T-Cell Receptor Vβ Repertoire CDR3 Length Diversity Differs within CD45RA and CD45RO T-Cell Subsets in Healthy and Human Immunodeficiency Virus-Infected Children

The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vβ gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vβ families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4⁺ and CD8⁺ T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8⁺ CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4⁺ or CD8⁺ T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vβ families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.     

Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division.

We recently showed that circulating human CD8(+) effector cells have a CD45RA+CD27(-) membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA+CD27(-) phenotype is dependent on antigenic stimulation and involves cellular division, TCR Vbeta usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8(+) subsets. FACS analysis showed that the TCR Vbeta repertoire of CD8(+)CD45RA+CD27(-) cells differed significantly from that of unprimed CD8(+)CD45RA+CD27(+) cells. Moreover, in two out of six individuals large expansions of particular Vbeta families were observed in the CD8(+)CD45RA+CD27(-) subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8(+)CD45RA+CD27(-) population most of the 22 tested Vbeta families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3+/-1.0 kb shorter in the CD8(+)CD45RA+CD27(-) subset compared with the unprimed CD8(+)CD45RA+CD27(+) population, but did not differ substantially from that of memory type, CD8(+)CD45RA-CD27(+) T cells. These findings indicate that the CD8(+)CD45RA+CD27(-) cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.     

Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8+ T cells with predominant Vbeta repertoires

CD8(+) T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-gamma) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8(+) T cells in humans. To this end, we determined the TCR beta chain (Vbeta) usage of CD8(+) T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vbeta usage varied among volunteers, we identified oligoclonal Vbeta subset expansions in individual volunteers (Vbeta 2, 5.1, 8, 17, and 22 in volunteer 1; Vbeta 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vbeta 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-gamma secretion assays on Vbeta sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7(-) CD27(-) CD45RO(+) CD62L(-)) and coexpressed gut homing molecules (e.g., high levels of integrin alpha4beta7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vbeta families. Moreover, these serovar Typhi-specific CD8(+) T cells bearing defined Vbeta specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.     

T-cell receptor repertoire in healthy Sardinian subjects

The T-cell receptor (TCR) Vbeta gene usage of CD4+ and CD8+ T-cell subpopulations was evaluated by flow cytometric analysis and by CDR3 spectratyping in healthy subjects belonging to Sardinian population, which is ethnically homogeneous and genetically distant from all other Italian and Caucasoid groups. As described in healthy Caucasian subjects, we found a nonrandom Vbeta gene usage and in some Vbeta families a significant skewed reactivity toward CD4+ T cells. Moreover, different subjects showed expansions in some Vbeta subfamilies, mainly in the CD8+ T cells. By CDR3 spectratyping analysis we found a significantly higher degree of skewness in the TCR Vbeta repertoire of CD8+ than in that of CD4+ T cells. The similarity found in the TCR Vbeta gene usage between the population examined and other Caucasoid groups suggest that the shape of the TCR repertoire is more influenced by rearrangement processes than ethnic background. However, genetic polymorphisms may condition the expression levels of some Vbetas, determining the variability of the TCR repertoire between different populations. Finally, the profound perturbations evidenced in the CD8+ T cell subpopulation could be related to a different response to the antigenic stimulation between CD8+ and CD4+ T lymphocytes.     

In Vitro Expansion of T-Cell-Receptor Vα2.3+ CD4+ T Lymphocytes in HLA-DR17(3), DQ2+ Individuals upon Stimulation with Mycobacterium tuberculosis

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.     

Alterations in T-cell receptor Vbeta repertoire of CD4 and CD8 T lymphocytes in human immunodeficiency virus-infected children

Perturbations in the T-cell receptor (TCR) Vbeta repertoire were assessed in the CD4 and CD8 T lymphocytes of human immunodeficiency virus (HIV)-infected children who were receiving therapy during the chronic phase of infection by flow cytometry (FC) and PCR analysis. By FC, representation of 21 TCR Vbeta subfamilies was assessed for an increased or decreased percentage in CD4 and CD8 T cells, and by PCR, 22 TCR Vbeta subfamilies of CD4 and CD8 T cells were analyzed by CDR3 spectratyping for perturbations and reduction in the number of peaks, loss of Gaussian distribution, or clonal dominance. The majority of the TCR Vbeta subfamilies were examined by both methods and assessed for deviation from the norm by comparison with cord blood samples. The CD8-T-lymphocyte population exhibited more perturbations than the CD4 subset, and clonal dominance was present exclusively in CD8 T cells. Of the 55 total CD8-TCR Vbeta families classified with clonal dominance by CDR3 spectratyping, only 18 of these exhibited increased expression by FC. Patients with high numbers of CD8-TCR Vbeta families with decreased percentages had reduced percentages of total CD4 T cells. Increases in the number of CD4-TCR Vbeta families with increased percentages showed a positive correlation with skewing. Overall, changes from normal were often discordant between the two methods. This study suggests that the assessment of HIV-induced alterations in TCR Vbeta families at cellular and molecular levels yields different information and that our understanding of the immune response to HIV is still evolving.     

Restriction in T-cell receptor repertoire in a patient affected by trichothiodystrophy and CD4+ lymphopenia

Molecular analysis of T-cell receptor (TCR) repertoire, by measuring the CDR3 heterogeneity length of beta-variable regions (spectratyping), is useful for acquiring novel information on the status of immune system in primary immunodeficiency. Here, we evaluate TCR repertoire in a child with trichothiodystrophy (TTD) and combined immunodeficiency (CID). Spectratyping revealed marked alterations of TCR repertoire distribution: 21 and 10 out of 27 TCR Vbeta (TCRBV) families and subfamilies were skewed in CD8+ and CD4+ subsets, respectively. These findings revealed, for the first time in a TTD patient with CID, a marked reduction in the TCR repertoire complexity, which may reflect alterations in the mechanisms regulating the generation and homeostasis of T cells.     

Antiretroviral Therapy Restores Diversity in the T-Cell Receptor Vβ Repertoire of CD4 T-Cell Subpopulations among Human Immunodeficiency Virus Type 1-Infected Children and Adolescents

Human immunodeficiency virus (HIV) type 1 infection perturbs the T-cell receptor (TCR) Vβ repertoire. The TCR CDR3 length diversity of individual Vβ families was examined within CD45RA and CD45RO CD4 T cells to assess the impact of the virus on clonality throughout CD4 T-cell activation and differentiation. A cross-sectional and longitudinal cohort study of 13 HIV-infected and 8 age-matched healthy children and adolescents examined the Vβ CDR3 length profiles within CD4 T-cell subsets by the use of spectratyping. HIV-infected subjects demonstrated higher numbers of perturbations in CD4 CD45RA T cells (5.8 ± 4.9 Vβ families) than healthy individuals (1.6 ± 1.8 Vβ families) (P = 0.04). Surprisingly, CD4 CD45RO central memory T cells from infected subjects showed no increased perturbations compared to the perturbations for the same cells from healthy subjects (2.9 ± 3.1 and 1.1 ± 1.8 Vβ families, respectively; P = 0.11). CD4 CD45RA TCR perturbations were higher among infected subjects with >25% CD4 cells than healthy subjects (mean number of perturbed Vβ families, 6.6 ± 5.4; P = 0.04). No correlations between perturbations in CD4 subsets and pretherapy age or viral load were evident. In contrast to CD8 T cells, HIV induces TCR disruptions within CD45RA but not CD45RO CD4 T cells. Therapy-induced viral suppression resulted in increases in thymic output and the normalization of the diversity of TCR within CD45RA CD4 T cells after 2 months of treatment. Perturbations occur prior to CD4 T-cell attrition and normalize with effective antiretroviral therapy. The impact of HIV on the diversity of TCR within naïve, central memory, and effector memory CD4 T cells is distinctly different from that in CD8 T cells.Human immunodeficiency virus type 1 (HIV-1) infection alters T-cell homeostasis by both impairing thymic output and inducing chronic T-cell activation. These disruptions are manifest by the increased level of expression of T-cell activation markers and decreased numbers of naïve T cells from the thymus (10, 12, 51). Oligoclonal T-cell expansion results in perturbations of the T-cell receptor (TCR) Vβ repertoire within both CD4 and CD8 T cells, with CD8 T cells being affected to a greater extent than CD4 T cells (7, 12, 16, 29, 50). Many of these abnormalities occur prior to CD4 T-cell attrition and are not fully reconstituted when viral replication is controlled by antiretroviral therapy (6, 17, 30). Multiple mechanisms have been postulated to contribute to this processes of aberrant T-cell activation and clonal expansion, including microbial translocation across the gastrointestinal tract as a result of virus-induced intestinal fibrosis (4, 5, 40) and the loss of immune regulation due to chronic HIV-induced antigenemia (8, 22).CD4 and CD8 T cells are heterogeneous populations that differ functionally and in their expression of activation and differentiation markers, forming the basis of their classification as naïve, central memory (CM), or effector memory (EM) T cells (42). Isoforms of CD45 (CD45RA and CD45RO) are frequently used to subdivide CD4 and CD8 T cells into functional subsets (1, 13, 25, 44, 45). Oligoclonal expansions and deletions within T-cell subpopulations can be measured by analysis of the hypervariable CDR3 region of the TCR (37). CDR3 length variation reflects changes within the TCR Vβ repertoire during antigen-induced T-cell activation (24, 25, 34). Differences in CDR3 length diversity within the CD4 or the CD8 CD45RA or CD45RO subset enable assessments of disruptions of the TCR repertoire and the detection of oligoclonal expansion that would have been missed if the analysis were limited to unfractionated T cells (25, 26). While optimal control of viral replication by antiretroviral therapy (ART) corrects many T-cell abnormalities and slows the progression to AIDS, it is not clear if therapy completely restores the TCR repertoire or fully diminishes T-cell activation (7, 14, 27, 51). In the present study, we examined the relationship of TCR diversity, thymic output, and the expression of T-cell activation markers within the CD45RA and CD45RO subpopulations of CD4 and CD8 T cells before and after the initiation of ART to determine the extent to which the control of viral replication restores the TCR repertoire.     

CD8+ T-cell subsets defined by expression of CD45 isoforms differ in their capacity to produce IL-2, IFN-gamma and TNF-beta.

Expression of different isoforms of CD45, the leucocyte common antigen (LCA), on T-cell subsets has permitted distinctions between the functional activities of subpopulations within the major CD4+ T-cell subset. With respect to cytokine production, the expression on CD4+ cells of CD45RA, a high molecular weight isoform, defines a population which produces only interleukin-2 (IL-2) and tumour necrosis factor-beta (TNF-beta) in quantity, with peak production of IL-2 occurring after 24-48 hr stimulation, while the CD4+ population bearing high levels of CD45RO, a low molecular weight isoform, can produce a wide range of cytokines within 24 hr of activation. The literature is conflicting on the capacities for cytokine production of CD8+ subsets divided on the basis of either CD45RA or CD45RO expression. The aim of this study was to attempt to clarify this area by determining the amount and kinetics of production of IL-2, interferon-gamma (IFN-gamma) and TNF-beta in CD8+ cells separated on the basis of both CD45RA and CD45RO isoform expression. The results showed that CD8+ CD45RA- and CD8+ CD45RO+ T lymphocytes produce significantly more of all three cytokines than do CD8+ CD45RA+ or CD8+ CD45RO- T cells. The kinetics for IFN-gamma and TNF-beta production were similar for both subsets, while IL-2 production was delayed by approximately 3 hr in the CD8+ CD45RO- population as compared to the CD8+ CD45RO+ subset. It is suggested that some of the confusion over cytokine production by these CD8+ subsets may be attributable to different conditions for isolation causing pre-activation of positively selected populations. It is also suggested that while CD8+ CD45RA+ cells are shown to acquire CD45RO upon activation, as do CD4+ CD45RA+ cells, the results of the present study argue for a different relationship between CD8+ subsets separated on the basis of CD45 isoform expression than between the corresponding CD4+ subsets.     

T cell receptor excision circles (TRECs), CD4+, CD8+, and their CD45RO+, and CD45RA+, subpopulations in hepatitis C virus (HCV)-HIV-co-infected patients during treatment with interferon alpha plus ribavirin: analysis in a population on effective antiretroviral therapy

Interferon (IFN)-alpha induced CD4(+) T lymphopenia is a toxic effect of the treatment of chronic hepatitis C virus (HCV) in human immunodeficiency virus (HIV)-co-infected patients. To increase the knowledge about this secondary effect, we performed an analysis of the evolution of the T cell receptor excision circles (TRECs), CD4(+) and CD8(+) T cells and of their CD45RO(+) and CD45RA(+) subpopulations during the treatment of chronic hepatitis HCV with peginterferon alpha (pegIFN-alpha) + ribavirin. Twenty HCV/HIV-co-infected patients, with undetectable HIV load after highly active antiretroviral therapy (HAART), were treated with pegIFN-alpha + ribavirin. TRECs were determined using real-time polymerase chain reaction. CD4(+) and CD8(+) T cells and their CD45RO(+) and CD45RA(+) subpopulations were analysed by two-colour flow cytometry. Median baseline CD4(+) and CD8(+) T cells were 592 mm(3) and 874 mm(3), respectively. Median baseline CD45RO(+) subpopulation was 48% for CD4(+) T and 57% for CD8(+) T lymphocytes. A progressive decrease in both T cell populations, as well as of their CD45RO(+) and CD45RA(+) subpopulations, was detected, with a difference between the baseline and nadir levels approaching 50%. The evolution of T cell populations and TRECs was independent of the response to the treatment. T lymphocytes and their subpopulations returned to baseline levels at 24 weeks after the end of treatment, with the exception of the T CD4(+) CD45RA(+) subpopulation. The ratio of CD4(+) CD45RO(+)/CD4(+) CD45RA(+) increased from 0.89 (baseline) to 1.44 (24 weeks after the end of the therapy). TRECs/ml did not return to the basal values. In conclusion, a significant reduction of CD4(+) and CD8(+) T cells, and of their CD45RA(+) and CD45RO(+) subpopulations, in HIV/HCV co-infected patients treated with pegIFN-alpha was observed. Both subpopulations increased after the suppression of treatment, but the CD4(+) CD45RA subpopulation did not reach the basal levels as a consequence, at least in part, of a decrease in thymic production.     

HIV-1感染者CD4+T细胞受体基因多样性特点及其与病毒载量的相关性

目的 分析HIV-1感染者CD4+T细胞受体(TCR)基因的多样性特征及其与病毒载量的相关性.方法 应用抗CD4单克隆抗体从25份HIV-1感染者和10份HIV-1阴性对照样本外周血单个核细胞(PBMC)中分离CD4+T细胞,提取细胞总RNA,然后通过逆转录及巢式多聚酶链反应(nestedPCR)对TCR 22个Vβ基因家族的互补决定区3(CDR3)进行扩增,利用ABI3700测序仪对扩增的PCR产物进行扫描,定最分析HIV-1感染者TCRCDR3区多样性变化特征及其与病毒载量的相关性.结果 HIV-1感染者CD4+T细胞TCR CDR3区平均D(distance)值显著高于正常对照组(P＜0 05),TCR Vβ基因各家族CDR3长度谱型成寡克隆分布,TCR CDR3区的紊乱与病毒载量呈正相关(r=0 494,P＜0 05);HIV-1感染引起TCR多样性的改变不仅表现在不同Vβ基因家族上,而且也表现在CDR3长度上,其中感染者Vβ8、Vβ22、Vβ23基因家族的变化与正常人差异有统计学意义.结论 HIV-1感染能引起CD4+T细胞TCR基因多样性的减少及高斯(Gaussian)分布的破坏,TCR CDR3区的紊乱与病毒载量呈正相关.

Abstract：

Objective To assess the impact of the virus on the complementary determining region 3 (CDR3) length diversity of T cell receptor(TCR) Vβ repertoires of CD4+ T lymphocytes and to explore its association with viral load in individuals with HIV-1 infection. Methods The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD4+ T cells in HIV infected and healthy adults. Separation of CD4+ T cells from peripheral blood mononuclear cells ( PBMCs) was carried out by using immunomagnetic beads coated with anti-CD4 antibody. Total RNAs from the purified CD4 + T lymphocytes were isolated and used to perform nested-PCR amplifications in CDR3 of 22 TCR gene families. CDR3 diversity and its association with viral load in individuals with HIV-1 infection were analyzed. Results An average diversity for all CDR3 profiles in CD4+ T cells from 25 HIV-infected individuals was significantly different as compared to 10 age-matched healthy donors (P＜0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles. There was positive correlation between changes in TCR CDR3 diversity and viral load (r = 0. 494, P ＜ 0. 05). The changes in CDR3 length diversity of Vβ families in HIV-infected individuals, particular in Vβ8, Vβ22, Vβ23 were statistically different from the healthy controls. Conclusion HIV-1 infection might induce the loss of TCR Vp repertoire diversity and disrupt the CDR3 Gaussian distributions within CD4 + T cells. There should be positive correlation between changes in TCR CDR3 diversity and the viral load in HIV-1 infected patients.     

Maternal and neonatal lymphocyte subpopulations at delivery and 3 days postpartum: increased coexpression of CD45 isoforms

PROBLEM: Huge physiologic changes during parturition involve immune cells. Alterations in maternal and neonatal lymphocytes postpartum might ascertain the subpopulations that are most affected and may possibly be of importance in the process. METHOD OF STUDY: Peripheral blood was taken from 20 healthy women at vaginal delivery and 3 days later, concomitantly with cord and peripheral blood from their newborns. Lymphocyte immunophenotyping was done by three-color flow-cytometry. RESULTS: Maternal T helper cells were decreased and natural killer (NK) cells were significantly increased during labor. Percentage of CD4(+) and percentage and absolute count of CD8(+) cells coexpressing CD45RA and CD45RO antigens were higher than 3 days later. In cord blood NK cells were considerably increased and more CD4(+) cells expressed CD45RO antigen. CONCLUSION: Coexpression of CD45RA and CD45RO molecules indicates activation of maternal CD4(+) and CD8(+) lymphocytes. NK cells increase suggests their possible association with parturition processes. Lymphocyte subsets in cord blood correspond to maternal subsets to some extent.     

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen- driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.      

Contributions of CD4+, CD8+, and CD4+CD8+ T cells to skewing within the peripheral T cell receptor beta chain repertoire of healthy macaques.

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.     

Loss of CD45RA and gain of CD45RO after in vitro activation of lymphocytes from HIV-infected patients.

T-lymphocyte activation is accompanied by a loss of CD45RA determinant and gain of CD45RO marker. Previous work suggests that the CD45RO population could represent the T-cell memory pool since it contains most of the cells that can respond to recall soluble antigens. A selective loss of memory T cells was recently found in early stages of human immunodeficiency virus (HIV) infection, which could contribute to the qualitative defects observed in HIV-infected patients. In order to investigate whether HIV infection could induce an alteration in the process of differentiation from naive cells to cells with a memory phenotype, we studied the expression of CD45RA and CD45RO in CD4+ and CD8+ lymphocytes, by flow cytometric analysis, after mitogenic stimulation of mononuclear cells from patients with HIV infection. These preliminary results suggest that after in vitro activation the CD4+ and CD8+ populations from HIV-infected patients lose the CD45RA marker and concomitantly acquire the CD45RO determinant in the same way as shown for the healthy control population studied simultaneously. Thus, it appears that an alteration in the process of conversion of CD45RA into CD45RO does not contribute to the defects observed in the memory T-cell population in HIV infection.     

Microbial colonization induces oligoclonal expansions of intraepithelial CD8 T cells in the gut

Two populations of CD8(+) IEL generally express restricted, but apparently random and non-overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8(+) IEL, i.e. that CD8alphabeta(+) IEL derive from a few peripheral CD8(+) T cell lymphoblasts stimulated by microbial antigens in gut-associated lymphoid tissue, whereas CD8alphaalpha(+) IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non-overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ-free (GF) rats, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed surprisingly diverse TCR Vbeta repertoires, although beta-chain diversity tended to be somewhat restricted in the CD8alphaalpha(+) subset. CDR3 length displays in individual Vbeta-Cbeta and Vbeta-Jbeta combinations generally revealed polyclonal distributions over 6-11 different lengths, similar to CD8(+) lymph node T cells, and CDR3beta sequencing provided further documentation of repertoire diversity. By contrast, in ex-GF rats colonized with normal commensal microflora, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed oligoclonal CDR3 length distributions for most of the Vbeta genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8alphabeta(+) and CD8alphaalpha(+) IEL locally in the gut.     

Distribution of Alloreactivity Amongst the CD45 Isoforms of Circulating CD4 and CD8 T Lymphocytes

The expression of different isoforms of the CD45 surface molecule allows lymphocytes to be divided into two nonoverlapping categories, CD45RA and CD45RO. Previous studies of CD4 T cells have shown that responses to soluble antigens are present predominantly in the RO subset and to mitogens in the RA, alloreactivity being present in both subsets. Responses of CD8 T cells have not been investigated in such detail, nor have responses been compared to CD4 cells. Here we report the alloreactive responses of both CD45RA and RO subsets amongst both CD4 and CD8 T lymphocytes. Following isolation of CD4 and CD8 cells with immunomagnetic beads, CD45 subsets were separated by negative depletion using specific monoclonal antibodies. CD45RA populations were greater than 97% pure and CD45RO cells greater than 91%. One-way primary mixed lymphocyte reactions were established using the purified responder cells with irradiated allogeneic peripheral blood mononuclear cells as stimulators; experiments were all repeated at least three times. In assays of CD4⁺ RA and RO subsets, reactivity was present in both isoforms, being consistently, but not significantly, greater amongst the RO subset. With CD8⁺ T cells, reactivity was also present in both isoforms, but was significantly greater in the CD45RA subset, with mean proliferation 2.5–3-fold that of the CD45RO cells ( P  < 0.05).     

CD45 isoform phenotypes of human T cells: CD4(+)CD45RA(-)RO(+) memory T cells re-acquire CD45RA without losing CD45RO

We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.     

Cytotoxic T lymphocyte activity and CD8 subpopulations in children at risk of HIV infection.

HIV-specific cytotoxic T lymphocytes (CTL) are thought to play a major role in viral control in HIV-infected adults. Changes in the relative proportions of CD8 lymphocyte subpopulations are also thought to be associated with disease progression. Less is known about the relative effectiveness of CTL against different HIV targets, or about the relationship, if any, between CTL activity and CD8 subpopulations. We have measured CTL activity against four HIV gene products (gag, tat, pol and env) and expression of CD45RO, CD45RA, HLA-DR, CD29, S6F1, and CD57 surface markers on CD8 cells from nine HIV-infected and 11 HIV-uninfected children. Of nine HIV-infected children, six showed antigen-specific CTL activity on at least one occasion: 4/6 directed against tat, 6/6 against pol, 1/6 against env, and 1/6 against gag. However, the specificity of the CTL activity varied between children and within individual children with time. Furthermore, two uninfected children showed CTL activity, one to HIV-gag, -pol and -tat, and the other to HIV-pol. All the HIV-infected and two uninfected children had abnormal proportions of CD8 subpopulations in whole blood compared with age-matched controls. There was no correlation between CTL activity and CD8 subsets in whole blood. Five children changed from CTL-positive to CTL-negative (or vice versa) during the study. In these, the occasions when CTL activity was detected coincided with an increase in CD8 cells, an expansion of HLA-DR+ CD8 cells and a loss of CD45RA+ CD8 cells.