Subset Of Memory CD4 Research Articles

Melanoma cell adhesion molecule identifies encephalitogenic T lymphocytes and promotes their recruitment to the central nervous system

In multiple sclerosis, encephalitogenic CD4(+) lymphocytes require adhesion molecules to accumulate into central nervous system inflammatory lesions. Using proteomic techniques, we identified expression of melanoma cell adhesion molecule (MCAM) on a subset of human effector memory CD4(+) lymphocytes and on human blood-brain barrier endothelium. Herein, we demonstrate that MCAM is a stable surface marker that refines the identification of interleukin 17(+), interleukin 22(+), RAR-related orphan receptor γ and interleukin 23 receptor(+) cells within the CD161(+)CCR6(+) subset of memory CD4(+) lymphocytes. We also show that MCAM(+) lymphocytes express significantly more granulocyte/macrophage colony stimulating factor and granzyme B than MCAM(-) lymphocytes. Furthermore, the proportion of MCAM(+) CD4(+) lymphocytes is significantly increased in the blood and in the central nervous system of patients with multiple sclerosis and experimental autoimmune encephalomyelitis animals compared with healthy controls or other neurological diseases, and MCAM expression is upregulated at the blood-brain barrier within inflammatory lesions. Moreover, blockade of MCAM or depletion of MCAM(+) CD4(+) T lymphocytes both restrict the migration of T(H)17 lymphocytes across blood-brain barrier endothelial cells and decrease the severity of experimental autoimmune encephalomyelitis. Our findings indicate that MCAM could serve as a potential biomarker for multiple sclerosis and represents a valuable target for the treatment of neuroinflammatory conditions.

The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin

Cytokine expression patterns of T cells can be regulated by pre-commitment to stable effector phenotypes, further modification of moderately stable phenotypes, and quantitative changes in cytokine production in response to acute signals. We showed previously that the epidermal growth factor family member Amphiregulin is expressed by T cell receptor-activated mouse CD4 T cells, particularly Th2 cells, and helps eliminate helminth infection. Here we report a detailed analysis of the regulation of Amphiregulin expression by human T cell subsets. Signaling through the T cell receptor induced Amphiregulin expression by most or all T cell subsets in human peripheral blood, including naive and memory CD4 and CD8 T cells, Th1 and Th2 in vitro T cell lines, and subsets of memory CD4 T cells expressing several different chemokine receptors and cytokines. In these different T cell types, Amphiregulin synthesis was inhibited by an antagonist of protein kinase A, a downstream component of the cAMP signaling pathway, and enhanced by ligands that increased cAMP or directly activated protein kinase A. Prostaglandin E2 and adenosine, natural ligands that stimulate adenylyl cyclase activity, also enhanced Amphiregulin synthesis while reducing synthesis of most other cytokines. Thus, in contrast to mouse T cells, Amphiregulin synthesis by human T cells is regulated more by acute signals than pre-commitment of T cells to a particular cytokine pattern. This may be appropriate for a cytokine more involved in repair than attack functions during most inflammatory responses.

The H1-isoform of the cell surface receptor Slamf6 suppresses expansion of PD-1+ OPN+ CD4+ T Follicular Helper cells in the lupus-prone B6.Sle1b mice. (123.5)

Abstract A major role for the Slamf6 gene in the pathogenesis of lupus-related autoimmunity has been revealed by the analysis of a number of congenic mutant mouse strains. Importantly, upon introduction of one copy of a BAC transgene expressing the protein isoform Slamf6-H1 markedly ameliorates T cell-dependent lupus in the Sle1b mouse, which lacks H1. Here we report that a PD-1+ subset of memory CD4+ T cells dramatically expands in Sle1b mice, but not in Sle1b mice, which express Slamf6-H1. Further analysis showed that this PD-1+ population mainly consists of CXCR5+ T follicular helper cells the expansion of which is prevented by Slamf6-H1 as well as by the absence of the adapter SAP. Surprisingly, these T follicular helper cells express the cytokine osteopontin, which is known to drive polyclonal B cell proliferation. As the human osteopontin gene have been linked to the development of SLE, the outcomes of the present studies could suggest novel therapeutic modalities for SLE patients.

A proteomic study of early signaling events regulating the Fas-FasL Death Inducing Signaling Complex (163.20)

Abstract Clearance of activated T cells by the Fas-FasL pathway is a critical mechanism of peripheral CD4 T cell tolerance. This process is preferentially higher in the effector subset of memory CD4+ T cells due to efficient recruitment and formation of the Fas death inducing signaling complex (DISC). In order to elucidate mechanisms of this heightened apoptosis sensitivity, we tested for differential DISC interacting proteins in Fas sensitive and Fas resistant CTCL cell lines using 2D-DIGE and Mass Spectrometry. We found several candidate proteins and invitro studies were done to confirm Fas receptor interactions through overexpression and Flourescence Energy Transfer (FRET) studies. One of these targets, Drebrin, is an actin binding protein previously found to play a role in T cell synapse formation by facilitating CXCR4 recruitment. Overexpression in 293 indicates that Drebrin interacts with Fas receptor. Further, endogenous drebrin preassociates with Fas receptor and remains associated with the Fas DISC even after Fas ligation only in Type I SKW cell line. Interestingly, Fas DISC associated Drebrin gets cleaved after Fas treatment and invitro caspase cleavage studies indicate that drebrin is a specific downstream target of caspase-8. Our studies propose Drebrin as a novel Fas interacting protein. Investigations are currently underway to study the mechanisms by which Drebrin modifies the Fas-DISC dynamics and the significance of the caspase-cleaved Drebrin in this process.

T inflammatory memory CD8 T cells participate to antiviral response and generate secondary memory cells with an advantage in XCL1 production

Besides the classically described subsets of memory CD8 T cells generated under infectious conditions, are T inflammatory memory cells generated under sterile priming conditions, such as sensitization to allergens. Although not fully differentiated as pathogen-induced memory cells, they display memory properties that distinguish them from naive CD8 T cells. Given these memory cells are generated in an antigen-specific context that is devoid of pathogen-derived danger signals and CD4 T cell help, we herein questioned whether they maintained their activation and differentiation potential, could be recruited in an immune response directed against a pathogen expressing their cognate antigen and further differentiate in fully competent secondary memory cells. We show that T inflammatory memory cells can indeed take part to the immune response triggered by a viral infection, differentiate into secondary effectors and further generate typical central memory CD8 T cells and effector memory CD8 T cells. Furthermore, the secondary memory cells they generate display a functional advantage over primary memory cells in their capacity to produce TNF-α and the XCL1 chemokine. These results suggest that cross-reactive stimulations and differentiation of cells directed against allergens or self into fully competent pathogen-induced memory cells might have incidences in inflammatory immuno-pathologies.

Intracellular detection of differential APOBEC3G, TRIM5alpha, and LEDGF/p75 protein expression in peripheral blood by flow cytometry

Expression studies on specific host proteins predominantly use quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based assay to study intracellular expression levels of three important host proteins involved in HIV-1 replication: apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and lens epithelium-derived growth factor (LEDGF/p75). An indirect intracellular staining (ICS) method was optimized using antibodies designed for other applications like enzyme-linked immunosorbent assay (ELISA), confocal imaging, and western blotting. The median fluorescence intensity (MFI) value – a measure for the protein expression level – increased upon higher antibody concentration and longer incubation time, and was reduced following preincubation with recombinant proteins. Staining of stably transfected or knock-down cell lines supported the method's specificity. Moreover, confocal microscopy analysis of peripheral blood mononuclear cells (PBMC), when stained according to the ICS method, confirmed the localization of APOBEC3G and TRIM5α in the cytoplasm, and of LEDGF/p75 in the nucleus. Also, stimulation with mitogen, interferon-alpha, or interferon-beta resulted in detectable, albeit weak, increases in intracellular expression of APOBEC3G and TRIM5α. After optimization, the method was applied to healthy control and HIV-1 infected subjects. For all subjects studied, the memory subset of CD4+ T cells showed significantly higher expression levels of APOBEC3G, TRIM5α, and LEDGF/p75, while the CD16+ subset of monocytes was characterized by higher expression levels of LEDGF/p75. In addition, we observed that therapy-naïve HIV-1 patients tended to have lower expression levels of APOBEC3G and TRIM5α than HIV-1 negative controls. In summary, our data provide proof-of-principle for the detection of specific host factors at the level of a single cell, which may prove useful for our further understanding of their role in virus-host interactions.

Altered T-cell and regulatory cell repertoire in patients with diffuse cutaneous systemic sclerosis

Objectives: The aim of this study was to evaluate a wide spectrum of peripheral immune-competent cell types, reflecting overall disturbances in immune homeostasis, characteristic of systemic sclerosis (SSc). We also assessed visceral organ involvement and evaluated the relationship between cell proportions and clinical symptoms of the disease.Methods: Twenty-one patients with diffuse cutaneous SSc (dcSSc) and 15 healthy individuals participated in the study. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed by enzyme-linked immunosorbent assay (ELISA), serum complement levels were measured by nephelometry, and autoantibodies were determined by indirect immunofluorescence staining and ELISA technique. Functional tests of regulatory T (Treg) cells were also carried out.Results: Patients with SSc had higher percentages of activated CD3+/HLA-DR+ T cells. Comparing naive vs. memory subsets of CD4+ and CD8+ T cells, a shift towards central memory phenotype was observed in SSc. Natural killer (NK) and T-helper (Th)17 cell percentages were increased, while NKT, Th1, Treg type 1 (Tr1), and CD4+CD25+ Treg cell percentages were decreased in patients. Moreover, the suppressor activity of CD4+CD25+ Treg cells was lower in SSc. Negative correlations occurred between modified Rodnan skin score (MRSS) and Tr1 cell percentages and between complement levels and CD4+CD25+ Treg cells. We also found decreased interleukin (IL)-10 levels in SSc.Conclusions: Our data suggest that the increased Th17/CD4+CD25+ Treg ratio and the altered regulatory function of CD4+CD25+ Treg cells play an important role in the development of SSc. Moreover, our study reveals the potential role of the decreased profile of IL-10-producing Tr1 cells in the progression of disproportionate immune responses in SSc.

IL-15 and IL-21 Mediated Homeostatic Proliferation of Human CD8 Memory T cells (96.7)

Abstract Accumulation of CD28- CD8 T cells is a hallmark of the age-related decline in immune response. IL-15, a common γ-chain (γc) cytokine, is critical for the homeostatic proliferation of memory CD8 T cells and is recently shown to be associated with the down-regulation of CD28 expression. IL-21 is a newly identified member of the γc family of cytokines that has been demonstrated to have pleiotropic effects, influencing T cell proliferation and function. In IL-15-stimulated human CD8 naive T cells, IL-21 has been shown to prevent down-modulation of CD28 expression and augment cell growth. Here, we analyzed the effects of IL-21 on IL-15-stimulated memory CD8 T cells. In comparison between CD28+ and CD28- memory CD8 T cells under IL-15 alone or with IL-21 in vitro in young and old donors, we found that IL-21 drives more IL-15-stimulated cells to enter cell division and undergo apoptosis and that IL-21 preferentially enhances IL-15-induced proliferation of the CD28+ subset of memory CD8 T cells over its CD28- counterpart. Furthermore, we showed that IL-21 delays the IL-15-induced CD28 down-regulation at the cell surface and mRNA level for memory CD8 T cells. These findings demonstrate that IL-21 can increase proliferation of CD28+ cells and delay the generation of CD28- cells in IL-15-stimulated memory CD8 T cells, suggesting a possible role of IL-21 as an antagonist to the IL-15-induced increase in CD28- CD8 T cells that occurs during aging.

Differential Effect of Human Herpesvirus 6A on Cell Division and Apoptosis among Naïve and Central and Effector Memory CD4 + and CD8 + T-Cell Subsets

The immune responses of naive and different memory subsets of CD4(+) and CD8(+) T cells to human herpesvirus 6 (HHV-6) have not been previously investigated. We show that HHV-6A induces cell division, as measured by 5,6-carboxyfluorescein succinimidyl ester dye and flow cytometry, predominantly in two populations of effector memory CD4(+) and CD8(+) T cells (T(EM) and T(EMRA)); naïve (T(N)) and central memory (T(CM)) CD4(+) and CD8(+) T cells showed almost no cell division. In contrast, HHV-6A induced apoptosis primarily in T(N) and T(CM) CD4(+) and CD8(+) T cells, whereas T(EM) and T(EMRA) CD4(+) and CD8(+) T cells were resistant to HHV-6A-induced apoptosis. HHV-6A-induced apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, suggesting the involvement of death receptor and mitochondrial signaling pathways. In addition, HHV-6A induced secretion of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-8, and gamma interferon by peripheral blood mononuclear cells; TNF-alpha secretion was observed exclusively from CCR7(+) (T(N) plus T(CM)) CD4(+) T cells. These data show that HHV-6 differentially influences the functions of naïve T cells and different subsets of memory CD4(+) and CD8(+) T cells, which in part may be due to differential susceptibility to HHV-6A-induced apoptosis.

Impaired proliferative capacity and abnormal cytokine profile of naive and memory CD4 T cells from HIV-seropositive patients.

Purified naive and memory CD4 T cells from healthy donors, HIV+ asymptomatic carriers and AIDS patients were examined for their proliferative activity and their pattern of cytokine secretion (IL-4, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)) upon stimulation with phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and cross-linked anti-CD3 MoAb, in the presence of recombinant IL-2 (rIL-2). We found a decrease in the proliferative capacity of naive CD4 T cells following stimulation with PHA and PMA, and a sharp decline in this response upon cross-linked anti-CD3 stimulation in both subsets, although it predominated in the naive subpopulation. In AIDS patients, less pronounced impairment of thymidine uptake by the naive subset was found upon PHA and cross-linked anti-CD3 MoAb stimulation. In addition, an altered secretion pattern of the different cytokines was observed, consisting of abnormal secretion of IL-6 by both naive and memory cells, an abnormal pattern of IFN-gamma secretion and frequent loss of detectable IL-4 production by HIV patients. These abnormalities were even more pronounced in AIDS patients than in the asymptomatic carriers. Overall, our results extend previous reports indicating functional impairment of memory CD4 subsets in HIV+ subjects by showing that this impairment involves naive CD4 T cells.

Immunological alterations in newly diagnosed primary Sjögren's syndrome characterized by skewed peripheral T‐cell subsets and inflammatory cytokines

Objectives: To describe how certain peripheral immune parameters reflect the inflammatory alterations in patients with primary Sjögren's syndrome (pSS). We determined lymphocyte subpopulations and their state of activation from peripheral blood, evaluating both soluble serum T‐helper (Th)1/Th2‐type cytokines and intracytoplasmic cytokines.Methods: Forty‐nine patients with newly diagnosed pSS and 40 healthy individuals, all free from immunomodulant or immunosuppressive medication, were studied. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed using enzyme‐linked immunosorbent assay (ELISA), and intracellular cytokine levels were measured after phorbol myristate acetate (PMA) stimulation by flow cytometry after staining of intracellular cytokines.Results: Patients with primary SS had higher percentages of activated CD3+/CD69+ T cells than controls. When comparing naïve vs. memory subsets of CD4+ and CD8+ T cells, a shift towards the memory phenotype was observed for both. Natural killer (NK) cell and NK T‐cell (NKT) percentages and Th0 and Th1 cell numbers were increased in patients compared to controls. Among circulating cytokines, interferon (IFN)‐γ was high, whereas interleukin (IL)‐10 was decreased in SS when compared to controls.Conclusions: SS, considered as a systemic autoimmune disease, is characterized by a complex interplay of various cytokines and immune cells. The skewed T‐cell subsets and cytokine imbalance play important roles in an orchestrated proinflammatory cascade.

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that naïve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in naïve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both naïve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human naïve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.

Activation phenotype, rather than central– or effector–memory phenotype, predicts the recall efficacy of memory CD8+ T cells

The contributions of different subsets of memory CD8+ T cells to recall responses at mucosal sites of infection are poorly understood. Here, we analyzed the CD8+ T cell recall responses to respiratory virus infection in mice and demonstrate that activation markers, such as CD27 and CD43, define three distinct subpopulations of memory CD8+ T cells that differ in their capacities to mount recall responses. These subpopulations are distinct from effector– and central–memory subsets, coordinately express other markers associated with activation status, including CXCR3, CD127, and killer cell lectin-like receptor G1, and are superior to CD62L in predicting the capacity of memory T cells to mediate recall responses. Furthermore, the capacity of vaccines to elicit these memory T cell subpopulations predicted the efficacy of the recall response. These findings extend our understanding of how recall responses are generated and suggest that activation and migration markers define distinct, and unrelated, characteristics of memory T cells.

Persistence and Function of Central and Effector Memory CD4+ T Cells following Infection with a Gastrointestinal Helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.

Accentuate the Positive

The cytokine interleukin-2 (IL-2) facilitates proliferation of naïve T cells, but several studies have shown that antibodies that bind IL-2, which at first glance should be inhibitory, can promote the expansion of subsets of memory CD8 + T cells. Thus, IL-2 might somehow be inhibiting suppressive T cell populations that would otherwise prevent memory CD8 + T cell expansion. Boyman et al . (see the Perspective by Prlic and Bevan) now show that, instead, binding of antibodies to IL-2 augments the direct activity of the cytokine on memory CD8 + T cells themselves. Immune complexes form that focus local levels of IL-2 through presentation by Fc receptors. These observations could be important to consider in therapies that involve the manipulation of IL-2 and other cytokines, such as bone marrow transplantation and tumor immunotherapy. O. Boyman, M. Kovar, M. P. Rubinstein, C. D. Surh, J. Sprent, Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311 , 1924-1927 (2006). [Abstract] [Full Text] M. Prlic, M. J. Bevan, An antibody paradox, resolved. Science 311 , 1875-1876 (2006). [Summary] [Full Text]

Paradoxic inhibition of human natural interferon-producing cells by the activating receptor NKp44

AbstractNatural killer (NK) cell-mediated cytotoxicity is triggered by multiple activating receptors associated with the signaling adaptor protein DNAX activation protein 12/killer cell-activating receptor-associated protein (DAP12/KARAP). Here, we show that one of these receptors, NKp44, is present on a subset of natural interferon-producing cells (IPCs) in tonsils. NKp44 expression can also be induced on blood IPCs after in vitro culture with interleukin 3 (IL-3). Crosslinking of NKp44 does not trigger IPC-mediated cytotoxicity but, paradoxically, inhibits interferon α (IFN-α) production by IPCs in response to cytosine-phosphate-guanosine (CpG) oligonucleotides. We find that IPCs in tonsils are in close contact with CD8+ T cells and demonstrate that a subset of memory CD8+ T cells produces IL-3. Therefore, IL-3-mediated induction of NKp44 on IPCs may be an important component of the ongoing crosstalk between the innate and adaptive immune response that allows memory CD8+ T cells to control the IPC response to virus. (Blood. 2005;106: 2076-2082)

Molecular mechanisms of apoptosis in the cells of the immune system in human aging

Aging is associated with progressive decline in immune functions and increased frequency of infections, autoimmunity, and cancer. Among immune functions, a decline in T-cell functions during aging predominates. In this review, I discuss the molecular signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the most recently described endoplasmic reticulum stress pathway, and the relative sensitivity of naive, central memory, and effector memory CD8(+) T-cell subsets to apoptosis. In addition, I review apoptosis, especially via death receptor pathway, in naive and various memory subsets of CD4(+) and CD8(+) T cells (with primary emphasis on CD8(+) naive and memory subsets) in human aging and discuss the role of apoptosis in immune senescence.

Differential Upregulation of CD38 on Different T-Cell Subsets May Influence the Ability to Reconstitute CD4+ T Cells Under Successful Highly Active Antiretroviral Therapy

Immune activation is an independent surrogate marker of CD4 T-cell depletion in HIV-infected patients. Highly active antiretroviral therapy (HAART) reduces disease progression as a direct consequence of suppressing HIV replication. Immune function does not normalize completely in most subjects on HAART, however, perhaps reflecting residual HIV replication. So far, it is unclear to what extent immune activation may influence the evolution of CD4 T-cell counts in patients on HAART. The expression of CD38 on naive and memory subsets of CD4+ and CD8+ T cells was measured quantitatively by flow cytometry in 62 drug-naive HIV-positive and 30 HIV-uninfected controls. In addition, the evolution of this marker as well as that of some virologic parameters (plasma viremia and proviral load) and CD4 counts were assessed in 25 HIV-infected individuals who initiated HAART and were followed for 12 months. The mean level of CD38 on memory CD4+ and CD8+ T cells as well as in naive CD8+ cells was significantly higher in drug-naive HIV-positive subjects than in HIV-negative controls. Moreover, it was highly correlated with viral load titers. In patients on successful HAART, immune activation declined in all T-cell subsets, particularly among memory CD8+ cells. It remained elevated with respect to HIV-negative controls, however, even after 12 months of HAART. There was a significant correlation between the CD8+ T-cell activation decay and the increase of CD4+ T cells on HAART. Patients with the highest decline in CD8 activation were those showing the highest CD4 T-cell gains after 12 months of therapy. The level of CD38 expression on different T-cell subsets is differentially upregulated in drug-naive HIV-infected patients. After successful HAART, immune activation decreases in all T-cell subsets, although it still remains elevated in most cases after 12 months of HAART. The extent of immune deactivation under successful HAART correlates with the ability to reconstitute CD4 counts.

Retrovirus mediated gene transduction of human T-cell subsets.

Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukaemia. Graft versus host disease (GVHD) is a potentially life threatening complication of AlloBMT mediated by the T cells contained within the graft. In order to be able to control GVHD, the allogeneic T cells may be transduced with a suicide gene such as herpes simplex virus thymidine kinase (HSV-tk). For this strategy to be successful, all subsets of T cells should be transduced to a similar extent. Also, the transduction protocol should not induce expression of unwanted homing receptors, nor should it lead to unwanted skewing of the T-cell receptor repertoire. We have studied the transduction efficiency of naïve and memory subsets of CD4+ and CD8+ T cells, and examined the transduced T-cell subsets for possible changes in T-cell receptor (TCR) repertoire and homing receptor expression. The cells were transduced using a Moloney murine retroviral vector carrying a conjugate of the genes encoding the truncated form of the cell surface marker, low affinity nerve growth factor receptor (DeltaLNGFR) and HSV-tk. Transduction efficiency and homing receptor expression were quantified by flow cytometry. TCR repertoire was determined by spectratyping. We obtained a transduction efficiency of 30-50% of the cells, with no difference between the T-cell subsets. Cell surface receptors responsible for homing to skin, gastrointestinal tract or lymph nodes were practically absent at the end of 2 weeks in culture. The activation procedure seemed to favour the expansion of certain T-cell clones over polyclonal populations. However, there was no difference in the TCR repertoire between transduced and non-transduced cells. Changes in the composition of the T-cell subsets at the end of the cell culture were the results of the activation, and not the suicide gene transduction. The transduced T cells did not express unwanted homing receptors.

The Acquired CD40L Deficiency in B-CLL Is Reversible and Due To Contact Dependant and Independent Factors.

Interactions between CD40L (CD154) and its receptor CD40, are of central importance in T-cell mediated B-cell activation, proliferation and isotype switching and the regulation of antigen presentation by dendritic cells. Peripheral blood T lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) have an acquired defect of activation induced CD40L expression that might contribute to the immunodeficiency characteristic of the disease. In view of recent reports that T-cells within bone marrow and lymph node pseudofollicles express CD40L we have re-examined the mechanism of the deficiency in PB cells. Activation of normal resting naïve and memory (CD45RA+ and −) T-cells with PMA and ionomycin increased the number expressing surface CD40L from a mean of 2 and 0% to 81.2 and 66.5% respectively. In line with previous reports, up-regulation of CD40L by CD4+45RA+ and CD4+45RA− T-cells from patients with B-CLL was reduced to a mean of 11.8% and 2.6%. This defect is reversible since removal of T-cells from the malignant clone by CD3 selection increased the number of cells able to up-regulate CD40L to 62.2% and 61.8% for naïve and memory CD4 subsets. To investigate whether this phenomenon is due to cell contact or soluble mediators, washed leukemic cells or supernatant (SN) harvested from the same cell number were incubated with normal donor T-cells for 48 hours then stimulated for 4 hours with PMA and Ionomycin. B-CLL SN reduced CD40L up-regulation by a mean of 51% (range 14–86, n=17, p<0.0001) but co-culture reduced this further to a mean of 17% (range 7–33%, n=8, p<0.0001). Other markers of T-cell activation were similarly affected, for example T-cell IL-2 production was reduced to 40.1% ( 6.2% SEM, p < 0.0001) of the level seen in the absence of B-CLL SN. Although SN and cell contact both prevented CD40L up-regulation, only cell contact caused its down-regulation in pre-activated T-cells (reduced to 94% of normal with SN, [range 93–96%, n=3, p=0.5] and 14% of normal with co-culture [range 4.3–21.7%, n=6, p p<0.0001]). The acquired CD40L deficiency observed in patients with B-CLL is thus reversible and mediated by contact with leukemic cells and soluble mediator(s). B-CLL cells are known to secrete a number of factors that might produce this effect. Studies using blocking monoclonal antibodies and immuno-adsorption excluded the most likely candidates including TGF-beta, soluble CD40 and soluble IL-2R. These findings indicate that regulation of the CD40/CD40L system in B-CLL is more complex than previously reported however the impact of this on disease pathogenesis needs to be determined.