Vida Hodara

Peripheral blood T cell expansions in patients with Behçet's disease

Behc¸ets disease (BD) is a chronic multisystemic inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Etiology and pathogenesis of BD remain unknown. T cell receptor (TCR) Vα/Vβ gene product expression as well as Jβ gene segment expression in peripheral blood of BD patients were analysed to investigate the possible role of T lymphocytes in the etiopathogenesis of BD. Flow cytometry with 12 TCR V‐specific MoAbs was used for TCRV analyses. Jβ gene segment usage by T cell populations expressing certain Vβs was determined by polymerase chain reaction (PCR) technique with Vβ‐ and Cβ‐specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabelled Jβ gene segment‐specific probes. Although 13 of the 23 BD patients exhibited increases in expression of one or more TCR V‐gene products, only expansions among the CD4+ T cell subset were significantly more frequent in BD patients (7/23) compared with healthy controls (0/15) (P = 0.019). Six out of eight cases followed for up to 20 months had at least one expansion correlated with disease activity. A strict preference for particular Jβ gene segments implicating clonality was apparent in all analysed T cell expansions and correlated well with disease activity. These results suggest a possible involvement of antigen‐specific T lymphocytes in the pathogenesis of BD.

Neutralizing antibodies and viral characteristics in mother-to-child transmission of HIV-1.

ObjectiveTo determine viral characteristics and the protective effect of virus neutralizing antibodies in mother-to-child transmission of HIV-1. Molecular studiesTen HIV-1-infected mother-child pairs were sampled within 4 months of delivery. Variable region 3 of the viral envelope was amplified by nested polymerase chain reaction and sequenced, directly and/or after cloning, by solid-phase DNA sequencing. The amino acid sequence of variable region 3 from all 10 children was homogeneous, whereas the mothers showed varying degrees of heterogeneity. Apparently, selection of an HIV-1 variant occurs either at transmission or during initial virus replication in the infected child. No characteristic molecular features of the transmitted virus were identified. Biological studiesVirus isolates from 13 mother-child pairs were characterized for replicative capacity in a variety of cell lines. Eight mothers from whom a virus with a slow/low replicative pattern was isolated transmitted the slow/low virus to their children, whereas mothers with a rapid/high virus transmitted either a rapid/high or a slow/low virus (two cases each). This indicates that viruses with rapid/high replicative capacity do not have a selective advantage during transmission. Virus neutralizingSera from 20 mothers were characterized for the ability to neutralize their own virus (autologous neutralization) and virus from other mothers (heterologous neutralization). The results showed that non-transmitting mothers had neutralizing antibodies against autologous virus more frequently than transmitting mothers. In addition, all mothers with autologous neutralizing antibodies also neutralized at least two heterologous primary isolates. This indicates that a broad neutralizing antibody response may be linked to a lower risk of mother-to-child transmission. ConclusionOn the basis of the variable region 3 loop sequence, HIV-1-infected infants harbour homogenous virus populations. Despite this, no molecular or biological markers for selective transmission could be identified. A maternal neutralizing antibody response with broad specificity may protect the child from HIV-1 infection.

HIV infection leads to differential expression of T-cell receptor V-beta genes in CD4+ and CD8+ T cells

ObjectiveTo analyse variation in T-cell receptor (TCR) Vβ gene expression in T cells in HIV-infected individuals. DesignBecause there are very few monoclonal antibodies available for studying TCR VP gene expression, we used polymerase chain reaction (PCR) to analyse the TCR Vβ repertoire in HIV-infected individuals using specific primers for 20 distinct families of TCR Vβ. MethodsEvaluation of TCR Vβ gene expression in peripheral blood from HIV-1-infected individuals [two in Centers for Disease Control (CDC) stage II, five in CDC stage III and four in CDC stage IV]. Complementary DNA was produced from CD4+ and CD8 + T cells, amplified by PCR and analysed after Southern blotting and hybridization with a Cp-specific oligonucleotide probe. ResultsVβ gene expression was dramatically modified, especially in AIDS patients. The CD4+ T-cell subset showed both overexpression (Vβ2) and deletions or underexpression (Vβ9-Vβ20), whereas these gene segments were expressed normally in the CD8+ subset. Only Vβ 3 was deleted or underexpression in both CD4+ and CD8+ populations in AIDS patients. ConlusionsHIV-1 infection induces CD4+ T-cell deficiency, both in total numbers and by causing a paucity of select Vβ gene expression in this subset. In addition, the Vβ3 gene family was deleted or underexpressed was observed in both CD4+ and CD8 + T-cell subsets from patients in CDC stage IV. These results are compatible with changes in Vβ gene expression known to occur under the action of endogenous or exogenous superantigens.

Interplay of HIV-1 phenotype and neutralizing antibody response in pathogenesis of AIDS.

A majority of human immunodeficiency virus type 1 (HIV-1) infected individuals display a rapid loss of CD4+ lymphocytes with fast progression towards overt acquired immunodeficiency syndrome (AIDS). However, a small proportion of individuals infected by HIV-1 remain immunologically intact for many years. In order to identify factors that might influence the pathogenesis of HIV-1 infection, 21 Italian mothers and 11 Swedish homosexual men were studied for the presence of autologous neutralizing antibodies in serum, biological phenotype of virus isolates and envelope variable region 3 (V3) sequences. The results were compared to the risk of mother-to-child transmission and progression of the disease. The presence of a neutralizing antibody response to the autologous virus as well as a virus with slow replicative capacity were linked both to low risk of mother-to-child transmission and non-progression of the disease. Patients whose peripheral blood mononuclear cells contained a mutation in the tip of the V3 loop (Arg318 to serine, lysine or leucine) significantly more often had neutralizing antibodies to autologous virus isolates containing arginine at this position. Thus, it appears that the interplay and balance between neutralizing antibody response of the host and the biological phenotype of HIV-1 strongly influence pathogenesis.

Nonrandom T-cell receptor Jβ usage pattern in human CD4+ and CD8+ peripheral T cells

Association frequencies of TCR J beta gene segments with six V beta families (V beta 3, 6.1-3, 8, 9, 12, and 18) were analyzed in T-cell populations obtained from healthy blood donors. The six selected V beta families are located at various chromosomal positions relative to other recombinatorial elements (D beta, J beta, C beta). We report here that in CD4+ as well as CD8+ T-cell subsets, all 13 J beta gene segments were used in combination with all the V beta s tested and that no correlation between the genomic position of the individual V beta s and J beta gene segment usage was observed. J beta gene segment usage was found to be nonrandom in general, with J beta 2.7 and J beta 2.4 exhibiting highest and lowest frequency of utilization, respectively. J beta family 2 was used more frequently than J beta family 1 by the two T-cell subsets. Some individual J beta gene segments were skewed toward either CD4+ or CD8+ T cells. Thus, J beta 1.3 and J beta 1.6 were consistently biased toward expression in CD4+ T cells. In contrast, when combined with V beta 8 or V beta 9, J beta 2.1 results were skewed dramatically toward expression in CD8+ T cells. We also found 70 cases of expanded individual V beta/J beta associations in a total of 1092 investigated combinations, 62 of which were confined to the CD8+ T-cell populations. CD8+ T-cell populations are thus much more likely to contain TCR V beta/J beta-restricted expansions than CD4+ T cells.

Detection of CD8 T-cell expansions with restricted T-cell receptor V gene usage in infants vertically infected by HIV-1.

Objective: To investigate the T‐cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV‐1 in order to discern possible perturbations in TCR usage as a consequence of HIV‐1 infection. Design: Blood samples from five HIV‐1 ‐infected and six non‐infected children born to HIV‐1‐seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. Methods: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V&agr; and V&bgr; gene products and antibodies to CD4 and CD8 was performed. Results: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V&agr; and V&bgr; gene products was seen in HIV‐1‐infected children (four out of five) but was rarely detected in uninfected children. Conclusion: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V&agr;/V&bgr; gene products in some HIV‐1 vertically infected infants similar to those observed during primary infection in adults.

Induction of apoptosis by primary HIV-1 isolates correlates with productive infection in peripheral blood mononuclear cells

Objective:To study the apoptosis-inducing capacity of HIV-1 primary isolates in human peripheral blood mononuclear cells (PBMC) in relation to the viral biological phenotype. Design and methods:Four HIV-1 primary isolates capable of replicating and inducing syncytia in the MT-2 cell line and two primary isolates lacking these properties were used to infect PBMC with the same infectious doses. The kinetics of virus production in the culture supernatants were followed in relation to apoptosis induction in PBMC as determined by intracellular labelling of apoptotic DNA strand breaks and flow cytometry analysis. Results:When low virus dose was used (0.001 m.o.i.), productive virus infection, with peak reverse transcriptase (RT) activity at days 5–7, was followed by high numbers of apoptotic cells at day 10 post infection. Tenfold higher inoculum dose (0.01 m.o.i.) resulted in enhanced virus production with peak RT activity at day 3 followed by high numbers of apoptotic cells at day 5 after infection. The apoptosis-inducing capacity of virus isolates was independent of their capacity to induce syncytia or replicate in the MT-2 cell line. However, upon cocultivation of infected PBMC with MT-2 cells, only virus with the MT-2 tropic phenotype initiated productive infection and induced apoptosis in MT-2 cells. Conclusions:These results show that apoptosis induction in PBMC by primary HIV-1 isolates is closely related to the kinetics of virus replication but is not influenced by other biological properties of the virus such as syncytium-inducing capacity and MT-2 tropism.

The Role of Virologic and Immunologic Factors in Mother-to-Child Transmission of HIV-1

PROBLEM: More than 90% of human immunodeficiency virus type 1 (HIV‐1) infection in children is acquired by mother‐to‐child transmission. However, infection of the child occurs in between 14 and 35% of cases.

T-cell receptor J-beta gene segment usage in immature and mature human thymocytes

Immature double positive (DP, CD4+CD8+) and mature single positive (SP, CD4+CD8− and CD4−CD8+) human thymocytes from nine thymi were analysed for their complete patterns of relative TCR Jβ multigene member usage in relation to six rearranged Vβ family exons (Vβ 5.1, 6.1–3, 8, 9, 12 and 18). Each sample tested contained mRNA transcripts corresponding to all potential Vβ(Dβ)Jβ combinations. Individual Jβ gene segments were expressed in a similar, highly non‐random manner both in SP and DP thymocytes, irrespective of original genomic position of the individual associated Vβ exon. In addition, ranges of family usage and frequency of individual over‐representations of Jβ gene segments, as determined in DP and SP thymocyte populations, displayed no significant differences. Upon comparison of DP and SP thymocytes, however, a discrepancy in one aspect of Jβ gene utilization was established: decreasing Jβ family 1/ Jβ family 2 ratios were determined to be positively correlated with increasing maturity of thymocytes, a condition further supported by data previously obtained from studies of PBL T cells. At the individual Jβ gene level, the observed gradual modification of the relative family usage can largely be explained by a significant shift from a higher Jβ 1.1 /Jβ 2.7 ratio in DP to a higher Jβ 2.7/Jβ 1.1 ratio in SP thymocytes. Altogether, the present results imply that selectional processes in the thymus appear to have only minor consequences on the distribution pattern of expressed Jβ exons. Hence, the disproportionate pattern of TCR Jβ gene usage seems to be established mainly at the recombinatorial level followed by minor adjustments during thymic and post‐thymic events.

Enhanced prevalence of T cell receptor Vβ7 gene family expression in human intestine-associated T lymphocytes

Relative levels of expression of T cell receptor variable (V) beta and joining (J) beta gene segments were determined in T cells derived from intestinal biopsies of healthy mucosal areas, mesenteric lymph nodes and peripheral blood of the same individuals. Samples taken from patients suffering from inflammatory (n = 8) and non-inflammatory (n = 8) bowel diseases were analyzed by semi-quantitative polymerase chain reaction-based methods. In the intestine, fewer (median = 3.5) V beta gene segments constituted more than 50% of the T cell receptor V beta repertoire compared to that of peripheral blood T cells (median = 7, P < 0.001). Interestingly, in all sixteen individuals studied, intestinal T lymphocytes (IL-T) expressed the V beta 7 gene family to a higher degree than did T cells in the paired peripheral blood and mesenteric lymph nodes (P < 0.001). T cell receptor J beta gene segment analyses of V beta 7+ T cells revealed no significant difference in oligoclonality rates between peripheral blood (4/16) and intestine (7/16) (P = 0.46). Hence, overexpression of intestinal TCR V beta 7 message does not seem to be due to oligoclonal expansions in the majority of the samples.