Borrelia burgdorferi stimulates in vitro a selective expansion of CD3-CD4-CD8- (triple negative) autoreactive cells in naive rhesus monkey lymphocytes

Autoreactive cell lines were generated from cell suspensions of freshly isolated naive monkey lymph node (LN) cells and peripheral blood mononuclear cells by cocultivation with freeze-thawed Borrelia burgdorferi spirochetes (Bb/FT). These cells produced interleukin (IL)-6 when cocultured with autologous antigen-presenting cells (APCs) alone without Bb/FT. IL-6 production was not observed when control cell lines were stimulated in the same fashion. CD4+-enriched T cell populations obtained from the LN autoreactive cell line also produced IL-6 when cultivated with APCs alone. When these cells were cultivated further in the presence of APCs, a population of cells whose phenotype was CD56+/-CD4-CD8-CD3- was predominantly selected. These cells both proliferated and produced IL-6 when cocultured with APCs alone. The possible relevance of these cells to Lyme disease pathogenesis remains to be determined.     

Pituitary epithelial cell implants reverse the accumulation of CD4-CD8- lymphocytes in thymus glands of aged rats.

Although implantation of GH3 pituitary epithelial cells has been shown to reverse thymic aging in rats, the differentiation pattern of T-lymphocytes within the reconstituted thymus glands has not been investigated. Syngeneic GH3 cells were implanted into 22-month-old female (old) Wistar-Furth rats. Eight weeks later, thymus glands and thymocyte subpopulations were compared to those in aged (24-month-old) and young (3-month-old) female Wistar Furth rats. We confirmed that implantation of GH3 cells increased (P less than 0.05) not only thymus size but also the number of thymocytes isolated from aged rats. Flow cytometric analysis using dual color direct immunofluorescence with fluorescein isothiocyanate-labeled anti-CD4 (W3/25) and phycoerythrin-labeled anti-CD8 (OX 8) monoclonal antibodies revealed that thymus glands from young rats had approximately 20% CD4-CD8- and 30% CD4+CD8+ cells. Thymus glands from old rats contained greater than 50% more CD4-CD8- cells and a reduced percentage of CD4+CD8+ lymphocytes compared to those of young rats (P less than 0.05). Moreover, both of these age-associated changes were reversed (P less than 0.05) by implanting GH3 cells. GH3-treated aged rats also had a significantly (P less than 0.05) greater proportion of CD4+CD8- thymocytes compared to aged control rats. There were no differences among the three groups of rats in the percentage of CD4-CD8+ thymocytes or in the percentage or intensity of cells expressing the T-cell markers CD3, T-cell receptor, or OX19. These results show that in aged rats, intrathymic maturation is inhibited at the key transitional stage where double negative cells differentiate into double positive cells, which may limit the diversity of the T-cell repertoire. The data also extend earlier results by demonstrating that GH3 epithelial cells promote not only growth, but also the differentiation of T-lymphocytes in the aging rat thymus.     

CD4-CD8- T-cell polymyositis in a patient with chronic active Epstein-Barr virus infection

We describe a 17-year-old woman with chronic active Epstein-Barr virus infection (CAEBV), who developed EBV+CD4-CD8- T-cell polymyositis. At 14 years of age, CAEBV was diagnosed with fever, cytopenia, liver dysfunction, and hepatosplenomegaly. Despite the transient remission of interferon-alpha therapy, migratory lesions emerged in back and extremities. MRI indicated polymyositis. Biopsy specimens revealed intramuscular infiltration of CD3+, CD4-, CD8-, CD56-, and EBV-encoded RNA 1+ cells. Circulating CD4-CD8-Vdelta2/Vgamma9 cells increased. gammadeltaT-cells contained 20-200 times higher EBV-DNA (2 x 10(4) copies/microgDNA) than alphabetaT-cells or NK-cells. The ominous polymyositis might denote the musculotropic invasion of EBV+gammadeltaT-cell lymphoproliferative disease as a consequence of CAEBV.     

Expansion of TcRalphabeta+CD3+CD4-CD8- (CD4/CD8 double-negative) T lymphocytes in a case of staphylococcal toxic shock syndrome

A 55-year-old woman presented with staphylococcal toxic shock syndrome (TSS). During the course of the disease a significant lymphocytosis appeared, and a high number of TcRalphabeta+CD3+CD4-CD8- (double-negative, DN) lymphocytes was observed both in bone marrow and in peripheral blood samples. Correction of the altered lymphocyte immunophenotype was observed only 6 weeks after recovery from TSS. The immunophenotype of circulating and bone marrow lymphocytes was also studied during a phase of an aspecific febrile episode observed 2 months after recovery, but no subset of DN lymphocytes was found. A small subset of DN lymphocytes can be found in normal bone marrow, liver, thymus, and skin. These cells show peculiar immune regulatory properties and can increase in certain autoimmune diseases. Our findings may represent a specific effect of lymphocyte stimulation by the staphylococcal exotoxin, which is the effector agent of TSS.     

Differentiation of thymocytes from CD3-CD4-CD8- through CD3-CD4-CD8+ into more mature stages induced by a thymic stromal cell clone.

We have investigated the capacity of our established thymic stromal cell clone (MRL104.8a) or its derived factor(s) to induce the differentiation of immature thymocytes. Culture of purified adult murine double-negative (CD4-CD8-, indicated here as CD4-8-) thymocytes on the MRL104.8a thymic stromal cell monolayer for 1 day resulted in the induction of an appreciable percentage of CD4-8+ thymocytes. A bone marrow-derived stromal cell monolayer or a L929 fibroblast monolayer failed to generate CD4-8+ cells. This differentiation could also be induced by a semipurified sample of the MRL104.8a culture supernatant, which contained a thymic stroma-derived T-cell growth factor capable of contributing to the growth of double-negative immature thymocytes. CD4-8+ thymocytes generated 1 day after coculture with the MRL104.8a cells or the sample containing thymic stroma-derived T-cell growth factor were found to be CD3- and J11d+, excluding the possibility of expansion of mature (CD3+4-8+) thymocytes present in the thymus. More importantly, when the culture period was extended to 2 or 3 days, an appreciable number of CD4+8+ and single-positive (CD4+) cells were generated on the MRL104.8a monolayer. Thus, these results provide the direct demonstration that CD3-4-8- immature thymocytes are promoted to differentiate through a rapidly cycling intermediate (CD3-4-8+) into double- and single-positive cells by a specialized thymic stromal component.     

Paraproteins and monoclonal expansion of CD3+CD8+ CD56-CD57+ T lymphocytes in a patient with HIV infection

An expansion of CD8+ lymphocytes associated with a monoclonal rearrangement of the T-cell receptor gamma locus was found in a woman with HIV-1 infection. A subpopulation of HIV-positive patients display an unusual response to HIV infection characterized by a persistent marked CD8+ lymphocytosis, the presence of which appears to be associated with an improved long-term prognosis. This condition is thought to represent a florid immune response to an ongoing viral infection which may be HIV itself, and suggests that monoclonal proliferation of CD8+ lymphocytes does not imply the presence of an underlying malignant process.     

Homeostatic cytokines orchestrate the segregation of CD4 and CD8 memory T-cell reservoirs in mice

Memory T cells (T(M)s) have been detected in many tissues but their quantitative distribution remains largely undefined. We show that in mice there is a remarkably biased accumulation of long-term CD4 T(M)s into mucosal sites (mainly gut, especially Peyer patches), and CD8 T(M)s into lymph nodes and spleen (in particular, peripheral lymph nodes [PLNs]). This distinction correlates with their differentiated expression of PLN- and gut-homing markers. CD8 and CD4 T(M)s selectively require the expression of PLN-homing marker CCR7 or gut-homing marker α4β7 for maintenance. PLNs and gut supply CD8 and CD4 T(M)s with their individually favored homeostatic cytokine, IL-15, or IL-7. Cytokine stimulation in turn regulates the different gut-homing marker expression on CD4 and CD8 T(M)s. IL-15 plays a major role in vivo regulating CD8 T(M)s homing to PLNs. Thus, the reservoir segregation of CD4 and CD8 T(M)s meets their individual needs for homeostatic cytokines and is under feedback control of cytokine stimulation.     

Molecular characterization of T-cell antigen receptor expression by subsets of CD4- CD8- murine thymocytes.

Precursors of all T-lineage cells are found in a population of thymocytes that lack the CD4 and CD8 surface glycoproteins. These "double-negative" thymocytes are markedly heterogeneous in their expression of other surface markers and include cells at various stages of development. In this study, CD4- CD8- adult murine thymocytes were separated into subsets based on the expression of the "heat stable antigen" (HSA) and of Ly 1 (CD5). The sorted subsets were analyzed directly (without prior expansion in culture) for T-cell antigen receptor (TcR) gene rearrangement and mRNA expression and for TcR and CD3 cell-surface protein expression. Very little surface CD3 or TcR expression was detected on the major HSA+ Ly 1low subset. However, the HSA+ Ly 1high, HSA- Ly 1high, and HSA- Ly 1low subsets all contained cells with surface expression of CD3 and TcR. In contrast to previous studies, we found no subset that exclusively expressed either the alpha beta or gamma delta heterodimer, although the ratio of alpha beta+ to gamma delta+ varied widely. Two of these three subsets (HSA- Ly 1low and HSA- Ly 1high) showed very high usage of V beta 8 gene products in the alpha beta heterodimer, but nevertheless included approximately equal to 15% non-V beta 8 alpha beta forms. All CD4- CD8- subsets were found to have extensively rearranged their TcR gamma genes and to express gamma mRNA. Expression of a high ratio of mature [1.3 kilobases (kb)] to truncated (1.0 kb) beta message and presence of alpha message was largely restricted to subsets with TcR alpha beta surface expression.     

Life after the thymus: CD31+ and CD31- human naive CD4+ T-cell subsets

Early in life, thymic export establishes the size and the diversity of the human naive T-cell pool. Yet, on puberty thymic activity drastically decreases. Because the overall size of the naive T-cell pool decreases only marginally during ageing, peripheral postthymic expansion of naive T cells has been postulated to account partly for the maintenance of T-cell immunity in adults. So far, the analysis of these processes had been hampered by the inability to distinguish recent thymic emigrants from proliferated, peripheral, naive T cells. However, recently, CD31 has been introduced as a marker to distinguish 2 subsets of naive CD4(+) T cells with distinct T-cell receptor excision circle (TREC) content in the peripheral blood of healthy humans. Here, we review studies that have characterized TREC(hi) CD31(+ thymic)naive CD4(+) T cells and have accordingly used the assessment of this distinct subset of naive CD4(+) T cells as a correlate of thymic activity. We will discuss further potential clinical applications and how more research on CD31(+ thymic)naive and CD31(- central)naive CD4(+) T cells may foster our knowledge of the impact of thymic involution on immune competence.     

Transient monoclonal expansion of CD8+/CD57+ T-cell large granular lymphocytes after primary cytomegalovirus infection

Lymphocytosis associated with viral infection is generally polyclonal or oligoclonal. In this article, we describe a case of transient monoclonal CD8+/CD57+ T-cell lymphocytosis with large granular lymphocyte (LGL) morphology occurring after primary CMV infection and review cases of virus-associated monoclonal CD8+ T-cell expansions reported in the literature. Several clinical features shared by virus-associated monoclonal CD8+ T-cell expansions suggest the reactive nature of the lymphocytosis. Based on this, our case report and those reported in the literature support the notion that T-cell receptor clonality per se is not necessarily indicative of malignancy. These observations further corroborate the need for a close follow-up before assigning the diagnosis of LGL leukemia to individuals developing monoclonal CD8+ T-cell expansions.     

CD4+ T lymphocytes mediate in vivo clearance of plasmid DNA vaccine antigen expression and potentiate CD8+ T-cell immune responses

There is evidence that the limited immunogenicity of plasmid DNA vaccines is the result, at least in part, of the rapid clearance of vaccine antigen expression by antigen-specific immune responses. However, the cell types responsible for the clearance of plasmid DNA vaccine antigens are not known. Here we demonstrate that macrophages, NK cells, and CD8(+) T cells did not significantly contribute to the DNA antigen clearance but CD4(+) T cells played the crucial role in attenuating plasmid DNA vaccine antigen expression. Adoptive transfer experiments demonstrate that CD4(+) T cells facilitated DNA vaccine antigen clearance in a Fas/FasL-dependent manner. Furthermore, we show that depletion of CD4(+) T cells prevented the clearance of vaccine antigen and the appearance of a CD8(+) T-cell immune response. Inoculation of major histocompatibility complex class II KO mice with the plasmid DNA led to persistent antigen expression and abolition of a CD8(+) T-cell immune response. Importantly, the prolongation of antigen expression by disrupting the CD4(+) T-cell Fas/FasL myocytes signaling led to a 3- to 5-fold increase of antigen-specific CD8(+) T-cell responses. These data demonstrate a dominant role of CD4(+) T cell-mediated cytotoxicity in plasmid DNA vaccine antigen clearance.     

Relative impact of HLA phenotype and CD4-CD8 ratios on the clinical expression of hemochromatosis

Hemochromatosis is a hereditary iron-overload disease linked to HLA. The clinical expression of hemochromatosis is influenced by sex and age. However, other factors must account for the notorious heterogeneity of expression of the disease independent of sex, age, and HLA phenotype. The present study attempts to clarify some of these additional factors based on exhaustive statistical analysis of data collected from 43 selected patients with hemochromatosis. The statistical analysis focused on three groups of variables: the first group included variables reflecting the clinical expression of the disease; the second group represented the biochemical and hematological values at the time of diagnosis; and the third group consisted of the independent variables sex, age, HLA phenotype, and T-cell subset profile, i.e., the percentages and total numbers of CD4+ and CD8+ cells and the CD4-CD8 ratios. The results show that the relative expansion of the two main T-cell subsets, in the context of the HLA phenotype, correlates significantly with the clinical expression of hemochromatosis and the severity of iron overload. The present findings substantiate further the postulate that T cells have a role in the regulation of iron metabolism.(Hepatology 1997 Feb;25(2):397-402)     

A CD8+ T-lymphocyte-mediated and CD4+ T-lymphocyte-independent autoimmune diabetes of early onset in transgenic mice

Summary While transgenic mice expressing tumour necrosis factor-alpha under the control of the beta-cell-specific insulin promoter display a marked lymphocytic infiltration of the islets, they never develop insulin-dependent diabetes mellitus (IDDM). In striking contrast, double transgenic mice whose beta cells express both tumour necrosis factor-alpha as well as the co-stimulatory B7-1 molecule all develop IDDM at an early age. Further, administration of anti-CD8 but not anti-CD4 immunoglobulins prevents diabetes onset. These results indicate that while tumour necrosis factor-alpha induced lymphocytic infiltration is not sufficient to effect beta-cell destruction, locally co-stimulated islet-infiltrating CD8⁺ T lymphocytes could play a critical role in the development of IDDM.Abbreviations FACS Fluorescence-activated cell sorter - IDDM insulin-dependent diabetes mellitus - i.p. intraperitoneal injection - mAb monoclonal antibody - NOD non obese diabetic - TNF tumour necrosis factor alpha