Molecular correlates in AIDS patients following antiretroviral therapy: diversified T-cell receptor repertoires and in vivo control of cytomegalovirus replication.

OBJECTIVES: To evaluate whether successful, long-term immune reconstitution in vivo can be achieved in end-stage AIDS patients following antiretroviral therapy (ART). METHODS: A 1-year prospective study of changes of CD4+ and CD8+ T-cell surface phenotypes, T-cell receptor (TCR) repertoires and capacity to control in vivo replication of cytomegalovirus (CMV) was performed in five treatment-naive end-stage AIDS patients (median CD4+ T-cell counts of 19 cells/microL) following therapy. Proportions of CD45RA+, CD45RO+ and CD28+ cells within the CD4+ and CD8+ subsets, were determined by flow cytometry. Changes in TCR Vbeta repertoires within the CD4+ and CD8+ T-cell compartments were evaluated using CDR3 spectratyping. CMV replication was determined by a sensitive polymerase chain reaction (PCR) assay using whole blood. RESULTS: Following ART, proportionate increases in 'naive' (CD45RA+) and 'memory' (CD45RO+) T cells were observed within both CD4+ and CD8+ T-cell subsets, while increased numbers of CD28+ T cells were mainly observed within the CD4+ subset. Diversification of CD4+ and CD8+ TCR repertoires was established concomitantly with renewed in vivo control of CMV replication. CONCLUSIONS: An important degree of molecular and functional immune recovery is possible in end-stage AIDS patients introduced to therapy. Diversification of TCR repertoires and the in vivo restoration of immunocompetence to control opportunistic infections clearly show that an important degree of molecular immune reconstitution is established following the initiation of ART even in late-stage AIDS.

Clonal evolution of CD8+ T-cell expansions in HIV-infected patients on long-term HAART.

HIV-1 continually replicates in spite of long-term highly active anti-retroviral therapy (HAART) and therefore, it is conceivable that the low level, persistent viral activity could continue to stimulate the hosts immune system despite remaining below the detection limit of the current assays. In this study, we performed a longitudinal analysis of the CD8+ T-cell receptor Vbeta repertoire in HAART-treated and untreated HIV patients. HAART-mediated control of viremia, for up to 18 months, did not prevent similar perturbations within the CD8+ Vbeta repertoire in both study groups as defined by CDR3 spectratyping. Oligoclonal Vbeta expansions, with new dominant CDR3 lengths, were observed throughout the study period. Our findings are compatible with antigen-driven CD8+ immune responses to bursts of replication from a continuously changing viral reservoir, regardless of HAART-mediated suppression of HIV-1 viremia.

T-cell re-population in HIV-infected children on highly active anti-retroviral therapy (HAART).

In this pilot study, we address the nature of the re-population of the T-cell compartment in HIV-1+ (Human Immunodeficiency Virus 1), vertically infected children placed on successful regimens of HAART (highly active anti-retroviral therapy) incorporating 2 NRTI and a protease inhibitor. The clonality of the T-cell compartment and the abundance of RTEs (Recent Thymic Emigrants) were determined 2 weeks before and 20 weeks after initiation of HAART in a subgroup of children taking part in the PENTA (Paediatric European Network for the Treatment of AIDS) 5 trial. Analysis of the clonality of the circulating T-cell compartment was assessed using CDR3 spectratyping and analysed using the Kolmogorov-Smirnov two sample test. This revealed that a high degree of T-cell clonal restriction still exists 5 months into therapy, despite the appearance of previously undetectable T-cell clones within the periphery. We detected no increase in RTE abundance in this 5 month period, as determined by PCR detection of TRECs (T-Cell Receptor Excision Circles). We conclude that the observed re-population of T cells within the periphery of treated children is heavily reliant upon the maintenance/expansion of pre-existing cells during the 5 month period immediately following the initiation of therapy.

H-2D(b-/-) mice are susceptible to persistent infection by Theiler's virus.

H-2(b) mice are resistant to persistent infection of the central nervous system by Theiler's virus. They clear the infection 7 to 10 days after intracranial inoculation. Resistance maps to the H-2D gene and not to the H-2K gene and is associated with a potent antiviral cytotoxic T-lymphocyte (CTL) response. We used H-2(b) mice in which the H-2D or the H-2K gene had been inactivated to dissect the respective roles of these genes in resistance. We report that H-2D(-/-) but not H-2K(-/-) mice were susceptible to persistent infection. Furthermore, whereas H-2K(-/-) mice mounted a vigorous virus-specific CTL response, similar to that of control C57BL/6 mice, the CTL response of H-2D(-/-) mice was nil or minimal. Using target cells transfected with the H-2D(b) or the H-2K(b) gene, we showed that the H-2K-restricted CTL response against the virus was minimal in H-2D(-/-) mice. These results demonstrate that the H-2D(b) and H-2K(b) genes play nonredundant roles in the resistance to this persistent infection.

'Naïve' and 'memory' CD4+ T-cells and T-cell receptor (TCR) V beta repertoire dynamics are independent of the levels of viremia following HIV seroconversion.

The progression of 'naive' and 'memory' T-cells and the T-cell receptor Vbeta (TCR Vbeta) repertoire dynamics within the peripheral CD4+ T-cell compartment were studied in individuals following HIV seroconversion. Profound TCR Vbeta repertoire perturbations were observed within the CD4+ T-cell pool in treatment-naive patients regardless of their levels of viremia during the first 6-8 months after seroconversion. The ratio of 'naive' to 'memory' CD4+ T-cells as well as the TCR Vbeta repertoire dynamics did not appear to correlate with absolute numbers of CD4 T-cells.

An early, abundant cytotoxic T-lymphocyte response against Theiler's virus is critical for preventing viral persistence.

In genetically susceptible strains of mice, the DA strain of Theiler's virus, a picornavirus, causes a persistent infection of the white matter of the spinal cord associated with chronic demyelination. In resistant strains, on the other hand, the infection is cleared within 1 to 2 weeks. In this article, we show that Theiler's virus induces a rapid and abundant cytotoxic T lymphocyte (CTL) response in resistant C57BL/6 mice, while the response remains low throughout infection in susceptible SJL/J mice. This difference can be referred to a higher number of virus-specific CTL precursors in C57BL/6 mice. These observations indicate that the efficient induction of virus-specific CTL precursors is critical for avoiding the establishment of a persistent picornaviral infection.

Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice.

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.

In vivo administration of interleukin-2 protects susceptible mice from Theiler's virus persistence.

In vivo administration of interleukin-2 (IL-2)-secreting tumor cells results in complete protection against persistent infection by Theiler's murine encephalomyelitis virus (TMEV) in susceptible DBA/2 mice. The IL-2-mediated protection was found to depend on the inoculum size as well as the timing of IL-2 administration. IL-2-treated and TMEV-infected mice displayed a three- to fourfold relative increase in virus-specific cytotoxic T-lymphocyte (CTL) precursors. Thus, we postulate that the persistence of TMEV infection in susceptible mice reflects limited numbers of relevant CTL precursors and their time course of induction and activation.

Co-expression of T-cell receptor beta and delta mRNA detected at high frequency in hybridomas derived from adult thymus.

Two different hybridoma collections from adult C3H/HeJ thymus were generated in order to analyse T-cell receptor (TcR) rearrangements, surface expression of T-cell receptors and differentiation markers as well as lymphokine production. Large, low density thymocytes were either directly fused to the thymoma BW 5147 alpha-beta- variant, or fused after stimulation with Concanavalin A in the presence of interleukin-2 for 48 h. The hybrids obtained from Concanavalin A-stimulated cells represented rather mature thymocytes, with regard to TcR rearrangements and surface T-cell receptor expression. The collection of hybrids derived from freshly isolated large thymocytes contained cells in various stages of T-cell development. An unexpectedly large number of hybrids (46 out of 84) from this group expressed full-length C beta together with full-length, or shorter, C delta mRNA. This finding suggests that a considerable proportion of alpha beta T cells proceeds through a stage in development where delta genes are being rearranged and transcribed.

Induction of CD8 molecules on thymic gamma/delta T cells in vitro is dependent upon alpha/beta T cells.

Thymocytes differentiate upon interactions with microenvironmental components, but the precise role of different stromal cells, or other T cells, in early differentiative events remains unclear. Here we have analyzed the in vitro differentiation of double-negative (DN) thymocytes from young adult mice. We demonstrate that a substantial proportion of DN thymocytes differentiate into CD8+ gamma/delta T cells upon stimulation with concanavalin A and recombinant interleukin 2. However, if alpha/beta T cells are excluded from the initial population of DN thymocytes, the CD8+ gamma/delta T cells do not appear in the cultures. These results suggest a role for T-T cell interactions in thymic differentiative events, and provide evidence for physiological interactions between the alpha/beta and gamma/delta T cell compartments within the thymus.

Interleukin 2-dependent activation of tumor-specific cytotoxic T lymphocytes in vivo.

We have quantitatively studied the effect of interleukin (IL) 2 on the cytotoxic T lymphocyte (CTL) response to tumor cells in vivo. Mastocytoma P815 was transfected with murine IL 2 cDNA (P815-IL 2) and injected into syngeneic mice. The anti-tumor response was analyzed and compared with the response induced by the non-transfected cells. P815 parental cells are highly tumorigenic, causing death within 20-30 days. In contrast, IL 2-transfected cells were totally rejected. Co-injection of IL 2-secreting and parental cells resulted in the inhibition of growth of both type of tumors. In addition, the response induced by IL 2-secreting cells protected the mice against a subsequent challenge with P815. Long-term memory persisted in treated mice 3 months after tumor rejection. Frequencies of CTL precursors and CTL specific for P815 increased as a result of IL 2 secretion by the target cells. Estimates of CTL frequency at days 8 and 12 after injection were 2 to 3 times higher in mice inoculated with P815-IL 2 cells, and this correlated with tumor rejection.

A novel approach to the induction of specific cytolytic T cells in vivo.

The induction of specific cytotoxic T lymphocytes (CTL) is one component in the immune response which can effectively protect the host against the progression of many viral infections. CTL are also known to play an important role in immune defence against tumour growth. CTL induction is dependent the presence of the specific antigen, appropriately presented, and interleukin-2 (IL2), provided by T helper lymphocytes. We studied the specific CTL response induced by tumour cells transfected with murine IL2. Our results show that tumour cells manipulated to secrete IL2 induce an improved specific anti-tumour response which results in tumour rejection in mice. To further investigate the effect of IL2 on the CTL response to different antigens, we introduced synthetic peptides into IL2-secreting tumour cells and determined the specific CTL induction in syngeneic mice immunized with these cells. We report here that such IL2-secreting cells can effectively prime peptide-specific CTL in vivo. Our data are relevant to immunotherapy and vaccine development and open up the possibility that autologous cells, manipulated to secrete IL2 and located with one or a cocktail of peptides, could be used to stimulate a specific CTL response.

Analysis of T cell receptor V beta gene usage in primary mixed lymphocyte reactions: evidence for directive usage by different antigen-presenting cells and Mls-like determinants on T cell blasts.

The usage of four different T cell receptor (TcR) V beta gene families within normal, non-primed T cell populations in response to various types of antigen-presenting cells (APC) in primary mixed lymphocyte reaction has been studied. We demonstrate that distinct patterns of V beta gene usage are obtained within a given T cell population in response to different types of APC with the same allo-H-2. When responder T cells are stimulated with one type of allogeneic APC, from various H-2-disparate mice, the same V beta gene preference is observed. Furthermore, when H-2- and Mls-mismatched APC gene used as stimulators, the Mls-associated V beta 6 and V beta 8.1 gene families are highly elevated in response to both B and T cell blasts from certain Mls-positive strains. The results demonstrate that different types of allogeneic APC have the capacity to generate biases in TcR V beta gene usage and imply that functional Mls-like determinants are presented by T cell blasts. The findings are discussed with respect to TcR-major histocompatibility complex interactions in allostimulation.

Amelioration of intrathymic T cell development and peripheral T cell reactivities in autoimmune mice undergoing therapy with a novel immunomodulator.

MRL lpr/lpr mice develop a generalized autoimmune disease associated with a massive accumulation in the peripheral lymphoid organs of abnormal, phenotypically immature T cells. Both the lymphoadenopathy and the autoimmunity are thymus-dependent and likely to arise from an aberrant pathway of intrathymic differentiation. We here present the marked beneficial effects acquired in MRL lpr/lpr mice after in vivo administration of a novel immunomodulator called linomide. The highly altered pattern of thymic subpopulations in MRL lpr/lpr mice is normalized after linomide-treatment. Concomitantly, the peripheral T cell compartment, which in MRL lpr/lpr is highly deficient in producing and responding to IL-2, gains substantial functional reactivities. We propose that linomide acts by correcting the abnormal T cell development in autoimmune MRL lpr/lpr mice. This new immunomodulator may be a useful tool for providing insight into both the pathogenesis of autoimmune disorders and intrathymic differentiation.

Decreased levels of pathogenic IgG anti-DNA antibodies in autoimmune mice after linomide treatment.

Treatment with a novel immunomodulator called linomide resulted in highly reduced levels of pathogenic anti-DNA antibodies in MRL-lpr/lpr mice compared to untreated mice. This reduction occurred without altering the total levels of serum immunoglobulins, implying that the compound does not have any direct B-cell suppressive effect. The possible role of T cells in regulating autoreactive B-cell clones is discussed.

A compartment of effector helper and suppressor T cells in the normal mouse thymus.

We have recently described an autonomously activated set of T cells in the spleens of normal and "antigen-free" mice which display effector T helper (TH) or T suppressor (TS) activities; we describe here an intrathymic effector T-cell compartment which directly helps or suppresses B-cell responses and appears to be distinct from the peripheral set of effector cells. Splenic effector T cells do not represent recent thymic migrants (because adult thymectomized mice have unaltered levels of effector TH and TS cells in the spleen), nor do intrathymic effector T cells represent circulating peripheral T cells (since thymic effector T cells are B2A2+, while splenic effector T cells are B2A2-). Furthermore, effector TH cells within the two compartments exert differential effector activities: splenic effector TH cells induce B cells to both proliferation and maturation, while thymic effector TH cells are defective in activating B-cell maturation. The present findings extend our studies on "natural" lymphocyte activities in the normal immune system, revealing the existence of two apparently distinct effector T-cell compartments. The potential significance of the intrathymic set of effector cells in repertoire selection is considered.