Preferential accumulation of mature NK cells during human immunosenescence.

The major histocompatibility complex-unrestricted, cell-mediated, constitutive anti-tumor cytotoxic function of natural killer cells is highly preserved in healthy elderly. A study of the dynamics of expression of natural killer cell-associated phenotypes during immunosenescence shows that selective, bidirectional, and disproportionate changes in certain natural killer cell subset number and ratio take place during aging. The mean natural killer cell subset ratio (%CD16+CD57+ over %CD56+CD57-) gradually increases from a young adult level of 0.7 to 4.6 with advancing age predominantly due to a tripling of %CD16+57+ cells as opposed to a moderate decrease (-54%) in %CD56+57- phenotype. The parallel increase in natural killer phenotype ratio and cytotoxic activity might represent a shift in the maturity status of these cells. Based on these findings, a model of natural killer cell immunosenescence is proposed. It is concluded that not all immunosenescent changes need be detrimental; some may even improve the potential for survival and represent an adaptational immunosenescent change.     

Human NK cells proliferate and die in vivo more rapidly than T cells in healthy young and elderly adults

NK cells are essential for health, yet little is known about human NK turnover in vivo. In both young and elderly women, all NK subsets proliferated and died more rapidly than T cells. CD56(bright) NK cells proliferated rapidly but died relatively slowly, suggesting that proliferating CD56(bright) cells differentiate into CD56(dim) NK cells in vivo. The relationship between CD56(dim) and CD56(bright) proliferating cells indicates that proliferating CD56(dim) cells both self-renew and are derived from proliferating CD56(bright) NK cells. Our data suggest that some dying CD56(dim) cells become CD16(+)CD56(-) NK cells and that CD16(-)CD56(low) NK cells respond rapidly to cellular and cytokine stimulation. We propose a model in which all NK cell subsets are in dynamic flux. About half of CD56(dim) NK cells expressed CD57, which was weakly associated with low proliferation. Surprisingly, CD57 expression was associated with higher proliferation rates in both CD8(+) and CD8(-) T cells. Therefore, CD57 is not a reliable marker of senescent, nonproliferative T cells in vivo. NKG2A expression declined with age on both NK cells and T cells. Killer cell Ig-like receptor expression increased with age on T cells but not on NK cells. Although the percentage of CD56(bright) NK cells declined with age and the percentage of CD56(dim) NK cells increased with age, there were no significant age-related proliferation or apoptosis differences for these two populations or for total NK cells. In vivo human NK cell turnover is rapid in both young and elderly adults.     

Negative modulation of human NK cell activity by purinoceptors. 1. Effect of exogenous adenosine triphosphate.

A variety of receptors, including purinergic receptors (for adenosine and adenosine nucleotides), are present on lymphoid cells. We have investigated the negative modulatory action of ATP, a high-energy purinergic compound, on the human peripheral blood natural killer (NK) cells. Micromolar doses of exogenous ATP produced a strong, direct, reversible, and dose-dependent (2.5 x 10(-5) to 8 x 10(-4) M) inhibition of NK cell-mediated cytotoxic activity in 98% of the 131 healthy donors tested. The inhibitory effect of ATP could be demonstrated in unseparated total lymphocytes and enriched NK cells (Percoll density gradient or negative selection by complement lysis) in 2- or 16-hr NK assays, at various effector:target ratios, at suboptimal and saturating concentrations of the K562 targets. The inhibitory constants ID20 and ID50 max for ATP (2 hr), which were 5.6 x 10(-5) and 8 x 10(-5) M, respectively, appear to be a relatively stable feature of an individual. The mechanism of action of ATP was also explored. It is not due to a simple chelation by ATP of the divalent cations Mg2+ and Ca2+ which play a role in the binding/lytic reactions during NK cell-mediated oncolysis. ATP treatment of lymphocytes neither nonspecifically decreased the number of viable cells nor specifically eliminated the NK cells, including their subsets, CD57+16-, CD57+16+, and CD57-16+ as revealed by immunofluorescence staining. Use of an enzyme-like kinetic approach to define the nature of ATP-induced inhibition suggests that it may be an "uncompetitive" type (Lineweaver-Burke reciprocal plot), because both Vmax (maximal velocity of oncolysis) and Km (Michaelis constant) were reduced simultaneously from 1.53 x 10(4) and 2.64 x 10(4) to 0.44 x 10(4) and 0.73 x 10(4), respectively, in presence of ATP. Other methods (Eadie-Hofstee's and Hane's equations) confirmed that ATP does elicit a qualitatively and quantitatively similar shift in these kinetic parameters. Finally, an 18-hr preactivation of effectors by interleukin 2 (IL2) resulted in partial protection from the negative modulation of NK activity by ATP, while the presence of ATP during preincubation did not interfere with the desensitizing effect of IL2. The biological relevance of these findings are discussed and it is concluded that by interacting with appropriate (non-FcRIII?) cell-surface binding sites such as P2 purinergic receptors, ATP is capable of negatively modulating the antitumor cytotoxic activity of human NK cells. This down-regulation can be partially overcome by IL2 that up-regulates NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Compensatory effect of TNFalpha on low natural killer activity in the elderly

Regulatory effect of CD25, an activation antigen the alpha subunit of interleukin 2 receptor (IL2R) on the activity of natural killer (NK) cells was studied in fifty elderly (57-70 years old) and fifty young people (19-35 years old). Cytotoxic NK activity was assessed by 51Cr release assay, the levels of interleukin 2 (IL2) and tumour necrosis factors alpha (TNFalpha) were measured using bioassays and expression of CD16 and CD25 proteins by flow cytometry. Low NK activity in the elderly was associated with decline of full health, lowered serum concentration of IL2 and increased production of TNFalpha during NK reaction. Inhibition of TNFalpha activity by anti-TNF monoclonal antibody suppressed exclusively NK activity of low NK responders. Moreover, stimulation in vitro of blood mononuclear cells, with TNFalpha induced in the elderly low NK responders a significantly higher increase of the CD25 expression on the surface of NK cells as compared with that in the elderly high responders. Since the CD25 molecule constitutes a subunit of the high affinity receptor, binding IL2 to immunocompetent cells, its increased expression on NK cells of low NK responders would enable them to bind even low amounts of the endogenous IL2 available in this group of the elderly. Thus, an overproduction of TNFalpha seems to be a mechanism compensating, in the non-fully healthy elderly, for the decreased IL2 production, promoting efficient cytotoxic reaction.     

CD56bright cells respond to stimulation until very advanced age revealing increased expression of cellular protective proteins SIRT1, HSP70 and SOD2

Background

NK cells are cytotoxic lymphocytes of innate immunity composed of: cytotoxic CD56dim and immunoregulatory CD56bright cells. The study aimed to analyze the expression of cellular protective proteins: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in CD56dim and CD56bright NK cells of the young, seniors aged under 85 (‘the old’) and seniors aged over 85 (‘the oldest’). We studied both non-stimulated NK cells and cells stimulated by IL-2, LPS or PMA with ionomycin. The expression level of proinflammatory cytokines TNF and IFN-γ was also assessed in NK cell subsets and some relationships between the studied parameters were analyzed.

Results

CD56bright cells showed sensitivity to most of the applied stimulatory agents until very advanced age in regards to the expression of SIRT1 and intracellular HSP70. On the contrary, CD56dim cells, sensitive to stimulation by most of the stimulatory agents in the young and the old, in the oldest lost this sensitivity and presented rather high, constant expression of SIRT1 and HSP70, resistant to further stimulation. With reference to SOD2 expression, CD56dim cells were insensitive to stimulation in the young, but their sensitivity increased with ageing. CD56bright cells were sensitive to most of the applied agents in the young and the old but in the oldest they responded to all of the stimulatory agents used in the study. Similarly, both NK cell subsets were sensitive to stimulation until very advanced age in regards to the expression of TNF and IFN-γ.

Conclusions

CD56bright cells maintained sensitivity to stimulation until very advanced age presenting also an increased expression of SIRT1 and HSP70. CD56dim cells showed a constantly increased expression of these cellular protective proteins in the oldest, insensitive for further stimulation. The oldest, however, did not reveal an increased level of SOD2 expression, but it was significantly elevated in both NK cell subsets after stimulation.The pattern of expression of the studied cellular protective proteins in ageing process revealed the adaptation of NK cells to stress response in the oldest seniors which might accompany the immunosenescence and contribute to the long lifespan of this group of the elderly.

     

Bone marrow-derived IFN-producing killer dendritic cells account for the tumoricidal activity of unpulsed dendritic cells

The CD11c(int)B220(+)NK1.1(+)CD49(+) subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11c(int)B220(+)NK1.1(+)CD49(+) cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 x 10(3) highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.     

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.     

Declined miR‐181a‐5p expression is associated with impaired natural killer cell development and function with aging

MicroRNAs (miRNAs) regulate gene expression and thereby influence cell development and function. Numerous studies have shown the significant roles of miRNAs in regulating immune cells including natural killer (NK) cells. However, little is known about the role of miRNAs in NK cells with aging. We previously demonstrated that the aged C57BL/6 mice have significantly decreased proportion of mature (CD27⁻CD11b⁺) NK cells compared with young mice, indicating impaired maturation of NK cells with aging. Here, we performed deep sequencing of CD27⁺ NK cells from young and aged mice. Profiling of the miRNome (global miRNA expression levels) revealed that 49 miRNAs displayed a twofold or greater difference in expression between young and aged NK cells. Among these, 30 miRNAs were upregulated and 19 miRNAs were downregulated in the aged NK cells. We found that the expression level of miR‐l8la‐5p was increased with the maturation of NK cells, and significantly decreased in NK cells from the aged mice. Knockdown of miR‐181a‐5p inhibited NK cell development in vitro and in vivo. Furthermore, miR‐181a‐5p is highly conserved in mice and human. MiR‐181a‐5p promoted the production of IFN‐γ and cytotoxicity in stimulated NK cells from both mice and human. Importantly, miR‐181a‐5p level markedly decreased in NK cells from PBMC of elderly people. Thus, our results demonstrated that the miRNAs profiles in NK cells change with aging, the decreased level of miR‐181a‐5p contributes to the defective NK cell development and function with aging. This opens new strategies to preserve or restore NK cell function in the elderly.     

Redistribution of natural killer (NK) cell frequency and NK cytotoxic activity in primary IgA nephropathy.

Recent findings have indicated an imbalance of immune responsiveness in primary IgA nephropathy (IgAN). Thus natural killer (NK) cell frequency and NK cytotoxicity were evaluated in fifteen IgAN patients. CD8+, CD11+, CD56+ and CD57+ lymphocyte percentages in IgAN individuals fell within normal values, while a significant decrease of CD16+ cells was observed in the same group of patients. In contrast, NK activity overlapped that seen in controls as assessed by an agarose-single cell cytotoxic assay. To further investigate the discrepancy between CD16+ cell level and NK cytotoxic activity in IgAN, the proportion of CD11+ CD57+, CD56+ CD16+ and CD57+ CD16+ lymphocytes was determined. In spite of the unaffected CD56+ CD16+ cell frequency, IgAN subjects exhibited a significant decrease of CD11+ CD57+ and CD57+ CD16+ lymphocyte percentages in comparison to controls. It is suggested that a redistribution of NK lymphocyte subsets occurs in IgAN. This may have an important role in the impairment of the immunoregulatory network.     

Reference ranges and age-related changes of peripheral blood lymphocyte subsets in Chinese healthy adults

This study was performed to build region-specific reference ranges of peripheral blood lymphocyte subsets for Chinese healthy adults from the young to the elderly and analyze the trends of changes in lymphocyte subsets for evaluating the impact of age on the values.151 healthy adults aged 19—86 were recruited based on the SENIEUR protocol.Three sets of reference ranges were finally built applicable for the healthy young(19—44 years),middle-aged(45—64 years) and elder adults(≥65).Comparisons in parameters am...     

Long-term culture growth of CD4-CD8- lymphocytes exhibiting elevated non-MHC-restricted cytotoxic activity.

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum anti-tumor cytotoxicity. The system allows the exponential growth of monocyte- and B-lymphocyte-depleted CD4-CD8- lymphocytes in the presence of human AB serum and recombinant human interleukin-2 (IL-2) (2 x 10(2) U/ml) combined with interleukin (IL-1) beta (50 ng/ml). After 21 days in culture, these cells undergo massive amplification (i.e., the cell yield rises up to 30-120 times the starting values), and exhibit a marked anti-tumor cytotoxic activity against a panel of natural killer (NK)-resistant tumor cell lines. Interestingly, this activity correlates with the high level of perforin RNA. The membrane phenotypes of the final cell population, assessed by a panel of monoclonal antibodies (MoAbs) indicate a mixed population comprising two cell types in variable proportions (i) NKH-1+, T cell receptor (TCR) alpha/beta-, TCR gamma/delta-, CD3-, Leu 23+; (ii) NKH-(+), TCR alpha/beta-, TCR gamma/delta+, CD3+, Leu 23+. This culture system may provide a tool for cellular and molecular studies on the mechanisms of anti-tumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced cancers.     

The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab

BACKGROUND: Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. METHODS: Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. RESULTS: When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. DISCUSSION: bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.     

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC.

     

A single bout of exercise influences natural killer cells in elderly women, especially those who are habitually active

The purpose of our study was to examine the effects of a single exercise bout on the natural killer (NK) cell count and activity in physically active elderly people, sedentary elderly people, and sedentary young people. Eight elderly women who trained by walking (age, 64 +/- 1 years; Vo(2peak), 32.2 +/- 1.1 ml.kg(-1).min(-1)), 8 age-matched untrained women (63 +/- 1 years, 28.8 +/- 1.0 ml.kg(-1).min(-1)), and 8 young untrained women (25 +/- 1 years, 37.6 +/- 1.6 ml.kg(-1).min(-1)) were studied. Blood samples were taken before, immediately after, and 2 hours after a 30-minute bout of exercise at an intensity equivalent to 70-75% of Vo(2peak). Peripheral blood mononuclear cells were isolated and the NK cell count and activity were analyzed. The NK cell count of the elderly sedentary group immediately after exercise was significantly higher than those of the elderly women who walked and young sedentary women, whereas no significant interaction was detected in NK cell activity and NK cell activity per cell number among the 3 groups. Consequently, an intrinsic defect in the cytotoxic ability of NK cells appeared in sedentary elderly people but not in active elderly people who perform habitual exercise.     

NK and LAK activities from human marrow progenitors. I. The effects of interleukin-2 and interleukin-1.

We have investigated the role of interleukin-2 (IL2) as a differentiation factor for human marrow-derived NK cell progenitors and have assessed the effects of interleukin-1 (IL1) on this activity. The effects of these cytokines on early NK cell precursors was determined by testing marrow which had been depleted of mature cells and of CD2+ cells by treatment with soybean agglutinin and sheep erythrocytes (SBA-E-BM). The cytolytic activities of the SBA-E-BM were tested in 51Cr release assays following 7-8 days of liquid culture. K562 targets were used to assess NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity were measurable in marrow incubated in medium without cytokines, or in medium containing IL1 alone. In contrast, culture in medium containing IL2 resulted in a dose-dependent development of lytic activity. NK and LAK activities could be differentiated by the percentage of cultures in which the activity developed, the dose of IL2 required, the time kinetics of induction, and the effect of depletion of residual cells with NK phenotype prior to culture. The most lytically active effectors of both activities, however, were CD56+. Immunofluorescence analyses before and after culture with IL2 revealed that Leu19+ (CD56) cells increased from less than 2% to as much as 17% of the total marrow cells and showed the appearance of a population of CD56+CD16- cells. The addition of IL1 to the marrow cultures increased NK activity when suboptimal amounts of IL2 were used (less than or equal to 100 U/ml), but did not increase LAK activity at any concentration of IL2. A higher number of NK cells, as well as MY7+(CD13+) myeloid cells were recovered from cultures containing IL1 plus IL2, indicating that NK cells as well as myeloid cells had a growth advantage in the presence of IL1. IL2 receptor (CD25) expression was low in all cultures but was consistently higher in cultures containing IL1 and IL2, however, CD25 was not coexpressed on NK cells. These studies indicate that early NK cell precursors can grow and differentiate in response to IL2 and that NK and LAK lytic activities may be acquired at different developmental stages. IL1 may serve to promote the responsiveness of NK cell progenitors to low concentration of IL2 by a mechanism which may not require expression of CD25.     

The Impact of Immunosenescence on Humoral Immune Response Variation after Influenza A/H1N1 Vaccination in Older Subjects

Background

Although influenza causes significant morbidity and mortality in the elderly, the factors underlying the reduced vaccine immunogenicity and efficacy in this age group are not completely understood. Age and immunosenescence factors, and their impact on humoral immunity after influenza vaccination, are of growing interest for the development of better vaccines for the elderly.

Methods

We assessed associations between age and immunosenescence markers (T cell receptor rearrangement excision circles – TREC content, peripheral white blood cell telomerase – TERT expression and CD28 expression on T cells) and influenza A/H1N1 vaccine-induced measures of humoral immunity in 106 older subjects at baseline and three timepoints post-vaccination.

Results

TERT activity (TERT mRNA expression) was significantly positively correlated with the observed increase in the influenza-specific memory B cell ELISPOT response at Day 28 compared to baseline (p-value=0.025). TREC levels were positively correlated with the baseline and early (Day 3) influenza A/H1N1-specific memory B cell ELISPOT response (p-value=0.042 and p-value=0.035, respectively). The expression and/or expression change of CD28 on CD4+ and/or CD8+ T cells at baseline and Day 3 was positively correlated with the influenza A/H1N1-specific memory B cell ELISPOT response at baseline, Day 28 and Day 75 post-vaccination. In a multivariable analysis, the peak antibody response (HAI and/or VNA at Day 28) was negatively associated with age, the percentage of CD8+CD28low T cells, IgD+CD27- naïve B cells, and percentage overall CD20- B cells and plasmablasts, measured at Day 3 post-vaccination. The early change in influenza-specific memory B cell ELISPOT response was positively correlated with the observed increase in influenza A/H1N1-specific HAI antibodies at Day 28 and Day 75 relative to baseline (p-value=0.007 and p-value=0.005, respectively).

Conclusion

Our data suggest that influenza-specific humoral immunity is significantly influenced by age, and that specific markers of immunosenescence (e.g., the baseline/early expression of CD28 on CD4+ and/or CD8+ T cells and T cell immune abnormalities) are correlated with different humoral immune response outcomes observed after vaccination in older individuals, and thus can be potentially used to predict vaccine immunogenicity.     

GMP-Compliant,Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3⁺ T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3⁻CD16⁺CD56⁺ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

Phenotypic analysis of bronchoalveolar lavage lymphocytes from acquired immunodeficiency patients with and without Pneumocystis carinii pneumonia.

A study was performed to reveal possible differences in lymphocyte subpopulations from bronchoalveolar lavage (BAL) of acquired immunodeficiency patients with and without Pneumocystis carinii pneumonia. Forty-one consecutive human immunodeficiency virus-seropositive patients were studied. Pneumocystis carinii infection was detected in the BAL fluid from 18 patients. The BAL lymphocyte subpopulations were determined by surface marker analysis with the immunoperoxidase slide assay. No significant differences in the percentage of CD4+ and CD8+ lymphocytes were found between the two groups. The percentage of CD57+ natural killer (NK) cells was significantly higher in the Pneumocystis carinii-negative group than in the -positive group. Since NK cells protect from microbial infections, it is conceivable that the loss of CD57+ NK cells may be one of the phenomena leading to the immunodeficiency state that underlies the pulmonary complications characteristic of the acquired immunodeficiency syndrome.