Differential propagation of stroma and cancer stem cells dictates tumorigenesis and multipotency.

Glioblastoma Multiforme (GBM) is characterized by high cancer cell heterogeneity and the presence of a complex tumor microenvironment. Those factors are a key obstacle for the treatment of this tumor type. To model the disease in mice, the current strategy is to grow GBM cells in serum-free non-adherent condition, which maintains their tumor-initiating potential. However, the so-generated tumors are histologically different from the one of origin. In this work, we performed high-throughput marker expression analysis and investigated the tumorigenicity of GBM cells enriched under different culture conditions. We identified a marker panel that distinguished tumorigenic sphere cultures from non-tumorigenic serum cultures (high CD56, SOX2, SOX9, and low CD105, CD248, αSMA). Contrary to previous work, we found that 'mixed cell cultures' grown in serum conditions are tumorigenic and express cancer stem cell (CSC) markers. As well, 1% serum plus bFGF and TGF-α preserved the tumorigenicity of sphere cultures and induced epithelial-to-mesenchymal transition gene expression. Furthermore, we identified 12 genes that could replace the 840 genes of The Cancer Genome Atlas (TCGA) used for GBM-subtyping. Our data suggest that the tumorigenicity of GBM cultures depend on cell culture strategies that retain CSCs in culture rather than the presence of serum in the cell culture medium.

Long-term results after intracoronary injection of autologous mononuclear bone marrow cells in acute myocardial infarction: the ASTAMI randomised, controlled study.

OBJECTIVE: To investigate long-term safety and efficacy after intracoronary injection of autologous mononuclear bone marrow cells (mBMCs) in acute myocardial infarction (AMI). DESIGN: Randomised, controlled trial. SETTING: Two university hospitals in Oslo, Norway. PATIENTS: Patients from the Autologous Stem cell Transplantation in Acute Myocardial Infarction (ASTAMI) study were re-assessed 3 years after inclusion. INTERVENTIONS: 100 patients with anterior wall ST-elevation myocardial infarction treated with acute percutaneous coronary intervention (PCI) were randomised to receive intracoronary injection of mBMCs (n = 50) or not (n = 50). MAIN OUTCOME MEASURES: Change in left ventricular (LV) ejection fraction (primary). Change in exercise capacity (peak VO(2)) and quality of life (secondary). Infarct size (additional aim), and safety. RESULTS: The rates of adverse clinical events in the groups were low and equal. There were no significant differences between groups in change of global LV systolic function by echocardiography or magnetic resonance imaging (MRI) during the follow-up. On exercise testing, the mBMC-treated patients had larger improvement in exercise time from 2-3 weeks to 3 years (1.5 minutes vs 0.6 minutes, p = 0.05), but the change in peak oxygen consumption did not differ (3.0 ml/kg/min vs 3.1 ml/kg/min, p = 0.75). CONCLUSION: The results indicate that intracoronary mBMC treatment in AMI is safe in the long term. A small improvement in exercise time in the mBMC group was found, but no other effects of treatment could be identified 3 years after cell therapy.

Bone marrow mesenchymal stem cells in a hyaluronan scaffold for treatment of an osteochondral defect in a rabbit model.

The purpose of this study was to evaluate the efficiency of using mesenchymal stem cells (MSC) in a hyaluronan scaffold for repair of an osteochondral defect in rabbit knee. Bone marrow was harvested from the posterior iliac crest in 11 New Zealand White rabbits. MSC were isolated and cultured in autologous serum for 28 days and transferred to a hyaluronan scaffold 24 h prior to implantation. A 4 mm diameter and 1.5 mm deep defect was created in the medial femoral condyle of both knees and the scaffold with MSC was implanted in one knee while an empty scaffold was implanted in the contra-lateral knee. After 24 weeks the rabbits were killed and histological sections were subjected to semiquantitative and quantitative evaluation by observers blinded regarding treatment modality. High degree of filling was obtained, but there was no statistically significant difference between the two treatments. However, there was a tendency for a better quality of repair in the MSC treated knees. No hypertrophy was observed by either method. MSC in a hyaluronan scaffold may be a promising treatment approach, but further studies are needed to determine the best combination of scaffold and cells.

Treatment with autologous bone marrow mononuclear cells in patients with critical lower limb ischaemia. A pilot study.

BACKGROUND AND AIMS: Treatment with autologous, bone marrow mononuclear stem cells has shown effects in patients with chronic limb ischaemia in one randomized clinical study. The aim of the study was to test the potential effect of stem cell treatment in a strict defined group of patients with stable critical limb ischaemia (CLI). DESIGN: A prospective, combined-centre pilot study. MATERIAL: Eight patients with CLI of the lower extremities, and without any other treatment options. METHODS: Bone marrow cells were harvested from the patient's iliac crest and, after separation, injected into the calf muscles of the affected leg. Outcome was evaluated by digital subtraction angiography (DSA), visual analogue scale (VAS) and several non-invasive circulatory physiological tests. RESULTS: There were no complications from the procedures. Two patients were amputated two months after cell injection. Five patients reported pain relief after four months. Five patients could be evaluated at eight months. According to VAS and physiological tests, they were all either stable or showed improvement. CONCLUSION: This method seems to be a safe option for treating patients with CLI. Inclusion of patients took a long time, mainly because many patients with CLI are offered endovascular treatment in our institution. While symptomatic improvement was found in individual patients, larger trials are required to investigate efficacy. This will probably require multi-centre participation.

Long-term proliferation and survival of in vitro-activated T cells is dependent on Interleukin-2 receptor signalling but not on the high-affinity IL-2R.

Optimization of T-cell-activation protocols is an important prerequisite for the use of populations of activated, polyclonal T cells for immunotherapeutic purposes. This study compares two activation protocols. Following initial CD3/CD28 activation, naïve and memory subsets of CD8+ and CD4+ T cells were either repeatedly stimulated or maintained in medium containing interleukin-2 (IL-2). Initially, activation-induced cell death (AICD) was observed in all subsets. After 2-3 days, death in the cultures maintained in IL-2 only dropped dramatically, while live cells increased logarithmically. Despite intense proliferation, these cells lost the expression of CD25, the alpha chain of the IL-2 receptor and CD71, the transferrin receptor. Functional blocking of CD25 caused minimal changes in proliferation and survival of these cells as long as IL-2 was present in the medium. Blocking of CD25 in combination with the removal of IL-2 caused rapid death of these cells. Restimulation every 3-4 days led to persistently high levels of AICD and lower live cell counts. Live cells maintained the expression of activation markers and a blastoid phenotype. Initial CD3/CD28 followed by maintenance in IL-2 for 2-3 weeks seems to be the best in vitro T-cell-activation strategy. Signalling through the IL-2 receptor is vital for these cells, despite their downregulation of CD25.

Uptake of Apoptotic K562 Leukaemia Cells by Immature Dendritic Cells is Greatly Facilitated by Serum.

Dendritic cells (DCs) are potent antigen-presenting cells and play an important role in T-cell-mediated immunity. DCs have been shown to induce strong antitumour immune responses both in vitro and in vivo. One way of providing the DCs with all relevant tumour antigens would be to incubate the DCs with material from dead tumour cells. We have examined the uptake of apoptotic and necrotic K562 leukaemia cells by DCs under different culture conditions. Results from coincubation experiments strongly suggested that uptake of apoptotic K562 cells was dependent upon the addition of autologous serum (AS). Under these conditions, 47-79% of all DCs were shown to ingest apoptotic material. AS also seemed to be important for the expression of functionally important markers, most notably HLA class I, CD86, CCR7 and CD83. The vast majority of DCs were shown to ingest necrotic material from K562 cells, with no additional effect of AS. The results suggest that incubation of DCs with apoptotic material for cell therapeutic purposes may best be performed in the presence of AS.

The T cell receptor repertoire of CD8+CD28- T lymphocytes is dominated by expanded clones that persist over time.

The costimulatory molecule CD28 is expressed on almost all CD4+ T cells, but on only a portion of CD8+ T cells in healthy human adults. alpha beta T cells may thus be divided into three phenotypically and functionally different subsets: CD4+, CD8+CD28+ and CD8+CD28-. Using peripheral blood lymphocytes from six healthy adults, we have studied the T cell receptor (TCR) repertoire within these subsets by analysis of the distribution of lengths of the complementarity determining region 3 (CDR3) of the beta variable (BV) transcripts and flow cytometric analysis of TCR V beta usage. Expanded CDR3 lengths were identified in 86% of BV families within CD8+CD28- T cells, but in only 4% within CD4+ T cells, and 35% within CD8+CD28+ T cells (P < 0.01). When sequenced, the majority of expanded peaks were found to be dominated by single clones. Identical expanded clones were found within both CD8+CD28+ and CD8+CD28- subsets, consistent with the belief that CD8+CD28- T cells descend directly from CD8+CD28+ T cells. Greatly expanded CD28- clones were found within both CD8+ and CD4+ subsets and persisted at the same magnitude for up to 4.5 years of observation. The finding of a small proportion of cells expressing Ki-67 showed that some of these clonally expanded cells were in the active stages of the cell cycle, but few of the cells expressed activation markers CD69, CD25, CD71 or CD122. One likely explanation for the persistence of expanded peripheral lymphocyte populations in healthy individuals is the presence of persistent antigen.

Molecular analysis of the complementarity determining region 3 of the human T cell receptor beta chain. Establishment of a reference panel of CDR3 lengths from phytohaemagglutinin activated lymphocytes.

The T cell receptor (TCR) repertoire of a lymphocyte population may be characterized by the distribution of lengths of the hypervariable fragment known as the complementarity determining region 3 (CDR3). Immunological activity leading to clonal predominance will result in an over-representation of given CDR3 lengths and a distortion of the CDR3 length distribution. CDR3 length distribution may be studied by the in vitro amplification of TCRB cDNA followed by gel electrophoresis of the resulting product. We have established a simple, robust method for the evaluation of CDR3 length distribution in human lymphocyte samples. The CDR3 length distribution in phytohaemagglutinin (PHA) activated lymphocytes from a large number of healthy donors was established as a reference panel for each of 22 human TCR beta variable (BV) families. We propose that an abnormal CDR3 length distribution be defined as one in which one or more CDR3 lengths exceed the upper confidence limit (5% significance, one-sided test) given by this PHA reference population. Using this criterion in titration experiments, we were able to identify a clone when it constituted 2% of the cells analyzed. Over-dilution of cellular material or cDNA may produce falsely abnormal CDR3 length distributions. A nested technique using two separate amplification steps was found to yield results comparable in quality to the single amplification technique. When few cells are available, the nested method gives more material for CDR3 length analyses. However, it does not reduce the likelihood of a falsely abnormal distribution being recorded when the cellular material is too scarce.

In vivo expansion coincident with excessive in vitro cell death within the memory subset of CD8+ T cells in HIV type 1 infection.

In HIV-1-infected individuals the CD8+ T cell subset is considerably expanded. This has been shown to be caused predominantly by an increase in the number of CD8+CD28- T cells. To characterize further the subsets of CD8+ T cells, we have performed analyses of cell surface phenotype, T cell receptor Vbeta usage, and ability to survive in unstimulated cultures. CD8+CD28- T cells frequently expressed CD45RA. Nonetheless, coincident expression of CD95 (Fas) and high levels of integrins suggested that these cells were immunologically experienced. Contrary to what has been observed in CD8+CD28- T cells from uninfected individuals, a common hierarchy of Vbeta usage was found in CD8+CD28- T cells from HIV-1-infected individuals, which was adhered to by all the study participants, and which was shown to be similar to that observed within CD8+CD28+ T cells. Cells from the memory subsets of CD8+ T cells showed a high incidence of spontaneous death in unstimulated cultures, indicating that these cells have received signals for death by apoptosis in vivo. In contrast, most naive CD8+CD28+CD45RA+ cells survived. The CD8+CD28- memory subset is expanded in vivo despite evidence for coincident excessive cell death in vitro. Our results are consistent with the notion that frequent transitions occur from the memory CD8+CD28+CD45RA- T cell subset to the end-stage CD8+CD28- subset during HIV-1 infection.

Normal CD4 T-cell receptor repertoire in tonsillar tissue despite perturbed repertoire in peripheral blood in HIV-1 infected individuals.

OBJECTIVE: To compare the T-cell receptor (TCR) repertoire of T-cell subsets in peripheral blood and lymphoid tissue from HIV-1 infected individuals. DESIGN: Biopsies of tonsillar tissue and samples of peripheral blood were obtained from 10, mostly treatment-naive, HIV-1-infected individuals. CD4 and CD8 T-cell subsets were quantified, the TCR repertoire was analysed within 'naive' and 'memory' subsets, and results compared between identical subsets in tonsillar tissue and blood. METHODS: Cell subsets were quantified by flow cytometry. CD4 T cells and CD8 T cells were isolated by immunomagnetic beads. Populations were in most cases further subdivided by immunomagnetic selection on the basis of CD45RO expression. TCR repertoire was studied by spectratyping of the TCR beta variable (BV) complementarity determining region 3 (CDR3) transcripts. RESULTS: Amongst CD4 T cells, an abnormal TCR repertoire was found in median 25% (range, 0-88%) of BV families in peripheral blood, but in 0% (0-7%) in tonsillar tissue (P<0.05). Large peaks suggestive of expanded clones were common within CD8 T-cells, both in peripheral blood and tonsillar tissue. However, the expanded clones were rarely identical in the two compartments. Expanded CDR3 peaks, suggesting the presence of clonally expanded cells, were observed within both CD45RO+ and CD45RO- cells from all T-cell subsets, but, again they were mainly of different lengths. CONCLUSION: CD4 T cells were preserved in number and TCR repertoire in tonsillar tissue compared with blood in HIV-1 infected individuals. T-cells collected from the peripheral blood may not be representative of those residing in lymphoid tissue.

Correlates of apoptosis of CD4+ and CD8+ T cells in tonsillar tissue in HIV type 1 infection.

The immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased death by apoptosis of T cell subsets. In the present study, we have examined correlates of apoptosis of CD4+, CD8S+CD28+, and CD8+CD28- T cells in tonsillar lymphoid tissue in persons with HIV-1. Single-cell suspensions of tonsillar lymphocytes were analyzed by flow cytometry to determine the fraction of cells showing typical characteristics of apoptosis as well as the expression of activation markers within the live and the apoptotic cell populations. The proportion of cells carrying infectious provirus was quantified by limiting dilution analysis. Compared with uninfected controls, apoptosis of both CD4+ and CD8+ T cells was enhanced in HIV-1 infection and was higher among CD8+ than among CD4+ T cells. Apoptosis of CD28-cells was more prevalent than apoptosis of CD28+ cells for both CD4+ and CD8+ T cells. Occurrence of apoptosis of CD4+ T cells correlated with provirus levels and proportional expression of the activation marker HLA-DR. Apoptosis of CD8+CD28+ cells correlated with expression of the activation markers CD69 and HLA-DR while apoptosis within CD8+CD28- cells did not correlate with any of the studied parameters. Although apoptosis was much more prevalent among CD8+ than CD4+ T cells, CD8+ T cells still accumulated in tonsillar lymphoid tissue in persons with HIV-1. Our data may be interpreted to suggest that apoptosis of CD4+, CD8+CD28+, and CD8+CD28- cells in tonsillar tissue is regulated by different mechanisms and the results are of importance to our understanding of the immunopathogenesis of HIV-1 infection.

Activation and proliferation of CD8+ T cells in lymphoid tissues of HIV-1-infected individuals in the absence of the high-affinity IL-2 receptor.

Activation of CD8+ T cells may have important pathogenic implications in HIV-1 infection. Studies of this process have so far been confined to cells from the peripheral blood. In the present study, we have examined molecules involved in activation and proliferation of CD8+ T cells in lymphoid tissues from HIV-1-infected patients. Tonsillar tissue and blood samples from 13 HIV-1-infected patients and 6 seronegative controls were examined for cell surface expression and the presence of mRNA for CD69, CD25, and HLA-DR. Intonsillar tissue, the number of CD8+ T cells was increased several fold in HIV-1-infected patients compared with controls. The majority of these cells expressed CD69 and HLA-DR, but virtually no tonsillar CD8+ T cells were found to express CD25 on the cell surface or at the mRNA level. Following in vitro activation, however, almost all activated CD8+ T cells were found to express CD25. Tonsillar CD4+ T cell numbers were maintained or reduced compared with controls, and a considerable proportion expressed CD25. The data suggest that CD8+, but not CD4+ T cells proliferate extensively in lymphoid tissues in HIV-1-infected patients in the absence of the high-affinity interleukin-2 (IL-2) receptor.

Allospecific helper T-lymphocyte repertoire in monozygotic twins.

In the course of maturation in the thymus there is a selection of T lymphocytes based on the avidity between their T-cell receptors and HLA/peptide complexes expressed on stromal thymic cells. The repertoire of mature T lymphocytes is further modulated by encounters with foreign antigens. Thus, the antigen specific repertoire of the peripheral T-lymphocyte pool is determined by genetic and environmental factors. We recently reported that pairs of monozygotic twins often display significant differences in their allospecific cytotoxic T-cell repertoire, suggesting an important role of confrontation with foreign antigens on the CD8+ T-cell repertoire. We have now performed similar studies on the repertoire of allospecific CD4+ T lymphocytes. Using positively selected CD4+ T cells in limiting dilution analyses we compared the differences in the allospecific helper T-lymphocyte precursor frequencies (HTLpf) between pairs of genetically identical monozygotic twins and pairs of unrelated, HLA disparate individuals. We found that all monozygotic twin pairs and most unrelated pairs had similar HTLpf to the same stimulator, i.e. the 95% confidence intervals were overlapping. However, when studied in greater detail, the differences in HTLpf within monozygotic twin pairs were found to be significantly smaller than the differences within pairs of unrelated responders. Thus, we find evidence of an influence by environmental antigens also on the repertoire of allospecific CD4+ T cells, but polymorphic genetic factors seem to be more important here than for the repertoire of allospecific CD8+ T cells.

Demonstration of identical expanded clones within both CD8+CD28+ and CD8+CD28- T cell subsets in HIV type 1-infected individuals.

In HIV-1-infected individuals, the CD8+CD28- T cell subset is considerably expanded and is frequently the largest subset of T cells found in peripheral blood. It has been assumed, but not proven, that CD8+CD28- T cells derive from CD8+CD28+ T cells in vivo. To further study the ontogeny of CD8+CD28- T cells, we have performed analyses of the complementarity determining region 3 (CDR3) of the TCRB of CD8+CD28+ and CD8+CD28- T cells from the peripheral blood of HIV-1-infected individuals. When cells from the same individual were compared, expanded peaks in CDR3 length analysis within a given BV family were frequently observed at the same location in both CD8+ subsets (p < 0.001). Sequencing of cDNA corresponding to dominant peaks revealed the presence of identical expanded CD8+ T cell clones within both the CD28+ and CD28- subsets on eight of nine attempts. Our results show that CD8+CD28+ and CD8+CD28- T cells are phenotypic variants of the same lineage, most likely evolving from CD8+CD28+ to end-stage CD8+CD28- T cells.

Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites.

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.

Correlates of latent and productive HIV type-1 infection in tonsillar CD4(+) T cells.

Correlates of virus load and characteristics of virus-producing cells in tonsillar tissue were investigated. Our results suggest that when less than 1:100 tonsillar CD4(+) T cells from individuals infected with HIV type-1 (HIV-1) contain replication competent provirus, the level of CD4(+) T cells in tonsils is comparable to that observed in uninfected individuals. Virus load at or above this level was associated with low CD4 cell numbers in tonsillar tissue. Only a few percent of all infected T cells in tonsillar tissue were active virus producers, with minor differences observed between individuals. Plasma viremia was found to correlate with infectious virus load in tonsillar tissue. With less than 1:1,000 of CD4 cells in lymphoid tissues being involved in active virus production, direct cytopathic effect by HIV-1 on infected CD4 cells is unlikely to fully explain the immunodeficiency seen in AIDS.

CD8+ T cells from HIV type 1-seronegative individuals suppress virus replication in acutely infected cells.

CD8+ lymphocytes (CD8 cells) have been shown to inhibit replication of the human immunodeficiency virus (HIV) in vitro when cocultured with HIV-infected CD4+ lymphocytes (CD4 cells). This suppressive effect on HIV replication in experimentally infected CD4 cells has so far been demonstrated only for CD8 cells from HIV-seropositive individuals. In the present study we have investigated if CD8 cells from HIV-negative individuals can also suppress HIV replication in experimentally infected CD4 cells. Positively selected CD4 cells were infected with phenotypically different primary isolates of HIV type 1 and 2 (HIV-1 and HIV-2). Graded numbers of CD8 cells were added to the infected cultures. The T cells were activated by antibodies directed against the CD3 molecule or the T cell receptor. Culture supernatants were harvested for HIV p24 quantitation and the CD8 suppression of HIV replication was calculated by comparing p24 levels from parallel cultures in the presence or absence of CD8 cells from different donors. We show that CD8 cells from unexposed HIV-seronegative blood donors are able to control HIV-1 and HIV-2 replication in experimentally infected autologous CD4 cells. The antiviral activity of CD8 cells from and HIV-naive individual was reproducible over time and the suppressive effect was comparable to that seen with CD8 cells from HIV-positive individuals. The infected cells were not eliminated from the cultures. The suppressive effect of CD8 cells varied depending on the dose and biological phenotype of the virus used for infection. Thus, exposure to HIV in vivo is not a prerequisite for CD8 cells to exert a suppressive effect on HIV replication in acutely infected cells.

Monozygotic twins display differences in their allospecific cytotoxic T-cell repertoire.

The antigen-specific repertoire of peripheral blood T lymphocytes is generated by selection in the thymus followed by peripheral immunological events. While thymic selection is predominantly under genetic control, i.e. self-HLA molecules + self-peptides, confrontation with foreign antigens will lead to expansion of given T-cells, thus modifying the T-cell repertoire. To evaluate the relative importance of such modifying events the precursor frequencies (pf) of cytotoxic T lymphocytes (CTL) against given allogeneic HLA molecules were compared in six pairs of monozygotic twins. Using limiting dilution analyses, experiments with several different allogeneic stimulator/target cell donors were performed for each twin pair. In 12 out of 17 experiments, when the twins were tested against the same stimulator/ target cell donor, the CTL pf differed and the 95% confidence intervals were non-overlapping. This suggests that modifying effects by confrontation with foreign antigens play an important role in shaping the allospecific T-cell repertoire.

Reduced CD4 cell counts in blood do not reflect CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1 infection.

OBJECTIVE: To investigate whether the loss of CD4 cells seen in peripheral circulation of HIV-1-positive individuals reflects a similar depletion of CD4 cells from lymphoid tissue. DESIGN: CD4 and CD8 cells in tonsillar mononuclear cell suspensions were quantified relative to tonsillar B cells, as these were thought to remain numerically unchanged in the course of HIV infection. Results were related to the CD4 cell counts in blood and to the clinical status of the patients. METHODS: Blood samples and tonsillar tissue were obtained from 13 HIV-1-seropositive individuals and six seronegative controls. B cells and T-cell subsets in mononuclear cells were quantified using a three-colour flow cytometry protocol. Histological sections were morphologically classified and B-cell areas were quantified by morphometry. RESULTS: The B-cell fraction was confirmed to be relatively unchanged in asymptomatic HIV-1-seropositive individuals compared with controls. The tonsillar CD4 : B-cell ratios in asymptomatic individuals was similar to those seen in controls, whereas the CD4 : B-cell ratios in symptomatic HIV-1-infected individuals were greatly reduced. The tonsillar CD4 : CD8 cell ratios in HIV-1-infected individuals were much lower than those seen in controls, in the asymptomatic group due to a considerable expansion of the tonsillar CD8 cell subset, and in the symptomatic group also due to a loss of CD4 cells. CONCLUSIONS: We found no evidence of CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1-infected individuals despite low CD4 cell counts in blood. Loss of CD4 cells from this lymphoid tissue seems to occur as a late-stage phenomenon correlated with the onset of clinical symptoms.