Lance E. Hultin

Generation of Functional Thymocytes in the Human Adult

Reconstituting the immune response will be critical for the survival of HIV-infected individuals once viral load is brought under control. While the adult thymus was previously thought to be relatively inactive, new data suggest it may play a role in T cell reconstitution. We examined thymopoiesis in adults up to 56 years of age and found active T cell receptor (TCR) rearrangement, generating a diverse TCR Vbeta repertoire. The resulting thymocytes are functional and are capable of responding to costimulatory signals. These data demonstrate that the adult thymus remains active late in life and contributes functional T cells to the peripheral lymphoid pool.

Predictive value of immunologic and virologic markers after long or short duration of HIV-1 infection.

Summary: Laboratory markers that predict HIV‐1 disease progression include plasma viral burden, CD4+ T‐cell count, and CD38 expression on CD8 T cells. To better understand whether the predictive value of these markers is dependent on how long an individual has been infected, we analyzed data from the Multicenter AIDS Cohort Study early (median = 2.8 years) and late (median = 8.7 years) in the course of infection. Overall, we found that HIV RNA and CD38 levels were similarly predictive of AIDS early on compared with a relatively weaker CD4 cell count signal. Later in the course of infection, CD38 level remained the strongest predictive marker and CD4 cell count registered a marked increase in prognostic power. Among untreated individuals, there was little difference in prognosis (median time to AIDS) associated with given marker values regardless of infection duration. The prognosis given a specific viral load level tended to deteriorate late in the course of infection among those undergoing treatment with monotherapy or combination therapy, however. These data provide a unique historical look at the predictive value and prognostic significance of HIV‐1 disease markers at different stages of infection in a large cohort, with direct relevance to current patients who are untreated or for whom treatment is ineffective.

Quality control in the flow cytometric measurement of T-lymphocyte subsets: The Multicenter AIDS Cohort Study experience

Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized. Interlaboratory proficiency testing utilizing identical specimens analyzed in the four laboratories was performed to evaluate the comparability of the data among the laboratories. Our results verify that such testing can identify technical bias in flow cytometric evaluations performed at different laboratories. Temporal laboratory consistency in flow cytometric measurements was evaluated using data from each sites HIV-seronegative homosexual reference group. Both sequential 95% confidence intervals (mean +/- 2 x SEM) and the within-person standard deviations of the immune measurements were considered. Significant variation in CD3, CD4, and CD8 lymphocyte subset percentages over time in the seronegative reference population was observed. Our observations indicate that the lymphocyte subset values of this seronegative group should be used to adjust those obtained on the seropositive study participants during a particular time period, thereby allowing improved discrimination of the effects of HIV on T cells in infected individuals. The data presented are of use for designing epidemiologic and intervention studies in HIV-1-infected individuals, especially for calculating sample sizes. The methods we have used to assess the quality of data in the MACS have general application to quality control programs in flow cytometry laboratories. In particular, comparison of sequential confidence intervals and within-person standard deviations for lymphocyte subset determinations on control populations are essential to a comprehensive proficiency testing program because they permit assessment of consistency within a laboratory over time.Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized. Interlaboratory proficiency testing utilizing identical specimens analyzed in the four laboratories was performed to evaluate the comparability of the data among the laboratories. Our results verify that such testing can identify technical bias in flow cytometric evaluations performed at different laboratories. Temporal laboratory consistency in flow cytometric measurements was evaluated using data from each sites HIV-seronegative homosexual reference group. Both sequential 95% confidence intervals (mean ± 2 × SEM) and the within-person standard deviations of the immune measurements were considered. Significant variation in CD3, CD4, and CD8 lymphocyte subset percentages over time in the seronegative reference population was observed. Our observations indicate that the lymphocyte subset values of this seronegative group should be used to adjust those obtained on the seropositive study participants during a particular time period, thereby allowing improved discrimination of the effects of HIV on particular time period, thereby allowing improved discrimination of the effects of HIV on T cells in infected individuals. The data presented are of use for designing epidemiologic and intervention studies in HIV-1-infected individuals, especially for calculating sample sizes. The methods we have used to assess the quality of data in the MACS have general application to quality control programs in flow cytometry laboratories. In particular, comparison of sequential confidence intervals and within-person standard deviations for lymphocyte subset determinations on control populations are essential to a comprehensive proficiency testing program because they permit assessment of consistency within a laboratory over time.

CD8+ T-lymphocyte activation in HIV-1 disease reflects an aspect of pathogenesis distinct from viral burden and immunodeficiency.

The CD8+ T-cell response is central to control and eventual elimination of persistent viral infections. Although it might be expected that CD8+ T-cell activation would be associated with a better clinical outcome during viral infections, in long-term HIV-1 infection, high levels of CD8+ T-cell activation are instead associated with faster disease progression. In this study, cell surface expression of CD38, a flow cytometric marker of T-cell activation of CD8+ T cells, had predictive value for HIV-1 disease progression that was in part independent of the predictive value of plasma viral burden and CD4+ T-cell number. Measurements of CD38 antigen expression on CD8+ T cells in HIV-1-infected patients may be of value for assessing prognosis and the impact of therapeutic interventions. The pathogenetic reason why CD8+ T-cell activation is associated with poor outcome in HIV-1 disease remains unknown. Possibly CD8+ T-cell activation contributes to immunologic exhaustion, hyporesponsiveness of T cells to their cognate antigens, or perturbations in the T-cell receptor repertoire.

Natural Killer Cell Immunodeficiency in Hiv Disease is Manifest by Profoundly Decreased Numbers of Cd16 + Cd56+ Cells and Expansion of a Population of Cd16dim Cd56- Cells with Low Lytic Activity

Natural killer (NK) cells were enumerated by three-color immunofluorescence in 255 uninfected and 399 human immunodeficiency virus-infected adults. Several dramatic alterations were observed. First, the median number and percentage of CD16+CD56+ NK cells, the subset that comprises > 90% of the NK cells in healthy adults, were severely decreased (median, 175/mm3 in uninfected controls; 63/mm3 in HIV-infected non-AIDS subjects). Even subjects with > 800 CD4+ cells/mm3 had decreased CD16+CD56+ NK cell levels (97/mm3). Second, the number of CD16+CD56- cells, an NK population that is rare in healthy adults, was elevated (median, 20/mm3 in uninfected controls; 64/mm3 in HIV-seropositive non-AIDS subjects). Third, the expression of CD16 on the NK cells was markedly reduced; some CD56+ cells and virtually all CD56- cells were CD16dim. Fourth, fluorescence-activated cell-sorting studies revealed little NK- or antibody-dependent cellular cytotoxic activity in the CD16dimCD56- cell population. These results indicate that the pathogenesis of HIV disease includes numerical alterations in subpopulations of NK cells. A better understanding of how HIV infection causes this aspect of pathogenesis is needed.

Homeostasis of the Naive CD4+ T Cell Compartment during Aging

Despite thymic involution, the number of naive CD4+ T cells diminishes slowly during aging, suggesting considerable peripheral homeostatic expansion of these cells. To investigate the mechanisms behind, and consequences of, naive CD4+ T cell homeostasis, we evaluated the age-dependent dynamics of the naive CD4+ T cell subsets CD45RA+CD31+ and CD45RA+CD31−. Using both a cross-sectional and longitudinal study design, we measured the relative proportion of both subsets in individuals ranging from 22 to 73 years of age and quantified TCR excision circle content within those subsets as an indicator of proliferative history. Our findings demonstrate that waning thymic output results in a decrease in CD45RA+CD31+ naive CD4+ T cells over time, although we noted considerable individual variability in the kinetics of this change. In contrast, there was no significant decline in the CD45RA+CD31− naive CD4+ T cell subset due to extensive peripheral proliferation. Our longitudinal data are the first to demonstrate that the CD45RA+CD31+CD4+ subset also undergoes some in vivo proliferation without immediate loss of CD31, resulting in an accumulation of CD45RA+CD31+ proliferative offspring. Aging was associated with telomere shortening within both subsets, raising the possibility that accumulation of proliferative offspring contributes to senescence of the naive CD4+ T cell compartment in the elderly. In contrast, we observed retention of clonal TCR diversity despite peripheral expansion, although this analysis did not include individuals over 65 years of age. Our results provide insight into naive CD4+ T cell homeostasis during aging that can be used to better understand the mechanisms that may contribute to immunosenescence within this compartment.

Enhanced levels of functional HIV-1 co-receptors on human mucosal T cells demonstrated using intestinal biopsy tissue

ObjectiveTo examine compartmental differences in co-receptor expression on CD4 lymphocytes between blood and gut using endoscopic biopsies. DesignMucosal and peripheral CD4 T cells from healthy controls were compared for co-receptor expression and vulnerability to infection by HIV-1. MethodsExpression of CCR5 and CXCR4 was quantified by flow cytometry on isolated mucosal CD4 lymphocytes obtained from endoscopic biopsies and blood from healthy controls. Vulnerability to in vitro infection by both R5 and X4 strains was assessed by measuring p24. ResultsBiopsies yielded sufficient lymphocytes for flow cytometric characterization and infectivity studies. The percentage of mucosal CD4 T lymphocytes that expressed CCR5 and the per cell expression of CCR5 were both significantly increased compared with that in peripheral blood CD4 T lymphocytes. CXCR4 was expressed on the majority of CD4 lymphocytes in both compartments. In vitro infection of mucosal mononuclear cells supported greater viral replication of both R5 and X4 strains than peripheral blood mononuclear cells. ConclusionsEnhanced expression of CXCR4 and CCR5 on CD4 lymphocytes in normal intestinal mucosa predicts increased vulnerability to infection by both R5 and X4 HIV-1. Endoscopic biopsies provide a useful mucosal tissue sampling technique to identify compartmental immunologic differences that may be exploited by HIV-1 in establishing initial mucosal infection.

Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ T cells is a marker of poor prognosis in HIV infection: Results of 6 years of follow‐up

Relative fluorescence intensity measurements from a flow cytometer were used to evaluate expression of CD38 and HLA-DR antigens. These molecules are associated with cellular activation and are present at increased levels on the CD8+ lymphocytes of HIV-infected subjects. In the current study, the prognostic value of mean fluorescence intensity measurements of CD38 and HLA-DR on CD8+ cells was compared to results from our previous study in which we reported prognostic value for an elevated percentage of CD8+ cells that were positive for expression of the CD38 antigen (Giorgi et al.: JAIDS 6:904-912, 1993). Using the proportional hazards model, elevated mean fluorescence intensity of CD38 expression on CD8+ cells had prognostic value for development of AIDS that was almost identical to the prognostic value of the percentage of CD8+ cells that were positive for expression of CD38. This prognostic value was in addition to that provided by the patients CD4+ cell measurement. To our knowledge, this is the first report that a measurement of fluorescence intensity can be used as a prognostic marker in an immunodeficiency disease. Efforts are needed to establish methods that will allow widespread application of this observation in the clinical management of HIV-infected subjects.

Premature aging of T cells is associated with faster HIV-1 disease progression.

Objective:To determine if untreated HIV-1 infection and progression is associated with premature aging of memory CD8+ and CD4+ T cells and naive CD4+ T cells. Methods:Twenty HIV-1-infected fast progressors and 40 slow progressors were included in our study, using risk set sampling. The expression of cell surface markers reflecting the differentiation stages of lymphocytes was measured using flow cytometry analyses performed on cryopreserved peripheral blood mononuclear cells. Results:We found that HIV-1 disease progression is associated with a decreased CD28 median florescence intensity on CD4+ and CD8+ T cells; an increased proportion of intermediate- and late-differentiated CD8+ T cells and a decreased CD31 median florescence intensity on naive CD4+ T cells of recent thymic origin. A selective depletion of peripherally expanded naive CD4+ T cells was found to be associated with HIV-1 infection but not with HIV-1 disease progression. Conclusions:The overall change during HIV-1 infection and progression is associated with a shift in the T-cell population toward an aged conformation, which may be further compromised by impaired renewal of the less-differentiated CD4+ T-cell population. Our results suggest that HIV-1 infection induces an accelerated aging of T lymphocytes, which is associated with the clinical progression to AIDS and death.

Optimization of methods to assess human mucosal T-cell responses to HIV infection

The majority of HIV-1 infections occur via sexual transmission at mucosal epithelia lining the vagina, cervix or rectum. Mucosal tissues also serve as viral reservoirs. However, our knowledge of human mucosal T-cell responses is limited. There is a need for reliable, sensitive, and reproducible methods for assessing mucosal immunity. Here we report on the collaborative efforts of two laboratories to optimize methods for processing, culturing, and analyzing mucosal lymphocytes. Rectal biopsy tissue was obtained by flexible sigmoidoscopy, which is rapid, minimally invasive, and well tolerated. Of the four methods compared for isolating mucosal mononuclear cells (MMC), collagenase digestion reproducibly yielded the most lymphocytes (4-7 x 10(6)). Furthermore, 0.5-1 x 10(6) MMC could be polyclonally expanded to yield 17 x 10(6) CD8+ T cells allowing mapping of responses to overlapping peptides spanning the HIV-1 genome using IFN-gamma enzyme-linked immunospot (ELISpot). Expansion also reduced the spontaneous IFN-gamma production normally detected in fresh MMC. Piperacillin-tazobactam and amphotericin B reduced contamination of MMC cultures to 4%. Taken together, these methods will be useful for studies of mucosal immunity to HIV-1 and other pathogens during natural infection and following vaccination.