Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

T cell receptor usage and fine specificity of human immunodeficiency virus 1-specific cytotoxic T lymphocyte clones: analysis of quasispecies recognition reveals a dominant response directed against a minor in vivo variant.

Numerous virus-specific, class I-restricted cytotoxic T lymphocyte (CTL) epitopes have been identified, yet little information is available regarding the specificity of the CTL response in persons of the same human histocompatibility leukocyte antigen (HLA) type. In this study, the human immunodeficiency virus (HIV) 1 envelope-specific CTL response was evaluated in five HLA-B14-positive persons. CTL responses specific for a previously described nine-amino acid epitope in gp41 (aa 584-592, ERYLKDQQL) could be identified in all subjects, and CTL clones specific for this epitope could be isolated from four persons. Despite heterogeneous T cell receptor usage, the fine specificity of the clones was similar, as defined by recognition of alanine-substituted peptides as well as peptides representing natural HIV-1 sequence variants. Correlation with in vivo virus sequences revealed that the dominant species in two of the subjects represented poorly recognized variants, with a K-->Q substitution at amino acid 588, whereas no variants were observed in the other two subjects. Although clonal type-specific responses to these dominant variants could be identified, the magnitude of these responses remained small, and the dominant CTL response was directed at the minor in vivo variant. These studies indicate that despite similar epitope-specific immunologic pressure in persons of the same HLA type, the in vivo quasispecies may differ, and that the major in vivo immune response to a given CTL epitope can be directed at a minor variant.

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.

Effects of intensified antihypertensive treatment in diabetic nephropathy: mortality and morbidity results of a prospective controlled 10-year study.

OBJECTIVE: The aim of this study was to describe the effect of intensified antihypertensive therapy based on a structured teaching and treatment programme on the prognosis of hypertensive type 1 (insulin-dependent) diabetic patients with kidney disease. DESIGN: The study was a controlled, prospective, parallel, 10-year follow-up trial. PATIENTS AND INTERVENTIONS: A sequential sample of 91 hypertensive type 1 diabetic patients with overt diabetic nephropathy was prospectively followed for 10 years. Forty-five patients (intensified antihypertensive therapy; IT group) participated in an intensified antihypertensive therapy programme and 46 patients received routine antihypertensive treatment as provided by family physicians, consultants and local hospitals (routine antihypertensive therapy; RT group). OUTCOME MEASURES: The main endpoint was death; secondary endpoints were renal replacement therapy, blindness and amputation. RESULTS: Blood pressure was reduced in the IT group and increased in the RT group. During the follow-up period, 29 patients died, seven in the IT group and 22 in the RT group. The survival curves were significantly different (P = 0.0008). The main causes of death were cardiac. In a multiple Cox proportional hazards model, allocation to the IT group reduced the mortality risk [relative risk (RR) = 0.213; 95% confidence interval 0.089-0.509, P = 0.00051, while age (P = 0.0039) and mean blood pressure (P= 0.0113) increased this risk. In multiple Cox or multiple logistic regression models, the risks of dialysis (RR = 0.269, 95% confidence interval 0.110-0.656, P = 0.0039), blindness (odds ratio = 0.158, 95% confidence interval 0.037-0.684, P= 0.0136), and amputation (RR = 0.181, 95% confidence interval 0.047-0.703, P= 0.0135) were significantly lower in the IT group compared with the RT group (log rank P = 0.0008). CONCLUSION: We conclude that intensified antihypertensive treatment, based on a hypertension teaching and treatment programme, reduces long-term morbidity and mortality in patients with diabetic nephropathy.

Detection of HIV-1-specific CTLs in the semen of HIV-infected individuals.

CTLs play an important role in controlling cell-associated HIV. Since the majority of HIV infections are acquired through sexual transmission, we investigated whether antiviral CTLs were present in the male urogenital tract using semen as a source of T cells. We were able to establish anti-HIV cytolytic lines in five of five HIV-infected men with CD4 counts of >500/microl, although cloning efficiencies were lower than with peripheral blood-derived T cells. CTLs generated from the semen of three men were analyzed in detail and showed a broadly active response, recognizing gag, env, and pol proteins. Detailed analysis of two gag-specific clones from one of the individuals demonstrated HLA class I restriction and recognition of the same p24 epitope (EQASQEVKNWMT). In summary, our results demonstrate the presence of a broad CTL response to HIV in the urogenital tract and provide a rationale for further studies of local enhancement of genital mucosal responses by anti-HIV immunization.

Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy.

Therapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38(+) activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.

Persistence of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte clones in a subject with rapid disease progression.

We longitudinally measured T-cell receptor transcript frequencies of human immunodeficiency virus type 1 (HIV-1) specific cytotoxic T lymphocytes (CTL) in an individual with rapidly progressive disease and high levels of viremia. CTL clones elicited during acute HIV-1 infection were present at the time of death, despite absent functional CTL responses, arguing against clonal deletion as a mechanism for the decline of CTL responses observed during HIV-1 infection.

Association between virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection.

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.