Central nervous system pathology caused by autoreactive CD8+ T-cell clones following virus infection

Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating disease in infected mice which has similarities to multiple sclerosis. Spleen cells from TMEV-infected SJL/J mice stimulated with antigen-presenting cells infected with TMEV resulted in a population of autoreactive CD8+ cytotoxic T cells that kill uninfected syngeneic cells. We established CD8+ T cell clones that could kill both TMEV-infected and uninfected syngeneic targets, although infected target cells were killed more efficiently. The CD8+ T-cell clones produced gamma interferon when incubated with either infected or uninfected syngeneic target cells. Intracerebral injection of the clones into na?ve mice induced degeneration, not only in the brain, but also in the spinal cord. This suggests that CD8+ Tc1 cells could play a pathogenic role in central nervous system inflammation.     

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.     

Follicular Dendritic Cells Retain Infectious HIV in Cycling Endosomes

Despite the success of antiretroviral therapy (ART), it does not cure Human Immunodeficiency Virus (HIV) and discontinuation results in viral rebound. Follicular dendritic cells (FDC) are in direct contact with CD4+ T cells and they retain intact antigen for prolonged periods. We found that human FDC isolated from patients on ART retain infectious HIV within a non-degradative cycling compartment and transmit infectious virus to uninfected CD4 T cells in vitro. Importantly, treatment of the HIV+ FDC with a soluble complement receptor 2 purges the FDC of HIV virions and prevents viral transmission in vitro. Our results provide an explanation for how FDC can retain infectious HIV for extended periods and suggest a therapeutic strategy to achieve cure in HIV-infected humans.     

Human T-cell leukemia virus type I infection of CD4+ or CD8+ cytotoxic T-cell clones results in immortalization with retention of antigen specificity.

The human T-cell leukemia virus type I (HTLV-I) is capable of chronically infecting various types of T cells and nonlymphoid cells. The effects of chronic infection on the specific functional activities and growth requirements of mature cytotoxic T lymphocytes (CTL) have remained poorly defined. We have, therefore, investigated the results of HTLV-I infection of both CD4+ and CD8+ human CTL clones. HTLV-I infection resulted in the establishment of functional CTL lines which propagated indefinitely in culture many months longer than the uninfected parental clone. The infected cells became independent of the need for antigen (target cell) stimulation as a requirement for proliferation and growth. Like their uninfected counterparts, however, these HTLV-I-infected clones remained strictly dependent on conditioned medium from mitogen-stimulated T lymphocytes for their growth. This growth factor requirement was not fulfilled by recombinant interleukin-2 alone. Furthermore, the infected lines remained functionally identical to their uninfected parental CTL clones in their ability to specifically recognize and lyse the appropriate target cells. Our findings indicate that the major effects of HTLV-I infection on mature CTL consist of (i) the capacity for proliferation in the absence of antigen stimulation and (ii) a prolonged or immortal survival in vitro, but they also indicate that the fine specificity and cytolytic capacity of these cells remain unaffected.     

Phenotypic and functional characterization of human T cell clones

The capacity of human peripheral blood-derived T cell clones to carry out a variety of functions was examined. T cell clones were generated by stimulating individual peripheral blood T cells with PHA by a procedure that yielded a growing clone from a mean of greater than 92% of the cultured cells. A total of 65 T cell clones (44 CD4+ and 21 CD8+) generated from two individual donors were examined for their functional capabilities. All T cell clones examined secreted IL-2, IFN-gamma, and lymphotoxin/tumor necrosis factor like activity when stimulated with immobilized mAb to the CD3 complex (64.1). When 54 additional T cell clones from a third donor were analyzed, all were found to produce IL-2. Upon activation with immobilized 64.1, all CD4+ clones and 91% of the CD8+ clones induced the generation of Ig-secreting cells from purified B cells. The CD8+ clones that did not serve as Th cells alone were able to augment the capacity of fresh CD4+ cells to generate Ig-secreting cells. Each of these clones was also found to effect MHC-unrestricted cytotoxicity upon activation with immobilized 64.1. The CD8+ clones were somewhat more effective killers than CD4+ clones, although there was considerable overlap. A total of 18 clones was analyzed for TCR beta-chain gene rearrangement. Of the clones exhibiting rearrangements of the beta-chain gene, 94% were found to have a single rearrangement pattern. Finally, the detailed phenotype of 15 (11 CD4+ and 4 CD8+) of these clones was examined. Variable numbers of cells of each of the clones expressed Ag identified by mAb 4B4 (CD29), Leu 8, Leu 15 (CD11b), and NKH1. Moreover, cells of 6 of 11 CD4+ clones and 4 of 4 CD8+ clones also expressed CD45R in addition to CD29; expression of CD45R and CD29 varied with the activation status of the clone. The current data demonstrate that nearly all of the T cell clones were able to accomplish each of the functions examined regardless of the surface phenotype. Inasmuch as the clones were generated using a technique that expanded more than 92% of the circulating T cells, the data imply that the progeny of the vast majority of T cells may have the inherent capacity to exert a wide array of functional activities.     

Human T lymphocyte clones specific for malaria (Plasmodium falciparum) antigens.

Peripheral blood mononuclear cells (PBM) from a patient who had lived in a malarial-endemic area were cultured in the presence of malarial antigens (a lysate of Plasmodium-infected erythrocytes). Responding cells were grown in IL-2-containing medium and then cloned, and subsequently subcloned, in the presence of phytohemagglutinin and allogeneic irradiated PBM. Ten clones were specific for malarial antigens. They proliferated in response to P. falciparum extract, but not to a lysate of uninfected erythrocytes. The response was HLA-restricted. All the clones tested responded to lysates of cells infected with parasites of either African or Asian origin. Six clones had the T4+/T8- phenotype and four the T4-/T8+ phenotype. Two of the T4+ clones recognised a parasite antigen of apparent mol. wt. approximately 50 000. All of the clones tested produced gamma-interferon following antigen stimulation.     

Comparison of lymphokine secretion and responsiveness of human T cell clones isolated in IL-4 and in IL-2.

Interleukin (IL)-4 has been shown to be secreted simultaneously with IL-2 and interferon (IFN)-gamma by the majority of CD4+ human T cell clones isolated and cultured using IL-2 as a growth factor. Moreover, IL-4 was found to be as efficient as IL-2 to promote the outgrowth of human T cell clones. In this study we have investigated the pattern of lymphokine production by human T cell clones isolated and cultured in IL-4. Most of the CD4+ T cell clones isolated in IL-4 were found to have the ability to simultaneously secrete IL-2, IL-4, and IFN-gamma upon activation. The T cell clones isolated in IL-4 produced, in general, more IL-4 and less IL-2 than the clones isolated and cultured in IL-2. This tendency did not appear to be a stable feature inasmuch as when representative CD4+ T cell clones were split and cultured in either IL-2 or IL-4, the clones in IL-2 secreted more IL-2 and less IL-4 than the same cells cultured in IL-4. These results indicate that the isolation and culture of human CD4+ T cells in IL-4 did not lead to an "irreversible" development of these cells into Th-1- or Th-2-like cells. Clones isolated in IL-4 responded better to IL-4 than they did to IL-2. On the other hand, T cell clones from the same donor isolated in IL-2 showed the reverse pattern since these latter cells were found to respond better to IL-2 than to IL-4. Furthermore, "nonresponsiveness" of a T cell clones in a [3H]TdR assay to either IL-2 or IL-4 is not a stable feature since clones, unresponsive to a particular lymphokine, could be adapted to become responsive.     

Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term stable expanded human CD4+ T cell clones specific for human cytomegalovirus are distributed in both CD45RAhigh and CD45ROhigh populations

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-gamma secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR beta-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3-1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28-CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.     

Analysis of host-virus interactions in AIDS with anti-gp120 T cell clones: effect of HIV sequence variation and a mechanism for CD4+ cell depletion

The primary human T cell response to HIV was analyzed by isolating from seronegative donors T cell clones specific for HIV gp120. T cell epitopes restricted by different MHC elements were identified within gp120, and synthetic peptides were used to address the fundamental problem of how HIV sequence variability affects T cell recognition. Even one conservative substitution can drastically reduce recognition; thus the interaction of gp120 epitopes with T cell receptors and MHC is precise and poorly crossreactive. Importantly, a subset of CD4+ gp120-specific clones manifest cytolytic activity and lyse uninfected autologous CD4+Ia+ T cells in the presence of gp120 in a process that is strictly dependent upon CD4-mediated uptake of gp120 by T cells. Assuming gp120 is shed from HIV-infected cells in vivo, this novel CD4-dependent autocytolytic mechanism may contribute to the profound depletion of CD4+ cells in AIDS.     

Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.

The lentiviruses of sheep, goats, and horses cause chronic multiorgan disease in which macrophages are highly permissive for viral replication. Monocytes, which mature into macrophages, are thought to be latently infected with lentivirus, but the extent to which other leukocytes are infected is unknown. Dendritic cells have not been studied separately from monocytes and T-cell subsets have not been examined in previous attempts to identify infected cells in peripheral blood mononuclear cells (PBMC). We found no evidence of T-cell tropism using an animal-passaged, pathogenic ovine lentivirus. Phytohemagglutinin-stimulated infectious PBMC produced 20-fold less virus than differentiated macrophages, and cocultivation of infectious PBMC with fresh, uninfected phytohemagglutinin blasts did not facilitate virus replication. Furthermore, central lymph cells, the best in vivo source of purified lymphocytes, lacked virus and did not yield virus upon in vitro cultivation. In contrast, cultivated blood-derived macrophages were highly permissive for viral replication. To identify the latently infected PBMC, PBMC from infected sheep were selectively depleted of monocytes and B cells by passage over nylon wool and then of nonadherent cells bearing CD4, CD8, T19, gamma delta T-cell receptor, CD45RA, or major histocompatibility complex class II antigens by panning. Removal of adherent monocytes and B cells or of adherent cells and the three major T-cell subsets (CD4+, CD8+, T19+) did not decrease the infectivity of PBMC. The richest sources of infected cells in fresh PBMC were CD45RA+ and major histocompatibility complex class II+ nonadherent cells, which are three characteristics of dendritic cells. Thus, the dendritic cell, and not the monocyte or the CD4+ cell, is probably the predominant infected cell type in blood.     

After 18 months of antiretroviral therapy,total HIV DNA decreases more pronouncedly in patients infected by CRF01_AE than in those infected by subtype B and CRF07_BC

Whether the amount of HIV DNA is associated with the subtype of HIV‐1 after antiretroviral therapy (ART) has not been reported. In the present study, the amount of HIV DNA and RNA and CD4+T counts in blood and semen prior to and after 18 months of ART were compared in 48 patients infected by CRF01_AE, subtype B or CRF07_BC of HIV‐1. Viral RNA was suppressed and CD4 cell count recovery achieved in all patients. The level of HIV DNA were similar before ART; however, patients with CRF01_AE had less HIV DNA after ART than those with subtype B and CRF07_BC infection. According to prediction of co‐receptor usage by Geno2Pheno and PSSM in combination, more than 35.6% of clones for CRF01_AE were predicted as CXCR4‐using before ART, whereas less than 6% of those for subtype B and CRF07_BC were predicted as CXCR4‐using. After 18 months of ART, no CXCR4‐using clones were predicted in any of the subtypes. Despite more HIV RNA and fewer CD4 + T cells in patients with CRF01_AE before therapy, no significant differences (P > 0.05) in viral RNA or CD4 cell counts were observed between the subtypes after 18 months of ART. Thus, 18 months of antiretroviral therapy was more efficient in patients with CRF01_AE. Considering that successful ART dramatically reduces the viral load in both blood and semen, risks of sexual transmission of HIV were reduced, contributing to prevention of rapid spread of HIV among men who have sex with men in the region.

     

Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones

We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.     

A Quantitative Comparison of Anti-HIV Gene Therapy Delivered to Hematopoietic Stem Cells versus CD4+ T Cells

Gene therapy represents an alternative and promising anti-HIV modality to highly active antiretroviral therapy. It involves the introduction of a protective gene into a cell, thereby conferring protection against HIV. While clinical trials to date have delivered gene therapy to CD4+T cells or to CD34+ hematopoietic stem cells (HSC), the relative benefits of each of these two cellular targets have not been conclusively determined. In the present analysis, we investigated the relative merits of delivering a dual construct (CCR5 entry inhibitor + C46 fusion inhibitor) to either CD4+T cells or to CD34+ HSC. Using mathematical modelling, we determined the impact of each scenario in terms of total CD4+T cell counts over a 10 year period, and also in terms of inhibition of CCR5 and CXCR4 tropic virus. Our modelling determined that therapy delivery to CD34+ HSC generally resulted in better outcomes than delivery to CD4+T cells. An early one-off therapy delivery to CD34+ HSC, assuming that 20% of CD34+ HSC in the bone marrow were gene-modified (G+), resulted in total CD4+T cell counts ≥180 cells/ µL in peripheral blood after 10 years. If the uninfected G+ CD4+T cells (in addition to exhibiting lower likelihood of becoming productively infected) also exhibited reduced levels of bystander apoptosis (92.5% reduction) over non gene-modified (G-) CD4+T cells, then total CD4+T cell counts of ≥350 cells/ µL were observed after 10 years, even if initially only 10% of CD34+ HSC in the bone marrow received the protective gene. Taken together our results indicate that: 1.) therapy delivery to CD34+ HSC will result in better outcomes than delivery to CD4+T cells, and 2.) a greater impact of gene therapy will be observed if G+ CD4+T cells exhibit reduced levels of bystander apoptosis over G- CD4+T cells.     

The contribution of parasite-specific T cells to isotype restriction in Mesocestoides corti-infected mice

Mice infected with the parasite Mesocestoides corti undergo a polyclonal antibody response that results in a hypergammaglobulinemia restricted to the IgM and IgG1 isotypes. It was found that a similar restriction to IgM and IgG1 could be observed in an in vitro lymphocyte culture system providing that the source of helper T cells was from infected animals. In order to characterize the helper T cells responsible for the restriction, helper T cell clones were generated. Attempts to obtain isotype-restricting helper T cell clones by using the intact, nonviable organism were unsuccessful in that these T cell clones promoted multiple antibody class expression. However, two types of CD4+ (cluster designation) T cell clones were generated by cultivation on the live organism that appeared relevant to the observed restriction. These T cells did not function as conventional carrier-specific helper T cells. Instead, they were shown to regulate T-dependent responses to 2,4-dinitrophenyl-keyhole limpet hemocyanin by 2,4-dinitrophenyl-specific B cells and keyhole limpet hemocyanin-primed T cells derived from uninfected mice. The helper phenotype of one regulatory clone enhanced the IgG1 response, whereas the other phenotype inhibited the production of the other non-IgM isotypes tested. It is concluded that the activities of these two prototype regulatory T cell clones may predominate in infected animals resulting in the IgM, IgG1 dominance of the antibody response.     

Adenovirus-specific CD4+ T cell clones recognizing endogenous antigen inhibit viral replication in vitro through cognate interaction

Human adenovirus (HAdV) infection is a frequent and potentially severe complication following allogeneic stem cell transplantation in children. Because treatment with antiviral drugs is often ineffective, adoptive transfer of donor-derived HAdV-specific T cells able to control viral replication of HAdV of multiple serotypes may be an option for therapy. In healthy donors, predominantly HAdV-specific T cells expressing CD4 are detected. In this study, a preclinical in vitro model was used to measure the antiviral effect of HAdV-specific CD4+ T cells. CD4+ HAdV-specific T cell clones restricted by HLA class II molecules were generated and most of these clones recognized conserved peptides derived from the hexon protein. These cross-reactive T cell clones were able to control viral replication of multiple serotypes of HAdV in EBV-transformed B cells (B-LCL), melanoma cells (MJS) and primary bronchial epithelial cells through cognate interaction. The HAdV-specific CD4+ T cell clones were able to specifically lyse infected target cells using a perforin-dependent mechanism. Antigenic peptides were also presented to the CD4+ T cell clones when derived from endogenously produced hexon protein. Together, these results show that cross-reactive HAdV-specific CD4+ T cells can control replication of HAdV in vitro and provide a rationale for the use of HAdV-specific T cells in adoptive immunotherapy protocols for control of life-threatening HAdV-infections in immunocompromised patients.