Effect of murine thymic epithelial cell line (MTEC1) on the functional expression of CD4(+)CD8(-) thymocyte subgroups

To determine the effect of thymic stromal cells on the functional maturation of CD4 single-positive (SP) thymocytes, the functional status of isolated CD4 SP thymocyte subgroups was investigated by means of cell proliferation and cytokine production in response to concanavalin A (Con A) prior and after co-culturing with a murine thymic epithelial cell line (MTEC1). Mouse medullary CD4 SP thymocytes were phenotypically divided into seven discrete subgroups predicted to reflect the maturation pathway from newly emerging CD4 SP thymocytes to terminally differentiated cells. For functional analysis, six major subgroups (6C10(+)CD69(+), 6C10(-)CD69(+), 6C10(-)CD69(-)3G11(+)Qa-2(-), 6C10(-)CD69(-)3G11(+)Qa-2(+), 6C10(-)CD69(-)3G11(-)Qa-2(-) and 6C10(-)CD69(-)3G11(-)Qa-2(+)) cells were isolated and their functional status in response to Con A stimulation assessed. A functional hierarchy is revealed among these subgroups, consistent with their phenotypic maturation status, which may imply that these cells undergo a functional maturation process within thymic medulla. The function of cytokine production by CD4 SP thymocytes is acquired in a stepwise manner from a low to high level and characterized by T(h)0-type cytokines in the main stream of differentiation pathway. However, a minor subgroup that appeared at the late stage as 3G11(-)6C10(-) cells was biased to produce T(h)2-type cytokines. Nevertheless, the functional capacity of the final two Qa-2(+) subgroups of CD4 SP thymocytes was still significantly lower than that of spleen CD4(+) T cells. After co-cultivation with MTEC1 cells, four subgroups of TCRalphabeta(+)CD4(+)CD8(-) thymocytes exhibited significantly higher levels of proliferation capability and modulation in cytokine production capability. However, co-culturing with MTEC1 cells did not change the pattern of T(h)0- or T(h)2-like cytokine production by respectively medullary CD4 SP thymocyte subgroups nor could MTEC1 induce CD4 SP thymocytes to secrete T(h)1-type cytokines. The results suggest that MTEC1 can regulate the functional status of these thymocyte subgroups.     

Thymic stromal cells produce soluble factors which increase polarization of chicken thymocytes.

The effects were examined of soluble factors present in culture supernatant (CS) of thymic stromal cells (TSC) on the differentiation and locomotor activity of chicken thymocytes. The locomotor activity of thymocytes was assessed by cell polarization assay. When thymocytes were incubated in the presence of TSC-CS, the proportion of polarized cells increased. This indicates that thymocytes acquired a potent locomotor activity. A high proportion of peanut agglutinin-positive (PNA + ) thymocytes, as well as CD4(-)CD8(-) and CD4(+)CD8(+) thymocytes showed polarization in TSC-CS. Bone marrow cells exhibited higher level of polarizing activity compared to CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes and spleen T cells. These results suggest that thymic stromal cells secrete a soluble factor(s) which enhances mobilizing activity of immature T cells. The factor may take part in the intrathymic migration of progenitor T cells into the site of differentiation.     

Peripheral human CD8(+)CD28(+)T lymphocytes give rise to CD28(-)progeny, but IL-4 prevents loss of CD28 expression.

At birth, virtually all peripheral CD8(+) T cells express the CD28 co-stimulatory molecule, but healthy human adults accumulate CD28(-)CD8(+) T cells that often express the CD57 marker. While these CD28(-) subpopulations are known to exert effector-type functions, the generation, maintenance and regulation of CD28(-) (CD57(+) or CD57(-)) subpopulations remain unresolved. Here, we compared the differentiation of CD8(+)CD28(bright)CD57(-) T cells purified from healthy adults or neonates and propagated in IL-2, alone or with IL-4. With IL-2 alone, CD8(+)CD28(bright)CD57(-) T cell cultures yielded a prevailing CD28(-) subpopulation. The few persisting CD28(dim) and the major CD28(-) cells were characterized by similar telomere shortening at the plateau phase of cell growth. Cultures from adults donors generated four final CD8(+) phenotypes: a major CD28(-)CD57(+), and three minor CD28(-)CD57(-), CD28(dim)CD57(-) and CD28(dim)CD57(dim). These four end-stage CD8(+) subpopulations displayed a fairly similar representation of TCR V(beta) genes. In cultures initiated with umbilical cord blood, virtually all the original CD8(+)CD28(bright) T cells lost expression of CD28, but none acquired CD57 with IL-2 alone. IL-4 impacted on the differentiation pathways of the CD8(+)CD28(bright)CD57(-) T cells: the addition of IL-4 led both the neonatal and the adult lymphocytes to keep their expression of CD28. Thus, CD8(+)CD28(bright)CD57(-) T cells can give rise to four end-stage subpopulations, the balance of which is controlled by both the cytokine environment, IL-4 in particular, and the proportions of naive and memory CD8(+)CD28(+) T cells.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

Heterogeneity within medullary-type TCRalphabeta(+)CD3(+)CD4(-)CD8(+) thymocytes in normal mouse thymus

The functional maturation process of medullary-type CD4(-)CD8(+) [CD8 single-positive (SP)] thymocytes remains largely uncharacterized. We describe a phenotypic analysis of CD8 SP medullary-type thymocytes and find a remarkable heterogeneity within this thymic cell population. While mature CD8(+) T cells in the periphery are relatively homogeneous (TCRalphabeta(+)CD3(+)Qa-2(+) HSA(-)3G11(-)6C10(-)CD69(-)), CD8 SP medullary-type thymocytes contain discrete subpopulations that can be identified by differential expression of several cell-surface markers. We have identified at least six discrete subpopulations in the subset of TCRalphabeta(+)CD3(+) CD8 SP cells in the thymus. According to the expressed phenotypes, a linear developmental pathway is predicted among these CD8 SP subpopulations as follows: 6C10(+)CD69(+)HSA(hi)3G11(+)Qa-2(-) --> 6C10(-)CD69(+)HSA(hi/int)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(int)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(lo)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(-/lo)3G11(-)Qa-2(-) --> 6C10(-)CD69(-)HSA(-/lo)3G11(-)Qa-2(+). This study provides a framework for understanding CD8 SP T cell maturation in the thymic medulla.     

CD8 alpha is an activation marker for a subset of peripheral CD4 T cells

Rat CD4 T lymphocytes express CD8 alpha upon activation. Here, we show that double-positive cells express CD8 alpha alpha homodimers, and we study their phenotype and function. Most activated CD4(+) lymphocytes expressing CD8 alpha are recent thymic emigrants. Accordingly, most activated CD4 single-positive thymocytes express CD8 alpha, and thymectomy and aging decrease the frequency of CD4(+)CD8 alpha(+) lymphocytes. However, CD8 induction is not restricted to CD4(+) recent thymic emigrants. CD4(+)CD8 alpha(+) and CD4(+)CD8 alpha(-)cells were generated in vitro from naive or from primed donors and, to study their function, were transferred to normal rats. Both cell types helped primary humoral responses, but only CD4(+)CD8 alpha(-) cells promoted secondary responses. Thus, memory CD4 T cells mediating antibody responses and some naive CD4(+) lymphocytes do not express CD8 alpha. In addition, CD4(+)CD8 alpha(+) cells produce mainly Th1 cytokines while CD4(+)CD8 alpha(-) cells produce IL-10 and showed a sustained proliferative response. Hence, CD8 alpha expression after activation distinguishes two distinct CD4 T cell subsets.     

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.     

4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.     

The survival and turnover of mature and immature CD8 T cells.

The present investigation has examined the phenotype, survival and fate of immature and mature CD8 T cells. CD4- CD8+ 'single positive' thymocytes (a model for recent thymic emigrants) were Thy-1+ CD45RC- RT6- before transfer to normal euthymic recipients, but changed phenotype within 7-10 days to become Thy-1- CD45RC+ RT6(+)--the phenotype of mature resting CD8 T cells. Following transfer to athymic nude recipients CD8 T cells from thoracic duct lymph of allotype-marked rats increased 12-17-fold during the first 2 months. Proliferation occurred in the complete absence of CD4 T cells and the donor CD8 T cells persisted [at 15-18% of peripheral blood lymphocytes (PBL)] for the life of the recipients. When combined with equal numbers of CD4 T cells, however, CD8 T cells occupied only 3-4% of PBL; in these animals CD4 T cells plateaued at 15-16% of PBL. The results suggested that CD8 T cells competed poorly with rapidly dividing CD4 T cells for limited space in a recirculating pool in which total T-cell numbers are homeostatically regulated. Although able to proliferate and self-renew in athymic nude recipients, when transferred to normal euthymic animals donor-derived mature CD8 T cells declined in number with time; their half-life was estimated to be 34 days. Similar studies with purified CD4- CD8+ 'single positive' thymocytes gave a comparable half-life of 37 days. The results indicated that lifespan was not due to an ageing process among CD8 T cells, but was rather a reflection of cell turnover dependent on thymic output.     

Attenuation of cytokine responsiveness during T cell development and differentiation.

Cytokines play critical roles during T cell development; however, it is unclear to what extent development is altered by the high levels of cytokines produced during immune responses. A potential mechanism to shield developing cells from cytokine influence is attenuation of cytokine signaling. Using intracellular staining and flow cytometry to detect cytokine-induced Stat phosphorylation, we analyzed the cytokine responsiveness of developmentally defined mouse T cells. We assessed CD4(-)CD8(-) (DN), CD4(+)CD8(+) (DP), CD4(+)CD8(-) (SP4), and CD4(-)CD8(+) (SP8) in the thymus, and CD4(+)CD44(lo) (naive), CD4(+)CD44(hi) (memory), CD8(+)CD44(lo) (naive), and CD8(+)CD44(hi) (memory) in the periphery for responsiveness to interleukin-2 (IL-2), IL-4, IL-6, IL-7, IL- 10, IL-15, interferon-alpha (IFN-alpha), and IFN-gamma. SP thymocytes responded to a wider range of cytokines than did the less mature DN and DP subpopulations. DP thymocytes were nonresponsive to all cytokines tested except for modest responses to IL-4 and IFN-alpha. Peripheral naive and memory T cells also displayed differential cytokine sensitivity. Memory T cells were less responsive to the proinflammatory cytokines IL-6 and IFN-gamma when compared with naive T cells, and the memory CD4(+) subset was less responsive to IL-4. In summary, developing thymocytes and memory T cells appear to be resistant to the influences of numerous cytokines produced during immune responses.     

Changes in circulating lymphocyte subpopulations following administration of the leucocyte function-associated antigen-3 (LFA-3)/IgG1 fusion protein alefacept

Alefacept, a recombinant leucocyte function-associated antigen-3 (LFA-3)/IgG1 fusion protein approved for the treatment of psoriasis, is reported to reduce selectively the numbers of circulating CD4(+) CD45RO(+) and CD8(+) CD45RO(+) T cells, while sparing the naive cells. The purpose of the present study was to elucidate further the effect of alefacept on various circulating lymphocyte subsets. Sixteen patients, 12 with chronic plaque psoriasis and four with pustular psoriasis, received alefacept 7.5 mg once weekly for 12 weeks. Blood samples collected at study entry and after 12 weeks of treatment were analysed by four-colour flow cytometry. There were statistically significant reductions in the total number of conventional memory (CD45RA(-) CD27(+)) and effector (CD45RA(-) CD27(-) or CD45RA(+) CD27(-)) T cells, including CD4(+) and CD8(+) T cells expressing CD161 and CD8(+) T cells expressing cutaneous lymphocyte-associated antigen (CLA). Natural killer (NK) T cells were also reduced significantly, while no statistically significant changes were seen in NK cells and CD4(+) CD25(high) cells. The affected subpopulations were all characterized by a high expression of CD2. However, CD4(+) CD25(low), and CD4(+) CLA(+) cells, which also expressed relative high levels of CD2, were not reduced significantly. Our results suggest a heterogeneous effect of alefacept on the circulating memory T cell population, indicating that high expression of CD2 may not, by itself, be sufficient to explain the reduction in cell count for a specific subpopulation.     

Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation

Multi-color flow cytometric analysis on human CD8(+) T cell subsets revealed that CXCR4 is predominantly expressed on CD8(+) T cells with the naive CD27(+)CD28(+)CD45RA(+) phenotype, and is down-regulated during differentiation into those with an effector phenotype. The down-regulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8(+) T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co-expression further showed that CD8(+) T cells expressing a high level of CXCR4 are CCR7(+)CCR5(-) naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7(-)CCR5(+/-) memory/effector and effector subsets. Epstein Barr virus-specific CD8(+) T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus-specific CD8(+) T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down-regulated during differentiation of viral antigen-specific CD8(+) T cells. The classification of human CD8(+) T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8(+) T cells.     

Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T-cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T-cell subpopulations in 17 CVID patients in comparison to age-matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA-CD62L+/RA+CD62L- were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA-DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T-cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA-DR+ and CD8+CD45RA-CD62L+/RA+CD62L- T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

Within the hemopoietic system, LAR phosphatase is a T cell lineage-specific adhesion receptor-like protein whose phosphatase activity appears dispensable for T cell development, repertoire selection and function

Expression of the receptor-type tyrosine phosphatase LAR was studied in cells of the murine hemopoietic system. The gene is expressed in all cells of the T cell lineage but not in cells of any other hemopoietic lineage and the level of expression in T cells is developmentally regulated. The CD4(-)8(-)44(+) early thymic immigrants and mature (CD4(+)8(-)/CD4(-)8(+)) thymocytes and T cells express low levels, whereas immature (CD4(-)8(-)44(-) and CD4(+)8(+)) thymocytes express high levels of LAR. Among bone marrow cells only uncommitted c-kit(+)B220(+)CD19(-) precursors, but not B cell lineage committed c-kit(+)B220(+)CD19(+) precursors, express low levels of LAR. In contrast to the c-kit(+)B220(+)CD19(+) pre-BI cells from normal mice, counterparts of pre-BI cells from PAX-5-deficient mice express LAR, indicating that PAX-5-mediated commitment to the B cell lineage results in suppression of LAR. During differentiation of PAX-5-deficient pre-BI cell line into non-T cell lineages, expression of LAR is switched off, but it is up-regulated during differentiation into thymocytes. Thus, within the hemopoietic system, LAR appears to be a T cell lineage-specific receptor-type phosphatase. However, surprisingly, truncation of its phosphatase domains has no obvious effect on T cell development, repertoire selection or function.     

Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro

The molecular interactions provided by the thymic microenvironment that predicate T cell development remain obscure. Here, we show that a bone marrow stromal cell line ectopically expressing the Notch ligand Delta-like-1 loses its ability to support B cell lymphopoiesis, but acquires the capacity to induce the differentiation of hematopoietic progenitors into CD4 CD8 double- and single-positive T cells. Both gammadelta-TCR(+) and alphabeta-TCR(+) T cells are generated, and CD8(+) TCR(hi) cells produce gamma-interferon following CD3/TCR stimulation. These results establish that expression of Delta-like-1 on stromal cells provides key signals for the induction of T cell lineage commitment, stage-specific progenitor expansion, TCR gene rearrangement, and T cell differentiation in the absence of a thymus. Thus, it is likely that Delta-like-1/Notch interactions by the thymus underpin its unique ability to promote lineage commitment and differentiation of T cells.     

Function of CD44(Pgp-1) homing receptor in human T cell precursors

T cell precursors migrate from extrathymic hematopoietic tissues and differentiate after encountering the thymic microenvironment. We asked whether human T cell precursors express the CD44(Pgp-1/gp90HR) class of homing receptors that have been implicated in the traffic of hematopoietic cells, such as lymphocyte entry to peripheral lymphoid organs. Flow cytometry and immunoprecipitation studies demonstrate that CD7+34+, CD1-2-3-4-8-14-16-20- cells in bone marrow and thymus, which have been shown to exhibit features of T cell precursors, bear CD44. Immunohistological studies show that clusters of thymocytes in the subcapsular and the inner cortex and most medullary thymocytes are clearly CD44+, whereas the expression of CD44 is selectively downregulated in CD3- and CD3low functionally incompetent cortical thymocytes. The expression of CD44 is not restricted to T cell precursors but also occurs in thymic stroma, which bear a different molecular species of CD44. CD44-specific antibodies exert stimulatory effects on T cell precursors, a process that is dependent on stromal cells. We postulate that CD44 might be an adhesion molecule for precursor homing to thymus and that it participates in cell-to-cell interactions within the thymic environment.     

Immunologic, virologic, and neuropsychologic responses in human immunodeficiency virus-infected children receiving their first highly active antiretroviral therapy regimen

Our objective was to measure the early dynamics, evolution, and durability over 96 wk of immunologic responses in children receiving their first highly active antiretroviral therapy (HAART) regimen. The study was designed as a prospective, single-arm study. Twelve human immunodeficiency virus (HIV)-infected children (median age, 11.8 yr) were enrolled. All subjects received stavudine, nevirapine, and ritonavir. Serial measurements included HIV viral load, lymphocyte subsets, thymic volume by computed tomography (CT), neurocognitive testing, and brain CT. Baseline median CD4(+) T cell count was 589 cells/mm(3) , viral load was 3.9 log(10) HIV RNA copies/mL, and thymic volume was 16.3 cm(3) . Ten children had an undetectable viral load at week 48. Eight maintained an undetectable viral load at 96 wk. The median increase in absolute CD4(+) T cell count was 225 cells/mm(3) by week 48, and 307 cells/mm(3) by week 96. The median increase in naive (CD45RA(+) CD62L(+) ) CD4(+) T cells was 133 cells/mm(3) by week 48, and 147 cells/mm(3) by week 96. The median number of naive CD8(+) T cells increased from 205 to 284 cells/mm(3) by week 24; this increase was sustained to week 96. The number of B cells increased and was associated with a decrease in immunoglobulin levels. The number of natural killer cells was stable. There were no significant changes in thymic volume. Most children exhibited stable cognitive function over the course of the study. We conclude that, in this cohort of relatively immunocompetent HIV-infected children, an initial HAART regimen was associated with rapid and sustained increases in total CD4(+) T cells, in naive CD4(+) and CD8(+) T cells, and in B cells through 96 wk.