections and intracellular parasites and account for the elevated IgE serum levels, eosinophilia, and the allergic-like clinical manifestations seen in a proportion of HIV-1-infected individuals.