Dysregulated lymphocyte proliferation and differentiation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory disease of the synovium of uncertain pathogenesis. A number of phenotypic and functional T-cell defects have been described in RA, including abnormal clonal expansions and suppressed proliferative responses, which suggest a defect in T-cell differentiation. Here, we show that RA patients possess fewer naive CD4(+) T cells than healthy controls. Furthermore, a smaller proportion of these cells contains a T-cell receptor excision circle (TREC). Patients with RA also have unusual populations of T cells. These include immature cells characterized as CD45RB(bright)CD45RA(+)CD62L(-) by flow cytometry and a large population that coexpresses CD45RA and CD45RO. These cells are hyperresponsive to mitogen and TCR stimulation when compared to naive cells. Additionally, an unusual putative central memory subset expressing CD62L, but not CD45RA, appears in RA patients at the expense of more typical cells. Levels of C-reactive protein correlate inversely with the TREC content of naive T cells and positively with the sizes of naive and immature atypical T-cell subsets. These data suggest that inflammation drives proliferation of naive T cells in RA and encourages their differentiation into atypical, hyperresponsive progeny. TREC content of individual naive and atypical T-cell subsets suggests an ontogeny consistent with this hypothesis. These studies provide further evidence of a T-cell differentiation defect in RA, which could explain some of the well-characterized immunologic features of the disease.     

Interleukin-7-treated naive T cells can be productively infected by T-cell-adapted and primary isolates of human immunodeficiency virus 1

Although human immunodeficiency virus (HIV) gag/pol DNA can be detected in naive T cells, whether naive T cells can be productively infected by HIV is still questionable. Given that interleukin-7 (IL-7) is a prospective therapeutic immunomodulator for the treatment of HIV, we evaluated the effect of IL-7 on promoting naive T-cell infection of laboratory-adapted (IIIB), M-tropic, and primary isolates of HIV. Initially, we determined that the 3 cell surface markers widely used to identify naive T cells (CD45RA(+)CD45RO(-), CD45RA(+)CD62L(+), and CD45RO(-)CD27(+)CD95(low)) are all equivalent in T-cell receptor excision circle content, a marker for the replicative history of a cell as well as for de novo T cells. We therefore used CD45RA(+)CD45RO(-) expression to define naive T cells in this study. We demonstrate that although untreated or IL-2-treated naive T cells are not productively infected by HIV, IL-7 pretreatment mediated the productive infection of laboratory-adapted, M-tropic, and primary isolates of HIV as determined by p24 core antigen production. This up-regulation was between 8- and 58-fold, depending on the HIV isolate used. IL-7 pretreatment of naive T cells also potently up-regulated surface expression of CXCR4 but not CCR5 and mediated the expansion of naive T cells without the acquisition of the primed CD45RO phenotype. Collectively, these data indicate that IL-7 augments naive T-cell susceptibility to HIV and that under the appropriate environmental milieu, naive T cells may be a source of HIV productive infection. This information needs to be considered in evaluating IL-7 as an immunomodulator for HIV-infected patients.     

Highly active antiretroviral therapy induces specific changes in effector and central memory T cell sub-populations

Highly active antiretroviral therapy (HAART) improves the immunodeficiency of HIV-infected individuals. In this report we show that HAART increases both naive (CD45RA+CD62L+) and central memory (CD45RO+CD62L+) CD4 lymphocytes. On CD8 lymphocytes, HAART induces an increase of naive cells associated with a consistent decrease of effector cells (CD45 RO+CD62L-). No specific differences in phenotypic changes were observed with different HAART regimens, suggesting that, once viral suppression is achieved, the pharmacological class of antiretroviral drugs does not affect immune reconstitution.     

Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals.

CD4+ T cells of patients with AIDS exhibit a qualitative defect in their ability to respond to soluble antigen while their responses to mitogens remain normal. CD4+ T cells can be broadly divided phenotypically into "naive" [CD45RA+ (2H4+)] and "memory" [CD29+ (4B4+) or CD45RO+ (UCHL1+)] cell subpopulations, which represent distinct maturation stages. To determine the human immunodeficiency virus type 1 (HIV-1) infectability of memory and naive CD4+ T-cell subsets in vitro and to determine the in vivo preference of HIV-1 in these subpopulations, we obtained highly purified CD4+ T-cell subsets from normal and HIV-1-infected individuals and studied them by viral cultivation, quantitative polymerase chain reaction, and functional assays. Polymerase chain reaction studies demonstrated that the memory cell subset of CD4+ T cells is preferentially infected (4- to 10-fold more than naive T cells) by HIV-1 in vitro, and these memory cells are the principal reservoir for HIV-1 within CD4+ T cells obtained from infected individuals. Functional abnormalities attributable to CD4+ T cells in HIV-infected individuals (failure to respond in vitro to soluble antigen or to anti-CD3 monoclonal antibodies) were shown to reside primarily within these memory cells. Thus, the present study suggests that the selective functional defects present in the memory CD4+ T-cell subset of HIV-infected individuals may be a direct result of the preferential infection and consequently greater viral burden within these cells.     

Impaired production of naive T lymphocytes in human T-cell leukemia virus type I-infected individuals: its implications in the immunodeficient state

Opportunistic infections frequently occur in patients with adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I) carriers. However, the underlying mechanisms of such infections remain unknown. To clarify the mechanism of immunodeficiency in those infected with HTLV-I, this study analyzed the T-cell subsets in HTLV-I carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis and ATL using 3-color fluorescence with CD62L and CD45RA coexpression either with CD4(+) or CD8(+) T cells. The number of naive T lymphocytes was markedly suppressed in patients with ATL, particularly in those with acute form, compared with uninfected control individuals. The number of naive T cells was low in HTLV-I-infected individuals under 50 years old compared with uninfected individuals, whereas the number of memory T lymphocytes was greater in HTLV-I-infected individuals. Although the increase of memory T lymphocytes correlated with HTLV-I provirus loads, no relationship was found between naive T-cell counts and provirus loads. T-cell receptor rearrangement excision circles (TRECs), which are generated by DNA recombination during early T lymphopoiesis, were quantified to evaluate thymic function in HTLV-I-infected individuals. TREC levels were lower in HTLV-I-infected individuals than in uninfected individuals. In HTLV-I carriers less than 70 years old, an increase of Epstein-Barr virus DNA in peripheral blood mononuclear cells was observed in 6 of 16 (38%) examined, whereas it was detectable in only 1 of 11 uninfected controls. These results suggested that the low number of naive T lymphocytes was due to suppressed production of T lymphocytes in the thymus, which might account for immunodeficiency observed in HTLV-I-infected individuals.     

T-cell dynamics during acute SIV infection

OBJECTIVES: To delineate T-cell dynamics during acute SIV infection, particularly of phenotypically defined memory T cell subsets. DESIGN: T cells are a heterogeneous mix of naive and memory subsets delineated by simultaneously measuring CD4, CD8, CD45RA/RO, CD11a, CD28, and CD27. The effects of SIV infection on these subsets was measured to evaluate the impact of changes in functionally distinct cell types during pathogenesis. METHODS: Peripheral blood was obtained from six SIV-infected macaques at multiple times before and after SIV infection and analyzed using 12-color flow cytometry. RESULTS: Acute infection was characterized by an initial lymphopenia caused by a decline in B cells. Total T-cell counts remained steady during the early acute phase; however, CD4 cell counts declined while CD8 T cells increased. The decline in CD4 T cells was a result of a decline in both naive and memory cells. CCR5+ or CD103+ subsets of CD4 T cells were depleted but only partially accounted for the decline of CD4 memory T cells, suggesting that acute infection was associated with a rapid redistribution of T cells from the periphery. Naive CD8 cell counts declined while memory CD8 cell counts increased. The increase coincided with declines in plasma viremia and was made up initially of CD27-CD28- (effector) cells; subsequently, the predominant phenotype became CD27+CD28-, akin to central memory cells. CONCLUSIONS: A complete understanding of the T-cell dynamics during acute SIV or HIV infection requires the simultaneous evaluation of a broad spectrum of T-cell subsets. Changes in homeostasis and associated immunopathogenesis can no longer be accurately described simply by measuring naive and memory T-cell subsets.     

IL-2 therapy and thymic production of naive CD4 T cells in HIV-infected patients with severe CD4 lymphopenia

OBJECTIVES: IL-2 therapy increases memory and naive CD4 T cells in HIV-infected patients, but its effect on thymopoiesis is unknown. To investigate this effect, we quantified T-cell receptor rearrangement excision circles (TREC) in CD4 T cells from lymphopenic AIDS patients treated with highly active antiretroviral therapy and IL-2. METHODS: CD4 cell subsets were evaluated by flow cytometry using anti-CD45RO/RA, CD62L, Ki67 and CD95 monoclonal antibodies. The proportion of recent thymic emigrant had been quantified by a real-time polymerase chain reaction assay for signal joint TREC in peripheral blood mononuclear and purified CD4 T cells. RESULTS: At initiation of IL-2, TREC copies/microl of blood were correlated with naive T cell numbers and age. Both naive and TREC numbers/microl significantly increased over time in all patients, with a wide range of TREC increases. Higher percentages of CD4+CD45RO-negative cells positive for the Ki67 cell-cycle marker were found in patients with a low TREC increase, but remained stable under IL-2. TREC and naive cell recovery were correlated; they also correlated with the numbers of TREC and naive cells at the start of IL-2, and with age, suggesting a thymic origin for naive T-cell recovery. A mathematical model showing the linear recovery of naive cells and TREC under IL-2 also strongly suggested that a naive T-cell increase reflects thymic export and involves little net death and proliferation. CONCLUSION: Although we cannot rule out a mechanism of altered proliferation or death rate, the thymus plays an important role in the long-term recovery of naive T cells under IL-2 therapy.     

IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro

Recent studies in mice have shown that although interleukin 15 (IL-15) plays an important role in regulating homeostasis of memory CD8+ T cells, it has no apparent function in controlling homeostatic proliferation of naive T cells. We here assessed the influence of IL-15 on antigen-independent expansion and differentiation of human CD8+ T cells. Both naive and primed human T cells divided in response to IL-15. In this process, naive CD8+ T cells successively down-regulated CD45RA and CD28 but maintained CD27 expression. Concomitant with these phenotypic changes, naive cells acquired the ability to produce interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), expressed perforin and granzyme B, and acquired cytotoxic properties. Primed CD8+ T cells, from both noncytotoxic (CD45RA-CD27+) and cytotoxic (CD45RA+CD27-) subsets, responded to IL-15 and yielded ample numbers of cytokine-secreting and cytotoxic effector cells. In summary, all human CD8+ T-cell subsets had the ability to respond to IL-15, which suggests a generic influence of this cytokine on CD8+ T-cell homeostasis in man.     

Segregation of R5 and X4 HIV-1 variants to memory T cell subsets differentially expressing CD62L in ex vivo infected human lymphoid tissue

OBJECTIVE: The mechanisms of HIV-triggered immunodeficiency were examined by determining the segregation of R5 and X4 HIV-1 variants into memory T cell subsets expressing differentially a homing receptor, CD62L-selectin, in human lymphoid tissue. METHODS: Subpopulations of CD3 and intracellular p24 gag-positive cells in human lymphoid tissue infected ex vivo with X4 HIV-1 variant NL4-3 and R5 HIV-1 variant AD8 were analysed for expression of the T cell memory markers CD45RO and CD45RA, the T cell homing receptor for lymphoid tissue CD62L, and the HIV-1 coreceptors CCR5 and CXCR4. RESULTS: Memory CD4 T cells were the predominant targets for productive infection of lymphoid tissue ex vivo with both R5 and X4 HIV-1. R5 HIV-1 predominantly infected CD62L-negative memory T cells, which selectively express CCR5. In contrast, X4 HIV-1 variants predominantly infected CD62L+ memory T cells, although CXCR4 coreceptor was equally expressed by memory T cells of both CD62L-positive and CD62L-negative subsets. A high proportion of X4 HIV-1, but not of R5 HIV-1, productively infected T cells, displayed a CD45RA+CD45RO+ phenotype. CONCLUSION: The selective expression of the CCR5 coreceptor by CD62L-negative terminally differentiated memory T cells correlates with the preferential productive infection of these cells with the R5 HIV-1 variant. The predominance of X4 HIV-1 variants in less-differentiated memory T cells may be related to their recent activation state, as suggested by the coexpression of both CD45RA and CD45RO molecules on their surface. Differential homing of CD62L-positive and CD62L-negative cells suggests different routes of dissemination of X4 and R5 viruses.     

Potent antiretroviral therapy of primary human immunodeficiency virus type 1 (HIV-1) infection: partial normalization of T lymphocyte subsets and limited reduction of HIV-1 DNA despite clearance of plasma viremia.

Antiretroviral therapy commenced during primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) may limit the extent of viral replication and prevent early loss of HIV-specific CD4 lymphocyte function. We studied the effect of current standard therapy (2 nucleoside analogues and a protease inhibitor) in 16 patients with symptomatic PHI. In the 13 patients who completed 1 year of treatment, plasma HIV RNA was <50 copies/mL and median CD4 cell counts were comparable to HIV-uninfected controls, with naive (CD45RA+CD62L+), primed (CD45RO+), and T cell receptor Vbeta subsets all within normal ranges. However, HIV-1 DNA levels in treated and untreated PHI patients were similar. Furthermore, CD8 cell counts remained elevated, including activated (CD38+HLA-DR+), replicating (Ki-67+), and cytotoxic (perforin+CD28-) lymphocytes. In conclusion, early antiretroviral therapy resulted in clearance of viremia and prevented loss of crucial CD4 subsets. The persistence of HIV-1 DNA together with increased CD8 T lymphocyte turnover and activation indicate continued expression of viral antigens.     

Low-level viremia and proviral DNA impede immune reconstitution in HIV-1-infected patients receiving highly active antiretroviral therapy

BACKGROUND: Immunological and virological consequences of low-level viremia in human immunodeficiency virus (HIV) type 1-infected patients receiving highly active antiretroviral therapy (HAART) remain to be determined. METHODS: For 24 months, 101 HAART-treated, HIV-1-infected patients with HIV RNA levels 20 copies/mL at >/=1 visit (dVL patients) (median increase, 81 copies/mL [interquartile range, 37-480 copies/mL]). dVL patients had higher concentrations of CD8 cells, activated and memory T cells, and proviral DNA, compared with uVL patients (P<.05). A higher HIV RNA level was independently associated with reduced CD4 gain (P<.001). A higher HIV RNA level also was associated with increases in activated CD8(+)CD38(+) and CD8(+)HLA-DR(+) cells (P<.05), and a higher level of activated CD8(+)CD38(+) cells was independently associated with reduced CD4 gain (P<.05). A higher proviral DNA level was associated with increases in CD4(+)CD45RA(-)CD28(-) effector cells and reductions in naive CD4(+)CD45RA(+)CD62L(+) and CD8(+)CD45RA(+)CD62L(+) cells (P<.05). Higher levels of activated CD4(+)HLA-DR(+) and early differentiated CD4(+)CD45RA(-)CD28(+) cells predicted increased risk of subsequent detectable viremia in patients with undetectable HIV RNA (P<.05). CONCLUSION: These findings indicate that low-level viremia and proviral DNA are intimately associated with the immunological and virological equilibrium in patients receiving HAART.     

Long-term effects of intermittent interleukin 2 therapy in patients with HIV infection: characterization of a novel subset of CD4(+)/CD25(+) T cells

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV(+)) patients receiving highly active antiretroviral therapy (HAART), and HIV(+) patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4(+)/CD25(+) T cells. Increased CD25 expression, especially in CD4(+) T cells but also in CD8(+) T cells, without increases in expression of the beta and gamma chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4(+) T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4(+)/CD25(+) T cells correlated positively with both the total and naive CD4(+) T-cell counts. A discrete population of CD45 double intermediate RA(+)/RO(+) CD4(+) cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4(+) count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4(+)/CD25(+) and CD4(+)/CD25(-) cells from IL-2-treated HIV(+) patients proliferated in response to mitogens, specific antigens, and T-cell-receptor-mediated stimuli. Thus, intermittent administration of IL-2 in HIV(+) patients leads to preferential expansion of a unique subset of CD4(+) T cells that may represent a critical population in T-cell homeostasis.     

Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells

Background

HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens.

Design and Methods

We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft-versus-host disease. T cells were stimulated in mixed lymphocyte reactions against HLA-deficient K562 cells transfected with single HLA-A/-B/-C/-DR/-DQ mismatch alleles. Alloreactivity was measured by interferon-γ spot production and cell proliferation.

Results

We observed that allogeneic HLA-reactivity was preferentially derived from subsets enriched for naïve T cells rather than memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD45RA (versus other markers) was used for sorting. The numbers of allogeneic HLA-reactive effector cells were in median 7.2-fold and 16.6-fold lower in CD45RA^neg memory CD8 and CD4 T cells than in entire CD8 and CD4 T cells, respectively. In contrast, proliferation of memory T cells in response to allogeneic HLA was more variably reduced (CD8) or equivalent (CD4) when compared to that of naïve T cells. We also demonstrated in HLA-matched donor-patient pairs that leukemia-reactive CD8 cytotoxic T-lymphocytes were mainly derived from subsets enriched for naïve T cells compared to memory T cells.

Conclusions

Memory T-cell subsets of most healthy individuals showed decreased allogeneic HLA-reactivity, but lacked significant anti-leukemia responses in vitro. The clinical use of memory or naïve-depleted T cells might be beneficial for HLA-mismatched patients at high risk of graft-versus-host disease and low risk of leukemia relapse. Preferred allografts are those which contain leukemia-reactive memory T cells. Alternatively, replenishment with leukemia-reactive T cells isolated from naïve subsets is desirable.     

Expansion of CD57 and CD62L-CD45RA+ CD8 T lymphocytes correlates with reduced viral plasma RNA after primary HIV infection.

OBJECTIVE: CD8 T cells, expressing cell surface molecules distinct from those on resting and naive T cells, are increased in HIV infection. The association of increased CD38 and human leukocyte antigen DR (HLA-DR) CD8 T cells with poor prognosis has suggested that activated CD8 T cells may aggravate HIV infection. We examined whether other immunological parameters might influence the viral setpoint. DESIGN: Peripheral T cells from nine untreated patients, obtained after primary HIV infection when plasma HIV had stabilized, were examined for proteins expressed in activated versus resting, memory versus naive, and cytolytic versus non-cytolytic T cells. METHODS: The proportion of CD8 T cells that stain for CD38 and HLA-DR, CD28 and CD57 was compared with plasma viraemia and CD4 cell count. These parameters were also compared with the proportion of CD4 and CD8 T cells that express CD62L and CD45RA, present on naive cells and down-modulated in memory cells. Internal staining for the cytotoxic protein granzyme A was also examined. RESULTS: An increase in CD38 and CD38 HLA-DR CD8 T cells correlated with increased plasma viral RNA (P < 0.00002, P < 0.03, respectively). An increase in CD8 T cells expressing granzyme A was associated with lower CD4 cell counts (P < 0.04). However, the expansion of CD57 and CD62L CD45RA+ CD8 T cells was associated with a lower viral setpoint (P < 0.01, P < 0.02, respectively). CONCLUSION: Phenotypically defined activated CD8 T cells may have different functions in HIV infection. Activated CD8 T cells that are CD57 or CD62L(-)CD45RA+ may be beneficial, because their expansion in untreated patients correlates with a reduced viral setpoint after primary infection.     

Poor lymphocyte recovery following CD34-selected autologous peripheral blood stem cell transplantation for non-Hodgkin's lymphoma

Positive selection of CD34+ cells in autologous grafts, designed to deplete tumour cells, also results in T-cell depletion. To assess the reconstitution of the different lymphocyte subsets and of the T-cell repertoire diversity following autologous transplantation of selected CD34+ peripheral blood stem cells (PBSC), we analysed sequential blood samples in eight patients autografted for advanced B-cell non-Hodgkin's lymphoma in a phase I-II pilot study. Although natural killer cell recovery was rapid, T- and B-cell recovery was delayed with a median of 110/microliters CD4+, 175/microliters CD8+ T cells and 45/microliters B cells at 12 months post-transplant. The naive CD45RA+ T-cell compartment was profoundly deficient up to 12 months for both CD4+ and CD8+ subsets. A transient expansion of memory CD8+CD45RO+ T cells consisting of an increased percentage of CD57+CD28- cells occurred within the first 3 months post-transplant, but the memory CD4+CD45RO+ T cells remained far below the normal value. The CD8+CD28+ T-cell subset did not recover. Using multiplex PCR analysis of the T-cell receptor gamma locus, we found that the repertoire diversity improved at 12 months after being poor and oligoclonal during the first 3 months post-transplant. As shown by monoplex PCRgamma analysis of every VJ combination, despite T-cell depletion of the graft, mature T cells were carried over with the selected CD34+ PBSC and contributed to the T-cell recovery after transplantation.     

Analysis of peripheral blood T-cell subsets in active thyroid-associated ophthalmopathy: absence of effect of octreotide-LAR on T-cell subsets in patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is thought to be a T-cell-mediated autoimmune disorder. We sought to characterize abnormalities in the peripheral blood T-cell subsets in patients with TAO, and examine whether the long-acting somatostatin analogue, octreotide-LAR, treatment affects these cells. We analyzed peripheral blood T-cell subsets by flow cytometry in 26 euthyroid patients with moderately severe active TAO and 24 controls. Twenty-five of the patients with TAO were enrolled in a randomized trial to receive either 30 mg of octreotide-LAR (n = 11) or placebo (n = 14) every 4 weeks for 16 weeks; all 25 patients subsequently received octreotide-LAR 30 mg every 4 weeks from week 16 to 32. T-cell subsets were analysed at baseline, week 16, and week 32. At baseline, the relative percentage of CD4+ helper T-cells (p = 0.0003) and the CD4+/CD8+ ratio (p = 0.008) were significantly higher in patients with TAO compared to controls. Patients with TAO had higher na?ve active T cells (CD45RA+, CD45RA+ CD4+) and lower memory T cells (CD45RO+, CD45RO+ CD4+) than controls. At weeks 16 and 32, there were no significant differences in any T-cell subsets between the octreotide-LAR-treated and placebo groups. These results support a role of T cell in the pathogenesis of TAO, and show that octreotide-LAR has no effect on T-cell subsets during 32-weeks of treatment.     

In vivo HIV-1 infection of CD45RA(+)CD4(+) T cells is established primarily by syncytium-inducing variants and correlates with the rate of CD4(+) T cell decline

Switch from non-syncytium-inducing (NSI) to syncytium-inducing (SI) HIV type 1 (HIV-1) is associated with accelerated CD4(+) T cell depletion, which might partially be explained by higher virulence of SI variants compared with NSI variants. Because NSI and SI variants use different coreceptors for entry of target cells, altered tropism might offer an explanation for increased pathogenesis associated with SI HIV-1 infection. To investigate whether SI and NSI HIV-1 variants infect different CD4(+) T cell subsets in vivo, the distribution of SI and NSI variants over CD4(+) memory (CD45RA(-)RO(+)) and naive (CD45RA(+)RO(-)) cells was studied by using limiting dilution cultures. In contrast to NSI variants that were mainly present in CD45RO(+) cells, SI variants were equally distributed over CD45RO(+) and CD45RA(+) cells. Infection of memory cells by both NSI and SI HIV-1 and infection of naive cells primarily by SI HIV-1 corresponded closely with the differential cell surface expression of CXCR4 and CCR5. The frequency of SI-infected CD45RA(+) CD4(+) T cells, but not the frequency of NSI- or SI-infected CD45RO(+) CD4(+) T cells, correlated with the rate of CD4(+) T cell depletion. Infection of naive cells by SI HIV-1 may interfere with CD4(+) T cell production and thus account for rapid CD4(+) T cell depletion.     

T-cell homeostasis in humans with thymic hypoplasia due to chromosome 22q11.2 deletion syndrome

Patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome) typically exhibit thymic hypoplasia, conotruncal cardiac defects, and hypoparathyroidism. The immunodeficiency that results from the thymic hypoplasia has been extensively described and consists primarily of T-cell lymphopenia. A curious feature of the T-cell lymphopenia is that the age-related rate of decline of T-cell numbers is slower in patients than controls. This leads to T-cell numbers in adulthood that are minimally decreased compared with controls. This suggests that homeostatic mechanisms might be acting to preserve the peripheral blood T-cell numbers in patients. We characterized changes in CD4/CD45RA and CD4/CD45RO T-cell populations in patients and controls of various ages and determined T-cell recombination excision circles and telomere length within the CD4/CD45RA population. Patients had evidence of accelerated conversion of naive to memory cells and had evidence of more extensive replicative history within the CD4/CD45RA compartment compared with controls. Oligoclonal T-cell receptor (TCR) Vbeta families and missing Vbeta families were seen more often in patients than controls. These data are consistent with homeostatic proliferation of T cells in patients with limited T-cell production due to thymic hypoplasia.     

Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity

To evaluate the importance of the thymus for the reconstitution of immunity in recipients of a T-cell-depleted bone marrow, we measured the appearance of CD4(+)CD45RA(+)RO(-) naive T cells (thymic rebound), restoration of the diversity of the T-cell-receptor (TCR) repertoire and the response to vaccinations with tetanus toxoid (TT). Repopulation by CD4(+)CD45RA(+)RO(-) thymic emigrants varied among patients, starting at approximately 6 months after transplantation. Young patients reconstituted swiftly, whereas in older patients, the recovery of normal numbers of naive CD4(+) T cells could take several years. Restoration of TCR diversity was correlated with the number of naive CD4(+)CD45RA(+)RO(-) T cells. Moreover, the extent of the thymic rebound correlated with the patient's capacity to respond to vaccinations. Patients without a significant thymic rebound at the moment of vaccination (CD4(+)CD45RA(+)RO(-) T cells less than 30 microL) did not respond, or responded only marginally even after 3 boosts with TT. We conclude that during the first year after transplantation, the absence of an immune response is due mainly to the loss of an adequate T-cell repertoire. Restoration of the repertoire can come only from a thymic rebound that can be monitored by measuring the increase of CD4(+)CD45RA(+)RO(-) naive T cells. This will allow postponing revaccinations to a moment when the patient will be able to respond more effectively. This may be particularly useful in the elderly patient who, owing to low thymic activity, might not yet be able to respond 1 year after transplant when revaccinations are usually scheduled.     

Immunophenotypic Analysis of Peripheral Blood Mononuclear Cells Undergoing In Vitro Apoptosis After Isolation From Human Immunodeficiency Virus-Infected Children

Lymphocytes of human immunodeficiency virus (HIV)-infectedindividuals undergo accelerated apoptosis in vitro, but the subsets ofcells affected have not been clearly defined. This study examined therelationship between lymphocyte phenotype and apoptotic cell death inHIV-infected children by flow cytometry. Direct examination of thephenotype of apoptotic lymphocytes was accomplished using a combinationof surface antigen labeling performed simultaneously with the Tdtmediated Utp nick end-labeling (TUNEL) assay. In comparison to live cells, apoptotic lymphocytes displayed anoverrepresentation of CD45RO and HLA-DR expressing cells, while CD28and CD95 expressing cells were underrepresented. Lymphocytes expressingCD4, CD8, and CD38 were equally represented in apoptotic and livepopulations. When percent lymphocyte apoptosis follow- ingculture was examined independently with lymphocyte subsets in freshblood, apoptosis was negatively correlated with the percentage of CD4cells, but not with specific CD4 T-cell subsets. Although notcorrelated with the percentage of total CD8 cells, apoptosis waspositively correlated with specific CD8 T-cell subsets expressingCD45RO and CD95 and negatively correlated for CD8 T cells expressing CD45RA. These results provide direct evidence that a population ofactivated lymphocytes with the memory phenotype lacking the costimulatory molecule CD28 are especially prone to undergo apoptosis. The findings related to CD95 expression in fresh and apoptotic cellsimplicate Fas-dependent and Fas-independent pathways of apoptosis inHIV disease in children.