Susceptibility to clinically manifest cyclosporine A (CsA)-induced autoimmune disease is associated with interferon-gamma (IFN-γ)-producing CD45RC+RT6− T helper cells

Lethally irradiated Lewis (LEW) rats reconstituted with syngeneic bone marrow and given CsA for a 4-week period, develop, upon withdrawal of CsA, a graft-versus-host-like disease, so-called CsA-induced autoimmunity (CsA-AI). This T cell-mediated autoimmune disease is thymus-dependent; it is generally held that this disease is a consequence of aberrant T cell recovery brought about by CsA. In this study we determined mononuclear cell subsets phenotypically by tri-colour flow cytometry. A strong decrease in recent thymic emigrants (Thy1.1⁺, TCR αβ⁺) was observed as a consequence of CsA treatment, eventually resulting in decreased absolute peripheral T cell numbers. In these rats no altered CD4:CD8 T cell ratio was observed before onset of CsA-AI; CD4⁺ and CD8⁺ cells consisted predominantly of monocytes (CD4^dim+, TCR αβ⁻) and natural killer cells (CD8⁺, TCR αβ⁻), respectively. LEW rats, x-irradiated, syngeneic bone marrow-reconstituted and treated with CsA, showed a marked and persistent, relative expansion of mature CD45RC⁺, RT6⁻ Th cells. In contrast, Brown-Norway rats treated in a similar fashion, or LEW rats subjected to either CsA treatment or x-irradiation, did not show a comparable expansion of mature CD45RC⁺, RT6⁻ Th cells, nor did these animals develop CsA-AI. The CD45RC⁺, RT6⁻ Th cells produced IL-2, and moreover constituted the only Th subset producing IFN-γ upon stimulation, and therefore were considered as Th1-like effector cells. These results are consistent with the view that a persistent preponderance of Th1 cells and not the mere presence of autoreactive cells determines whether or not clinically manifest CsA-AI will occur.     

Diabetes-prone BB rats express the RT6 alloantigen on intestinal intraepithelial lymphocytes.

The RT6 alloantigen of the rat is expressed on most peripheral T cells but not on thymocytes and thus represents a marker for postthymic T lymphocyte maturation in this species. Diabetes-prone (DP) BB rats exhibit a genetically determined T cell lymphopenia associated with a deficiency of RT6+ T cells. In this study we have analyzed the expression of RT6 on lymph node (LN) cells and intestinal intraepithelial lymphocytes (IEL) in two DP BB strains (BB/OK and BB/Mol) and two control strains (non-lymphopenic BB/PhiK and LEW) by flow cytometry. In the DP BB rats the number of LN T cells was substantially reduced (less than 25% TcR2+ cells) and completely lacked RT6 expression. The IEL population was also reduced in number and in marked contrast to normal rats consisted predominantly of CD4+ cells. The majority of IEL, however clearly expressed RT6. Treatment with a phosphatidylinositol (PI)-specific phospholipase C markedly reduced the RT6 density showing that PI-mediated anchoring of RT6 in the cell membrane also applies to IEL of DP BB rats. The results demonstrate that the DP BB strains possess a functional RT6 gene and are also able to generate the PI anchor. The defect in RT6 expression is thus unlikely to be the primary cause of the T cell lymphopenia.     

Evolution of a CD3+/CD+ α/ β T-Cell Receptor+ Mature T-Cell Clone from CD3-CD7+ Sorted Human Bone Marrow Cells

In order to study extrathymic differentiation in vitro, CD7⁺CD3^- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCRδ1 (TCRγ/δ-specific) or WT31 (TCR2, α/β-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3⁺, CD4⁺, CD8^-, TCR2⁺, TCR1^-, and was further characterized as CD2⁺, CD45RO⁺, CD16^-, and CD56^-. The presence of mRNA for TCR α and γ but not ,and γ chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted “natural killer (NK)-like” lysis of K562 target cells, with no autocytotoxicity detected. Tle NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimulation, the cells secreted tumor necrosis factor α and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-δ, and GM-CSF in these cells after stimulation with PHA and B-LCL.These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.     

Establishment and characterization of αs1-casein–specific T-cell lines from patients allergic to cow’s milk: Unexpected higher frequency of CD8 T-cell lines

To study cow’s milk allergy at the cellular level, we assessed the reactivity of peripheral blood mononuclear cells from patients allergic to cow’s milk to α_s1-casein, which is one of the major allergens in cow’s milk. Proliferation of the cells to α_s1-casein activation showed a rather weak response. Therefore to understand T-cell reactivity to α_s1-casein in more detail, we prepared α_s1-casein–specific T-cell lines from patients allergic to cow’s milk and established 26 T-cell lines. These T-cell lines could be classified into three groups by analyzing their surface marker expression: those containing predominantly CD4⁺CD8^- T cells, those containing both CD4⁺CD8^- and CD4^-CD8⁺ T cells, and those containing predominantly CD4^-CD8⁺ T cells. The CD8⁺ T cells were obtained at an unexpectedly higher frequency from the patients. These T-cell lines produced interferon-γ and IL-4. These results suggest that CD8⁺ T cells specific for α_s1-casein and CD4⁺ T cells were primed by the stimulation with α_s1-casein in patients allergic to milk and that both T cells may play a key role in the onset, progression of, or recovery from cow’s milk allergy. (J ALLERGY CLIN IMMUNOL 1996;97:1342-9.)     

Both allelic forms of the rat T cell differentiation marker RT6 display nicotinamide adenine dinucleotide (NAD)-glycohydrolase activity,yet only RT6.2 is capable of automodification upon incubation with NAD

The finding that recently cloned mono-ADP-ribosyltransferases show sequence similarity to the rat T cell differentiation marker RT6 has led us to investigate the enzymatic activity of this alloantigenic system. To search for ADP-ribosylation of cell surface proteins, T cell populations from RT6.1- and RT6.2-expressing rat strains, as well as RT6.1⁺ and RT6.2⁺T-T hybridoma cell lines, were incubated with [³²P]nicotinamide adenine dinucleotide (NAD). All RT6.2⁺, but no RT6.1⁺ or RT6^? cells, show incorporation of radioactivity into a single protein which could be identified as RT6.2 by immunoprecipitation with monoclonal antibodies. This automodification of RT6.2 is covalent, requires intact NAD as substrate, and displays characteristics typical for linkage of ADP-ribose to arginine. The alloantigens RT6.1 and RT6.2 differ in ten amino acids, RT6.2 having two arginine residues not present in RT6.1. Both alloantigens were found to display potent NAD-glycohydrolase activity.     

Early Deletion and Late Positive Selection of T-Cells Expressing a Male-Specific Receptor in T-Cell Receptor Transgenic Mice

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic αβ heterodimer, specific for the male (H-Y) antigen in association with H-2D^b, was determined. The transgenic α chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both α and β transchains and virtually all fetal thymocytes expressed the transgenicαβ heterodimer by day 17. The early expression of the transgenic TCR on CD4^-8^- thymocytes prevented the development of γδ cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4^-8^- thymocytes than the female transgenic thymus. The preponderance of CD4^-8^- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4⁺8⁺ thymocytes and their CD4^-8⁺ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16.The preferential accumulation of CD4^-8⁺ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was .obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4⁺8⁺ thymocytes to the CD4^-8⁺ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4⁺8⁺ thymocytes into CD4^-8⁺ T cells.In normal mice, the precursors of CD⁺4⁺8 and single positive thymocytes have the CD4^-8^- CD3^-J11d⁺ (or M1/69 ⁺) phenotype. Because of the early expression of the transgenic αβ heterodimer, this population was not detected in adult transgenic mice. All CD4^-8^- M1/ 69⁺ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.     

Stratification of alopecia areata reveals involvement of CD4 T cell populations and altered faecal microbiota

Alopecia areata (AA) is an immune-mediated disease that causes non-scarring hair loss. Autoreactive CD8 T cells are key pathogenic effectors in the skin, and AA has been associated both with atopy and with perturbations in intestinal homeostasis. This study aimed to investigate mechanisms driving AA by characterizing the circulating immunophenotype and faecal microbiome, and by stratifying AA to understand how identified signatures associated with heterogeneous clinical features of the condition. Flow cytometric analyses identified alterations in circulating B cells and CD4 T cells, while 16S sequencing identified changes in alpha and beta diversity in the faecal microbiome in AA. The proportions of transitional and naïve B cells were found to be elevated in AA, particularly in AA samples from individuals with >50% hair loss and those with comorbid atopy, which is commonly associated with extensive hair loss. Although significant changes in circulating CD8 T cells were not observed, we found significant changes in CD4⁺ populations. In individuals with <50% hair loss higher frequencies of CCR6⁺CD4 (“Th17”) and CCR6⁺CXCR3⁺CD4 (“Th1/17”) T cells were found. While microbial species richness was not altered, AA was associated with reduced evenness and Shannon diversity of the intestinal microbiota, again particularly in those with <50% hair loss. We have identified novel immunological and microbial signatures in individuals with alopecia areata. Surprisingly, these are associated with lower levels of hair loss, and may therefore provide a rationale for improved targeting of molecular therapeutics.     

Anaplastic lymphoma kinase-positive large B-cell lymphoma: a case report with emphasis on the cytological features of the pleural effusion

Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (ALK-positive LBCL) is an extremely rare distinct clinicopathological subtype of LBCL, characterized by the presence of ALK-positive monomorphic large immunoblast-like neoplastic B cells. Herein, we describe the first cytological report on ALK-positive LBCL in the pleural effusion. A 69-year-old Japanese male with a past history of malignant lymphoma of the cecum presented with progressive dyspnea and pleural effusion. Removal of the pleural effusion and aspiration of bone marrow were performed. May-Grünwald-Giemsa stain of the pleural fluid revealed abundant single or small aggregates of large-sized round cells. These cells had centrally-located large round to oval nuclei. The peculiar finding was the presence of pseudopodial cytoplasmic projections, and some neoplastic cells had eosinophilic pseudopodial cytoplasmic projections, which resembled “flaming plasma cells”. Histopathological and immunohistochemical studies of the bone marrow demonstrated CD138⁺, ALK1⁺, CD20^-, CD79a^-, CD30^-, and IgA⁺ large-sized neoplastic cells. Therefore, a diagnosis of ALK-positive LBCL was made. The peculiar finding of the present case was that most of the neoplastic cells had pseudopodial cytoplasmic projections, and some of them had eosinophilic pseudopodial cytoplasmic projections that resembled “flaming plasma cells”, which has been recognized as the characteristic finding of IgA myeloma. Therefore, tumor cells that resembled “flaming plasma cells” in the pleural effusion may have had IgA in the cytoplasm. Albeit extremely rare, ALK-positive LBCL shows aggressive clinical course, thus, recognition of the cytomorphological features of this type of malignant lymphoma is important for early and correct diagnosis.     

Proinflammatory and prothrombotic status in emphysematous rats exposed to intermittent hypoxia

Objectives: To develop an “overlap syndrome (OS)” rat model by intermittent hypoxia (IH) exposure on the base of pre-existing emphysema, and to explore whether “OS” exposure results in more severe systemic inflammation, and whether the inflammation changes levels of coagulant/anticoagulant factors and oxidative stress status. Methods: Sixty Wistar rats were put into 4 groups: Control group; IH group, IH exposure; Emphysema group, smoke exposure; Overlap group, smoke exposure and IH exposure. We obtained peripheral blood for apoptosis of CD3⁺CD4⁺, CD3⁺CD8⁺ T lymphocytes and neutrophils, and for endothelial progenitor cell (EPC) counts. Tumor necrosis factor (TNF)-α, interleukin (IL)-6 and coagulant/anticoagulant factors [antithrombin (AT), fibrinogen (FIB), Factor VIII (FVIII) and von Willebrand factor (vWF)] were evaluated. We also obtained tissue blocks of lung, liver, pancreas, and right carotid artery for pathologic scoring and measurements of liver oxidative stress [superoxide dismutase (SOD) activity, catalase (CAT) activity and malondialdehyde (MDA) concentration]. Results: The levels of TNF-α and IL-6, CD3⁺CD4⁺ T lymphocyte apoptosis, EPC counts, coagulant factors and MDA are the highest in Overlap group, the lowest in Control group, when the levels of neutrophil apoptosis, CD3⁺CD8⁺ T lymphocyte apoptosis, AT, SOD and CAT are the lowest in Overlap group, the highest in Control group (all P values < 0.05). Conclusion: In model animals, when IH is combined with emphysema, there will be a more severe or an “overlapped” systemic/multiple organic inflammation, oxidative stress and hyper-coagulability. And the pro-inflammatory and pro-thrombotic status resulted from “OS” exposure may elicit a robust EPC mobilization, which needs further investigation.     

CD4+ T cell-mediated rejection of major histocompatibility complex class I-disparate grafts: A role for alloantibody

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)-disparate grafts have generated controversy over both the autonomy of CD8⁺ T cells and the mechanism whereby CD4⁺ T cells are able to independently mediate rejection. In this study of rejection of RT1A^a class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1^u recipients, we show that elimination of CD8⁺ T cells [by anti-CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4⁺ T cells (by anti-CD4 mAb treatment) markedly delays rejection of class I-disparate heart grafts, and marginally prolongs survival of skin grafts. Anti-CD4-treated PVG-RT1^u athymic nude rats reconstituted with CD8⁺ T cells failed to reject class I-disparate skin grafts for several weeks and eventual rejection correlated with re-emergence of a small number of donor derived CD4⁺ T cells. Conversely, anti-CD8-treated nude rats reconstituted with CD4⁺ T cells alone rapidly rejected class I-disparate skin grafts. Passive transfer of anti-class I immune serum to anti-CD4-treated euthymic recipients promptly restored their ability to specifically reject a class I-disparate heart graft. Similarly, passive transfer of immune serum to PVG-RT1^u nude rats bearing skin allografts caused destruction of class I-disparate but not third-party grafts. These results demonstrate that CD4⁺ T cells are both necessary and sufficient to cause rejection of class I-disparate heart and skin grafts in this model and that CD4⁺ T cell-dependent alloantibody plays a decisive role in effecting rejection.     

Appearance and Maturation of T-Cell Subsets During Rat Thymus Ontogeny

In previous papers, we have described the ontogenetical development of thymic stromal-cell components (epithelium, macrophages, dendritic cells) of Wistar rats. Here, we correlate those results with the maturation of rat T-cell precursors along the fetal and postnatal life. First T-cell precursors, which colonize the thymus anlage around days 13-14 of gestation, largely express CD45, CD43, CD53, and Thy 1 cell markers, and in a lesser proportion the OX22 antigen. Rat CD3^-CD4^-CD8^- thymocytes present in the earliest stages of gestation could be subdivided in three major cell subpopulations according to the CD44 and CD25 expression: CD44^-/+CD25^- → CD44⁺CD25⁺ → CD44⁺CD25^- On fetal days 17-18, a certain proportion of CD4^-CD8^-cells weakly,express the TcRβ chain, in correlation with the appearance of the first immature CD4^-CD8⁺ thymocytes. This cell subpopulation, in progress to the CD4⁺CD8⁺ stage, upregulates CD8α before the CD8β chain, expresses the CD53 antigen, and exhibits a high proliferative rate. First mature thymocytes arising from the DP (CD4⁺CD8⁺) cells appear on fetal days 20-21. Then, the CD4⁺:CD8⁺ cell ratio is ≤1 changing to adult values (2-3) just after birth. Also, the percentage of VβTcR repertoire covered in adult thymus is reached during the postnatal period, being lower during the fetal life. Finally, in correlation with the beginning of thymocyte emigration to the periphery a new wave of T-cell maturation apparently occurs in the perinatal rat thymus.     

A T cell activation antigen,Ly6C,induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8⁺ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8⁺ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C⁺ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G₀/G₁ to S+G₂/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4⁺ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4⁺ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

T-Lymphocyte Subsets in the Embryonic Spleen Undergoing a Graft-Versus-Host Reaction

Allogeneic immunocompetent T cells injected into chicken embryos induce a graft-versushost reaction (GVHR) whose most prominent manifestation is splenic hyperplasia. The highly inbred CC and CB strains of chickens used here are, respectively, homozygous for the B4 or B12 MHC haplotypes. By means of a panel of immunological reagents, including alloantisera and monoclonal antibodies against public domains of the T-cell receptor, CD4, CD8, and the inducible interleukin-2-receptor light chain (CD25), it is shown that the bulk of cells in the enlarged spleen are of host origin and do not express markers typical of mature T or B lymphocytes. Among recipient splenocytes, the quantitatively most important population consists of TCRαβ^-TCRγδ^-CD4^-CD8⁺CD25⁺ (TCR0) lymphocytes. Donor cells encountered in the spleen prevalently exhibit a TCRαβ⁺CD4⁺CD8^-CD25⁺ phenotype and proliferate in vivo. The data demonstrate that nonspecific host and potentially specific donorderived cellular elements contribute to splenomegaly.     

Enhancement of CD8+ T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor

Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8⁺ T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8⁺ T-cell populations, increased CD8⁺ T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8⁺ memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8⁺ T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8⁺ T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8⁺ T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety.     

T cell regeneration after allogenic bone marrow transplantation

Various T cell subsets were characterized by double immunofluorescent staining using monoclonal antibodies (MoAb) in blood, bone marrow (BM) and tissues of 29 patients after allogeneic BM transplantation (BMT). In an attempt to prevent graft versus host disease (GvHD), 15 patients received cyclosporin A (Cy A). In the remaining 14 patients the BM was pre-incubated with a MoAb, OKT3. The regeneration of T4⁺ subset was delayed and the level of T8⁺ cells was abnormally high even 1 year after engraftment. This did not have any predictive value for the appearance of complications such as GvHD or severe viral infections. The number of T8⁺ cells was lower in the group of patients who received Cy A than in the OKT3 group (0·7±0·2 vs 1·5±0·3×10⁹/1 at day 90). In contrast to normal individuals, the T4/T8 ratio in both blood and regenerating BM of BMT patients was <1. A sizeable subset of circulating T cells expressed the phenotype T8⁺,T10⁺,HNK-1⁺,DR⁺. Circulating cells of this phenotype were transiently very high (up to 50%) in patients with active GvHD or suffering from severe viral infection. This subpopulation of lymphocytes was not found in the epidermal infiltrate that accompanied GvHD where the predominant phenotype was T8⁺,T1^-,T10^-,HNK-1^-,DR^-. We conclude therefore that after BMT the number and phenotype of circulating T cells reflects the T cell distribution seen in the regenerating BM.     

CD4+ CD25high Foxp3+ regulatory T cells downregulate human Vδ2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen

Vδ2⁺ T cells, the major circulating T-cell receptor-γδ-positive (TCR-γδ⁺) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vδ2⁺ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4⁺ T cells. CD4⁺ CD25^high Foxp3⁺ T cells (Tregs) comprise 5–10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-αβ⁺ lymphocytes, Tregs suppress Vδ2⁺ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vδ2⁺ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vδ2⁺ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-γ secretion by Vδ2⁺ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vδ2⁺ T-cell interferon-γ responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vδ2⁺ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vδ2⁺ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vδ2⁺ T-cell functional deficiencies observed in TB.     

Role of Prolactin in the Recovered T-Cell Development of Early Partially Decapitated Chicken Embryo

Although different experimental approaches have suggested certain regulation of the mammalian immune system by the neuroendocrine system, the precise factors involved in the process are largely unknown. In previous reports, we demonstrated important changes in the thymic development of chickens deprived of the major neuroendocrine centers by the removal of embryonic prosencephalon at 33-38 hr of incubation (DCx embryos) (Herradón et al., 1991; Moreno et al., 1995). In these embryos, there was a stopping of T-cell maturation that resulted in an accumulation of the most immature T-cell subsets (CD4^-CD8^- cells and CD4^-CD8^1o cells) and, accordingly, in decreased numbers of DP (CD4⁺CD8⁺) thymocytes and mature CD3⁺TcRαβ ⁺ cells, but not CD3⁺TcRγδ lymphocytes. In the present work, we restore the thymic histology as well as the percentage of distinct T-cell subsets of DCx embryos by supplying recombinant chicken prolactin, grafting of embryonic pituitary gland, or making cephalic chick-quail chimeras. The recovery was not, however, whole and the percentage of CD3⁺TcRαβ thymocytes did not reach the normal values observed in 17-day-old control Sham-DCx embryos. The results are discussed on the basis of a key role for prolactin in chicken T-cell maturation. This hormone could regulate the transition of DN (CD4^-CD8^-) thymocytes to the DP (CD4⁺CD8⁺) cell compartment through its capacity for inducing IL-2 receptor expression on the former.     

Killer cell immunoglobulin receptor profile on CD4+ CD28− T cells and their pathogenic role in non‐dialysis‐dependent and dialysis‐dependent chronic kidney disease patients

There is a progressive increase in cardiovascular disease with declining renal function, unexplained by traditional risk factors. A CD4⁺ T-cell subpopulation (CD4⁺ CD28⁻), activated by human heat-shock protein 60 (hHSP 60), expands in patients with acute coronary syndrome and is associated with vascular damage. These cells exhibit cytotoxicity via expression of activating killer cell-immunoglobulin-like receptor KIR2DS2, mainly in the absence of inhibitory KIR2DL3. We investigated expansion of these cells and the pathogenic role of the KIR in non-dialysis-dependent chronic kidney disease (NDD-CKD) and end-stage haemodialysis-dependent renal disease (HD-ESRD) patients. CD4⁺ CD28⁻ cells were present in 27% of the NDD-CKD and HD-ESRD patients (8–11% and 10–11% of CD4⁺ compartment, respectively). CD4⁺ CD28⁻ cells were phenotyped for KIR and DAP12 expression. Cytotoxicity was assessed by perforin and pro-inflammatory function by interferon-γ expression on CD4⁺ CD28⁻ clones (NDD-CKD n = 97, HD-ESRD n = 262). Thirty-four per cent of the CD4⁺ CD28⁻ cells from NDD-CKD expressed KIR2DS2 compared with 56% in HD-ESRD patients (P = 0·03). However, 20% of clones expressed KIR2DL3 in NDD-CKD compared with 7% in HD-ESRD patients (P = 0·004). DAP12 expression in CD28⁻ 2DS2⁺ clones was more prevalent in HD-ESRD than NDD-CKD (92% versus 60%; P < 0·001). Only 2DS2⁺ 2DL3⁻ DAP12⁺ clones were cytotoxic in response to hHSP 60. CD4⁺ CD28⁻ cells exhibited increased KIR2DS2, reduced KIR2DL3 and increased DAP12 expression in HD-ESRD compared with NDD-CKD patients. These findings suggest a gradual loss of expression, functionality and protective role of inhibitory KIR2DL3 as well as increased cytotoxic potential of CD4⁺ C28⁻ cells with progressive renal impairment. Clonal expansion of these T cells may contribute to heightened cardiovascular events in HD-ESRD.