Decreased thymic output accounts for decreased naive T cell numbers in children with Down syndrome

Children with Down syndrome (DS) have low numbers of naive T cells and abnormal thymus development and function. Because next to thymic production, peripheral proliferation greatly contributes to naive T cell generation in healthy children, we examined the cause of reduced naive T cell numbers in children with DS. Compared with aged matched controls, the total number of signal joint TCR excision circles (sjTREC) per ml blood was reduced in DS. Reduced frequencies and absolute numbers of protein tyrosine kinase 7-positive recent thymic emigrants, but similar levels of naive T cell apoptosis and Ag-driven activation in DS, suggested that reduced thymic output and not increased peripheral loss of naive T cells caused the reduced sjTREC numbers. We found no support for defective peripheral generation of naive T cells in DS. In DS the naive T cells responded to IL-7 and, based on Ki-67 expression, had similar proliferation rates as in healthy controls. sjTREC content per naive CD8(+) T cells was not increased, but even decreased, pointing to increased survival or peripheral generation of naive T cells in DS. In conclusion, we show in this study that reduced thymic output, but not reduced peripheral generation nor increased loss of naive T cells, results in the low naive T cell numbers found in DS.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Dynamics of T cell responses in HIV infection

Cytotoxic CD8(+) T cells play a major role in the immune response against viruses. However, the dynamics of CD8(+) T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8(+) T cells, in relation to Ag-specific CD4(+) T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8(+) T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.     

Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.     

Adoptive transfer of costimulated CD4+ T cells induces expansion of peripheral T cells and decreased CCR5 expression in HIV infection.

To study the safety and feasibility of T-cell reconstitution in HIV-infected individuals, we adoptively transferred activated autologous CD4+ T cells. Polyclonal peripheral blood CD4+ cells were costimulated ex vivo and subjects were given infusions of up to 3 x 1010 activated CD4+ cells. Dose-dependent increases in CD4+ cell counts and in the CD4:CD8 ratio were observed. Sustained increases in the fraction of cytokine-secreting T cells and decreases in the percentage of CD4+CCR5+ cells were noted in vivo, suggesting enhanced function and resistance to HIV infection. The frequency of CD4+Ki-67+ cells increased whereas CD4+ T cells containing T cell-receptor rearrangement excision circles (TRECs) decreased. These findings indicate that expansion of the peripheral T-cell pool mediated the increase in CD4 counts and suggest that approaches to reconstitute CD4 helper cell activity and decrease CCR5 expression may augment natural immunity to HIV infection.     

T cell recognition of HIV synthetic peptides in a natural infection

Because T cell responses are critical for defense against viral infections, a series of synthetic peptides derived from the predicted sequence for HIV-1 proteins gp41, pg120, gag, and viral polymerase were used to test the T cell proliferative response of HIV-1 seropositive individuals. Of HIV-1-infected donors from various clinical categories 90% (27/30) had sensitized cells that proliferated in response to at least one of 21 HIV peptides tested. Cells from HIV seronegative controls did not proliferate (0/9) in response to these HIV peptides. Individuals with fewer clinical manifestations of HIV-1 disease responded to a greater number of peptides (average for asymptomatic seropositives = 8.1 peptides; AIDS patients averaged 2.0). The number of peptides recognized also correlated with absolute number of CD4+ cells, but not with delayed cutaneous hypersensitivity to a (non-HIV) battery of Ag. However, clinical stage at no time correlated with the response to any particular peptide. Response patterns differed considerably among individuals, and some peptides stimulated proliferation in many (48%) HIV-infected donors (peptides gp41-2 and pol-3), whereas another peptide elicited no T cell response in any donor tested (peptide gp120-8). We have also begun to investigate the basis for individual heterogeneity of T lymphocyte proliferative responses of HIV-infected donors to the 21 HIV synthetic peptides. Peptide structure and HLA class II determinants both influenced patterns of lymphocyte responses. Reactivity correlated with peptide size, the presence of alpha and beta secondary structure and lack of reverse turn potential. Hydropathy and charge had no predictive value. Peptides derived from HIV sequences that vary highly among strains tended to be recognized less frequently. HIV-infected lymphocyte donors were HLA typed to examine the influence of the MHC on T lymphocyte proliferation. Analysis of the frequencies of individuals reacting to specific peptides, when compared to the allele frequencies in the population at large, indicated association of some responses to DR alleles. More DR association was observed with peptides that showed "moderate" reactivity than with those that were "highly" reactive. We suggest that highly reactive peptides are capable of forming a structure closer to an "ideal" T cell epitope that can associate with many DR alleles. In contrast, "moderately" reactive determinants have less favorable structures for interaction, are more limited in their ability to interact and therefore show more restriction to specific class II alleles.     

Relationship between CD4 T cell turnover,cellular differentiation and HIV persistence during ART

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.     

Cytotoxic T lymphocytes in HIV infection

Infection with human immunodeficiency virus (HIV) induces potent cytotoxic T lymphocyte (CTL) response. Their level of activity is often extraordinary high, which in the presence of normal precursor levels suggests that effector CTL may be overstimulated and terminally differentiated. Late in HIV infection the CTL response declines. Possible mechanisms for this are discussed, including clonal exhaustion, lack of T cell help and virus escape by mutations involving epitope sequences. Evidence for the last is presented. Such escape potential implies that CTL are important in controlling the virus infection and that HLA type could have an effect on outcome. Escape mutation also has implications for vaccine design.     

Interferon-alpha administration enhances CD8+ T cell activation in HIV infection

Background

Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.

Methods

To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.

Results

The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006). These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy). As plasma HIV levels fell with interferon therapy, this was correlated with a “paradoxical” increase in CD8+ T cell activation (p<0.001).

Conclusion

Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection.     

Evidence for differential induction of helper T cell subsets during Trichinella spiralis infection

The H-2-compatible mouse strains, AKR and B10.BR, exhibit disparate responses to infection with the parasitic nematode Trichinella spiralis. The resistant AKR mice expel intestinal adult worms faster than susceptible B10.BR mice. We tested antibody and lymphokine responses in these strains. With respect to antibody responses, the B10.BR mice had 3- to 10-fold more serum IgE and T. spiralis-specific IgG1 and IgA than AKR mice. The B10.BR mice also had greater numbers of IgG and IgA plaque-forming cells than AKR mice. In contrast, AKR mice produced T. spiralis-specific IgG2a, whereas the B10.BR mice did not. The antibody response kinetics of these strains were similar. We also analyzed lymphokine secretion after restimulating lymphocytes in vitro with T. spiralis Ag. The AKR mesenteric lymph node cells produced more IFN-gamma and less IL-4 than the B10.BR mesenteric lymph node cells. The B10.BR splenocytes produced more IL-4 than the AKR splenocytes, although splenocyte IFN-gamma production was not different. The kinetics of IL-4 production also differed between the two strains. In summary, resistant AKR mice produced more IFN-gamma and T. spiralis-specific IgG2a than susceptible B10.BR mice, which produced more IL-4, IgE, and T. spiralis-specific IgG1. Our results are consistent with differential activation of Th cell subsets in T. spiralis-infected AKR and B10.BR mice.     

Cell turnover and cell tropism in HIV-1 infection

Early infection with HIV-1 is dominated by CCR5-tropic (R5, non-syncytium-inducing) viruses. The evolution of CXCR4-tropic (X4, syncytium-inducing) viruses occurs later in the infection and is associated with rapid disease progression. Here, we propose that the tropism of X4 viruses for naive CD4+ T cells is disadvantageous in early infection owing to the low division rate of these cells. In healthy individuals, the division rate of memory cells is nearly ten times higher than that of naive cells and thus the memory-cell tropism of R5 viruses could account for their dominance early in infection. As the division rate of naive T cells increases with CD4+ depletion, X4 viruses come to dominate in late disease.     

CD4(+) T cell levels are decreased during active CMV infection in kidney transplant recipients

The numbers of T lymphocytes and T cell subsets (CD2(+), CD3(+), CD4(+), CD8(+)), activated T cells (CD26(+)), B cells (CD19(+)), granulocytes (CD15(+)) and natural killer cells (CD16/56) were monitored by flow cytometry in 79 kidney transplant recipients, 35 of whom had cytomegalovirus infection. The percentages of these cells were correlated with viral load, as determined by cytomegalovirus antigenemia. Development of cytomegaloviral infection coincided with a significant reduction in the percentages of CD4(+) (P < 0.005) and CD3(+) (P < 0.05) cells. Monitoring of lymphocyte subsets may provide useful information on immunological events during cytomegaloviral infection.