Long-term decrease of CD4+CD45RA+ T cells and impaired primary immune response after post-traumatic splenectomy

Authors


Dr Hermann M. Wolf Immunology Outpatient Clinic, Schwarzspanierstrasse 15/1/9, A-1090 Vienna, Austria. e-mail: hermann.wolf@itk.at.

Abstract

Congenital or acquired absence of the spleen and functional hyposplenism are associated with abnormalities of host defence such as an increased susceptibility to infection with encapsulated bacteria. The effects of the lack of the spleen on cell-mediated immunity are largely unknown. In the present study we have investigated peripheral blood lymphocyte subpopulations in healthy adults who had undergone splenectomy because of severe abdominal trauma > 4 years before the study. The results show a significant reduction in the percentage of CD4+ T cells due to a selective and long-term decrease in the percentage of CD4+CD45RA+ lymphocytes, the CD4+ T-cell subset mainly involved in primary immune responses to newly encountered antigens. Levels of the reciprocal CD45RO+CD4+ T-cell subset were comparable between splenectomized and control individuals, as were lymphoproliferative responses and IFN-gamma production to recall antigens. Decreased levels of CD4+CD45RA+ cells were accompanied by an impairment in primary immune responsiveness, as assessed by investigating T-cell proliferation to stimulation with keyhole limpet haemocyanin and by measuring antibody responses following primary immunization with a clinically relevant T-dependent antigen, hepatitis A vaccine, in vivo. These findings suggest a possible role of the spleen in the generation, maintenance and/or differentiation of naive, unprimed T cells or their precursors, which might have a possible functional relevance for primary immune responses following splenectomy.

Congenital asplenia, aquired loss of the spleen or functional hyposplenism (e.g. in patients with congenital erythrocyte abnormalities) result in increased susceptibility to systemic overwhelming infections. The most important aetiologic microorganisms in these infections are encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae type b or Neisseria meningitidis. Less common causes of bacteraemia include other streptococci, Staphylococcus aureus, Escherichia coli, and other Gram-negative bacteria such as Klebsiella species, Salmonella species and Pseudomonas aeruginosa. In addition, splenectomy also predisposes to babesiosis, infection by Capnocytophaga canimorsus (DF-2 bacillus) and clinically severe forms of malaria ( Working Party of the British Commitee for Standards in Haematology Clinical Haematology Task Force, 1996). Absence of the spleen probably also leads to increased susceptibility to disease complications associated with certain viral infections such as viral hepatitis ( Stone et al, 1967 ).

A variety of abnormalities in host defence have been described in asplenic or functionally hyposplenic patient populations and are believed to contribute to an undue susceptibility to infectious diseases and their complications. The spleen acts as a filter for the blood to remove foreign particles as well as senescent, defective or antibody-coated blood cells under normal or pathological conditions. The clearance of antibody- and complement-coated microorganisms by phagocytic cells of the spleen is very rapid and normally prevents the dissemination of infectious organisms to several distant and important organs such as the central nervous system, the kidney and the lungs. In the absence of the splenic function there is a compromised ability to clear microorganisms from the blood stream via the reticuloendothelial system ( Hosea et al, 1981a ). Other defects of innate host defence that have been described in the absence of the spleen include decreased levels of serum opsonins (e.g. properdin) and a resulting defective alternative pathway complement activation ( Corry et al, 1979 ), as well as decreased NK cytotoxicity and antibody-dependent cellular cytotoxicity ( Müller et al, 1988 ).

In addition to its role in innate host defence, the spleen is also the greatest single secondary lymphoid organ. The immune response to foreign antigens takes place primarily in the secondary lymphoid organs such as the lymph nodes, the mucosa-associated lymphoid tissue and the spleen. During a primary immune response, pathogens and their constituents are transported to these tissues where microbial antigens are presented to naive, unprimed lymphocytes that constantly enter and leave the secondary lymphoid organs. Depending on the localization of the antigen, different lymphoid organs are involved, e.g. bacteria that invade the body by penetrating the skin are drained to regional lymph nodes to stimulate an immune response, whereas microbial and dietary antigens that enter the body through the gastrointestinal tract stimulate an immune response in the mucosa-associated lymphoid tissue. An impairment in antibody production to intravenously injected antigens has been described following splenectomy ( Sullivan et al, 1978 ), a finding that reflects the importance of the spleen for immune response to antigen present in the circulation. Other abnormalities of the humoral immune system that have been reported in splenectomized patient populations include decreased levels of serum IgM ( Eibl, 1985) and impaired B-cell function in vitro (i.e. decreased pokeweed mitogen-induced immunoglobulin production in splenectomized individuals) ( Drew et al, 1984 ; Müller et al, 1984 ). Whether splenectomy has an effect on immune responsiveness to vaccination with antigens injected subcutaneously or intramuscularly is controversial and might depend on the type of antigen applied and/or whether underlying disease is present ( Sullivan et al, 1978 ; Hosea et al, 1981b ; Siber et al, 1978 ; Ahonkhai et al, 1979 ).

The effect of splenectomy on cell-mediated immunity is largely unknown. Although functional irregularities of the T-cell system such as a decreased lymphoproliferative response to mitogenic stimulation have been reported in splenectomized or asplenic patient populations ( Downey et al, 1987 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991), the effect of absence of the spleen on the distribution of lymphocyte subpopulations was variable, with some studies reporting a decrease in peripheral blood T cells or certain T-cell subpopulations ( Wang et al, 1988 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991; Westermann et al, 1990 ) and other studies showing no effect ( Balsalobre & Carbonell-Tatay, 1991; Passlick et al, 1991 ). The present study was performed to further clarify the long-term immunologic consequences of the absence of the spleen. To rule out that the immunologic abnormalities detected could be affected by an underlying pathologic condition and/or by the form of therapy applied, otherwise healthy adults were studied many years after post-traumatic splenectomy. In this uniform population we show that post-traumatic splenectomy results in a significant and long-term reduction of the levels of CD4+ T cells coexpressing the high molecular weight isoform of CD45, CD45RA, which characterizes the CD4+ T-cell subset critically involved in responsiveness to a novel antigen ( Plebanski et al, 1992 ; Mehta-Damani et al, 1995 ; Young et al, 1997 ). A possible functional significance of such immunologic abnormalities was suggested by the further finding that the capacity to mount a primary T-cell response to a neoantigen such as keyhole limpet haemocyanin (KLH) in vitro was significantly reduced following splenectomy, and that antibody responses following primary immunization with a clinically relevant, T-cell-dependent antigen, hepatitis A vaccine, were significantly lower as compared to age- and sex-matched controls vaccinated in parallel.

STUDY POPULATIONS AND METHODS

Patients and controls

12 adults with a history of previous post-traumatic splenectomy (10 males and two females, age 32 ± 10 years, mean ± SD), 11 age- and sex-matched adults (surgery control group, nine males and two females, age 28 ± 7 years, mean ± SD) who underwent abdominal surgery because of severe abdominal trauma but in whom the spleen could be preserved, e.g. due to the use of fibrin sealant, and 14 sex- and age-matched controls (study control group, 13 males and one female, age 29 ± 9 years, mean ± SD) were included in the study after informed consent was obtained. All study individuals were healthy as determined by physical examination and routine laboratory parameters and showed no sign of primary immunodeficiency. Splenectomy had been performed more than 4 years before the study (range 4 years and 1 month to 26 years and 6 months, mean 7 years and 8 months). Individuals included in the surgery control group underwent abdominal surgery in the same time period. All blood samples were obtained between 8 and 10 a.m., and groups of splenectomized individuals and controls were examined in parallel.

Hepatitis A immunization and determination of vaccination antibodies

Splenectomized individuals and controls were tested for serum antibodies against hepatitis A using a commercially available ELISA kit (ENZYGNOST Anti-HAV, Behringwerke AG, Marburg, Germany). Study subjects with negative or borderline hepatitis A antibody levels (leqslant R: less-than-or-eq, slant 33 IU/ml) were vaccinated with a commercially available vaccine against hepatitis A (Havrix 720 EL U/ml for adults, SmithKline Beecham Biologicals, Rixensart, Belgium), a whole-virus, formalin-inactivated, aluminium hydroxide adjuvanted product. Two immunizations were given by i.m. injection, with an interval of 4–6 weeks between the immunizations. Serum antibodies against hepatitis A were assessed by ELISA before vaccination and 4–6 weeks after the first and after the second vaccination. Serum antibodies against tetanus toxoid were determined using a commercially available ELISA kit (IMMUNOZYM Tetanus, Immuno GMBH Heidelberg, Germany).

Isolation of peripheral blood mononuclear cells and stimulation of lymphocyte proliferation and IFN-gamma production by mitogens, CD3 mAb and recall antigens

Mononuclear cells (MNC) were isolated from heparinized peripheral blood (containing 7.5 IU of preservative-free heparin per ml) by buoyant density gradient centrifugation as previously described ( Wolf et al, 1992 ). The isolated MNC were suspended in RPMI-1640 medium (Flow Laboratories, Irvine, U.K.) supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml), l-glutamine (2 m m) (RPMI suppl.) and 10% pooled, heat-inactivated (30 min, 56°C) human AB serum (complete medium). For assessment of lymphocyte proliferative responses, cell cultures were set up in triplicate in flat-bottomed microtitre plates (Falcon 3070, Microtest III, Becton Dickinson Labware, Lincoln Park, N.J.), containing 1 × 105 MNC in a final volume of 0.2 ml per well in complete medium containing antigen or mitogen at the following final concentrations: heat-inactivated (80°C, 3 h) E. coli 089:H10 at 5 × 106/ml (kindly provided by Immuno AG, Vienna, Austria); pokeweed mitogen (PWM, GIBCO, Grand Island, N.Y.) at a final dilution of 1:1000; PHA (Phytohaemagglutinin P, DIFCO Laboratories, Detroit, Mich.), final dilution of 1:1250; ConA (Sigma Chemical Company, St Louis, Mo.), final concentration of 12 μg/ml; OKT3 (Ortho-mune, Ortho Diagnostic Systems Inc., Raritan, N.J.), at 10 ng/ml; Tetanus Toxoid (Swiss Serum Institut, Bern, Switzerland), at a final concentration of 10 Loeffler flocculation units (Lf )/ml. Cell cultures were continued for 3 d (PHA, ConA, OKT3) or 7 d (PWM, Tet Tox, E. coli) at 37°C in a CO2 incubator. The resulting MNC proliferation was assessed by measuring 3H-thymidine incorporation. For the last 16 h of the incubation period the cell cultures were pulsed with 7.4 kBq 3H-thymidine per well (ICN Radiochemicals, Costa Mesa, Calif.) before the cells were harvested onto glass-fibre filter plates (Printer Filtermat A, Wallac Oy, Turku, Finland) using a semi-automatic cell harvester (Harvester 96 Mach III M, Tomtech Inc., Orange, Ct.) and melt-on scintillator sheets (MeltiLex A, Wallac Oy, Turku, Finland). The incorporated radioactivity was measured with a liquid scintillation counter (1450 MicroBeta Trilux, Wallac Oy, Turku, Finland). Results are expressed as delta disintegrations per minute (delta dpm, average of triplicate cultures).

For stimulation of IFN-gamma production, MNC were cultured for 1 and 3 d (OKT3, PHA, ConA) or 3 and 7 d (Tet Tox, PWM, E. coli) in flat-bottomed 24-well tissue culture plates (Falcon, Becton Dickinson Labware, Lincoln Park, N.J.), containing 1 × 106 MNC in a final volume of 1 ml per well in complete medium containing antigen or mitogen at the final concentrations given above. Then the MNC supernatants were aspirated and centrifuged at 9000 g for 3 min to remove contaminating cellular material. If the determination of cytokine content could not be performed on the same day, the supernatants were divided into aliquots which were kept frozen at −20°C until IFN-gamma concentrations were measured using a commercially available ELISA kit (IFN-gamma-EASIA, Medgenix Diagnostics, Fleurus, Belgium). Results are expressed as IU/ml of IFN-gamma as calculated from a standard curve derived by linear regression of the log-transformed concentrations of the cytokine standards supplied with the ELISA kit versus the respective log-transformed ELISA OD's.

Generation of monocyte-derived dendritic cells and measurement of primary T-cell response in vitro

Monocyte-derived dendritic cells were generated as previously described ( Jonuleit et al, 1997 ). In brief, peripheral blood mononuclear cells were resuspended in complete medium supplemented with 1% heat-inactivated (30 min, 56°C) pooled human AB serum and cultured in six-well tissue culture plates (Greiner, Kremsmünster, Austria) at a density of 5 × 106 cells per well in a final volume of 3 ml for 45 min at 37°C in a CO2 incubator. The non-adherent cells were then removed by careful washing the cells twice with warm PBS without Ca2+ and Mg2+, taking care not to remove loosely attached monocytes. The plastic-adherent cells (consisting mainly of CD14+ monocytes) were further incubated in RPMI suppl. containing 1% AB serum, GM-CSF (recombinant human GM-CSF, rhGM-CSF, Genzyme, Cambridge, Mass., final concentration 800 U/ml) and IL-4 (recombinant human IL-4, Genzyme, 103 U/ml). Fresh cytokines were added every 48 h without changing the culture medium. After 6 d most cells had developed a dendritic cell-like morphology; to induce further maturation the cells were cultured for 24 h in the presence of rh-IL-1β (10 ng/ml), rh-IL-6 (1000 U/ml), rh-TNFα (10 ng/ml) (all cytokines purchased from Genzyme) and PGE2 (Calbiochem-Novabiochem Corp., La Jolla, Calif.). On day 7, the cells could easily be removed from the plastic tissue culture plate with a rubber policeman.

The primary T-cell response to KLH in vitro was examined in splenectomized individuals and healthy controls using two different experimental systems. First, primary T-cell stimulation was induced in the presence of isolated dendritic cells as previously described ( Sallusto & Lanzavecchia, 1994; Mehta-Damani et al, 1995 ). Briefly, monocyte-derived dendritic cells (104 cells/well, generated as described above) were cocultured in triplicate with autologous, highly purified CD4+ T cells [105 cells/well, isolated using immunomagnetic purification with beads (Dynal AS, Oslo, Norway) as previously described ( Thon et al, 1997 ) and kept at −80°C during the 8 d culture required for the generation of dendritic cells] in the presence of keyhole limpet haemocyanin (KLH, Sigma-Aldrich Corporation, St Louis, Mo., Cat. No. H7017, 18H4835, final concentration 100 μg/ml) in flat-bottomed microtitre plates (Falcon 3070, Microtest III, Becton Dickinson Labware, Lincoln Park, N.J.) containing a total volume of 0.2 ml of complete medium supplemented with β-mercaptoethanol (50 μm, Sigma-Aldrich Corp.). Control cultures of dendritic cells and T cells were set up in the presence of recall antigen (tetanus toxoid, Swiss Serum Institute, Bern, Switzerland, final concentration 10 LF/ml) or medium alone; furthermore, parallel cultures contained dendritic cells alone or purified T cells alone. After 7 d of incubation, the proliferative T-cell response was assessed by measuring 3H-thymidine incorporation (7.4 kBq 3H-thymidine/well). Second, primary T-cell responses to KLH were examined according to the method as described by Plebanski et al (1992 ). In brief, peripheral blood T cells were isolated from splenectomized individuals and healthy controls by depletion of plastic-adherent cells and rosetting with 2-aminoethylisothiouronium bromide (Sigma-Aldrich Corp., Cat. No. A-5879)-treated sheep erythrocytes ( Fischer et al, 1993 ) and cocultured for 9 d with autologous mitomycin C (Sigma-Aldrich Corporation, Cat. No. M-0503, final concentration 50 μg/ml) treated peripheral blood mononuclear cells as antigen presenting cells in the presence of KLH (20 μg/ml) or medium alone before the resulting proliferative response was assessed by measuring 3H-thymidine incorporation (10.73 kBq 3H-thymidine/well).

Flow cytometry

The percentage of peripheral blood lymphocyte subpopulations was determined by conventional dual-colour direct immunofluorescence staining of freshly drawn whole blood using commercially available monoclonal antibodies purchased from Becton Dickinson and evaluated with a FACScan cytofluorograph (Becton Dickinson, San Jose, Calif.) according to standard methodology. The following directly conjugated monoclonal antibodies were used: anti-human Leu-2a (CD8, mouse IgG1, clone SK1), anti-Leu-3a (CD4, mouse IgG1, clone SK3), anti-human Leu-4 (CD3, mouse IgG1, clone SK7) ( Ledbetter et al, 1981 ); anti-leu-12 (CD19, clone 4G7) ( Meeker et al, 1984 ); anti-leu-18 (CD45R(A), clone L48, mouse IgG1, reactive with the high molecular weight, 220 kD isoform of CD45, the same antigen as detected by monoclonal antibody 2H4) ( Ledbetter et al, 1985 ; Morimoto et al, 1985 ; Trowbridge & Thomas, 1994); anti-human Leu-45RO (CD45RO, clone UCHL-1, mouse IgG2a, detecting the lower molecular weight, 180 kD isoform of CD45) ( Norton et al, 1986 ; Akbar et al, 1988 ; Smith et al, 1986 ; Trowbridge & Thomas, 1994); anti-HLA-DR (monomorphic, clone L243, mouse IgG2a) ( Lampson & Levy, 1980; Robbins et al, 1987 ); anti-Leu-19 (CD56, clone MY31, mouse IgG1) ( Lanier et al, 1986 ; Hercend et al, 1985 ). For use as a staining control, directly conjugated mouse monoclonal antibodies of the respective isotype (IgG1 or IgG2a, clones X40 or X39, respectively) directed against KLH, an antigen not expressed on human cells, were purchased from Becton Dickinson. Specific and control monoclonal antibody concentrations used for direct immunofluorescence staining were matched. Cells positive for a given antigen were defined by immunofluorescence staining intensity on a logarithmic scale that was higher than that of > 99% (typically higher than that of 99.5–99.9%) of cells incubated with the respective isotype control, and horizontal and vertical markers were set accordingly on dual-colour immunofluorescence dot plots. White blood cell counts were determined using a Coulter Z1 cell counter (Coulter Electronics Ltd, Luton, Beds., U.K.), and a white cell differential count was done by microscopy or flow cytometry to calculate the absolute numbers of the respective lymphocyte subpopulations per litre.

Statistics

Statistically significant differences among the three study groups were determined by calculating the nonparametric Kruskal-Wallis one-way ANOVA by ranks. For statistical evaluation of the difference between two study groups, the nonparametric Mann-Whitney U-test was employed. A difference was considered to be statistically significant at a level of P < 0.05. Spearman analysis of correlation was calculated to analyse the relationship between levels of CD4+CD45RA+ cells and serum levels of hepatitis A antibodies, time since splenectomy, age at examination or age at splenectomy.

RESULTS

Decreased levels of CD4+CD45RA+ T cells after post-traumatic splenectomy

The results presented in Table I confirm and extend previous studies in asplenic children ( Wang et al, 1988 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991) by showing a significant effect of the absence of the spleen on the distribution of peripheral blood lymphocytes in otherwise healthy individuals. Healthy adults investigated many years after post-traumatic splenectomy showed a decrease in the percentage of CD4+ cells as compared to age- and sex-matched healthy volunteers (study controls) or age- and sex-matched healthy individuals who had undergone abdominal surgery because of severe abdominal trauma, but without resection of the spleen (surgery controls). This decrease in CD4+ cells was accompanied by a corresponding decrease in the percentage of CD3+ cells. In contrast, leucocyte counts and percentages of B cells, NK cells, activated (HLA-DR-expressing) T cells and CD8+ T cells were comparable in splenectomized and control individuals ( 1 Table I). Although the difference was statistically not significant as examined by multivariate analysis, there were slightly higher levels of lymphocyte counts in the splenectomized individuals as compared to the controls, and, accordingly, absolute numbers of CD3 and CD4 positive cells were comparable in splenectomized individuals and controls, whereas absolute numbers of other lymphocyte subsets examined (i.e. lymphocytes expressing CD8, CD19 or CD56) were even slightly increased.

Table 1. Table I. Distribution of lymphocyte subpopulations in healthy adults examined many years after post-traumatic splenectomy. Lymphocyte subpopulations were determined in whole blood by dual-colour immunofluorescence flow cytometry. Values given represent mean ± SD. n.s. = the difference between splenectomized individuals and controls was statistically not significant as evaluated using the Kruskal-Wallis one-way ANOVA by ranks.* Normal values were obtained in 239 healthy adult blood donors.† For statistical analysis the three groups were compared using the non-parametric Kruskal-Wallis one-way analysis of variance. Non-parametric comparison of two study groups, healthy controls and splenectomized individuals, was performed using the Mann-Whitney U-test (P values in parentheses).Thumbnail image of

The results presented in Fig 1 further extend previously described effects of splenectomy on the distribution of T-cell subpopulations ( Wang et al, 1988 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991) by demonstrating for the first time that the reduction in the percentage of CD4+ T cells observed following splenectomy is mainly due to a decrease in the subset of CD4+ cells coexpressing CD45RA, the CD4+ T-cell subset mainly involved in response to a novel antigen. The reduction in the percentage of cells expressing CD4+CD45RA+ was statistically significant when compared to either the study controls or the surgery controls examined in parallel, and was observed on two independent occasions more than a year apart, thus indicating a true long-lasting reduction in this T-cell subset (Fig 1). Absolute numbers of CD4+CD45RA+ cells were slightly decreased following splenectomy, but the difference to the controls was statistically not significant (CD4+CD45RA+ cells × 109/l, mean ± SD: study controls 0.53 ± 0.31, surgery controls 0.57 ± 0.16, splenectomy 0.43 ± 0.24, P > 0.05). In contrast to CD45RA+ CD4+ cells, levels of CD4+ T cells coexpressing CD45RO were comparable in splenectomized individuals and controls (Fig 1). No statistically significant correlation was found between the level of CD4+CD45RA+ cells and the time since splenectomy, the age at examination or the age at splenectomy (Fig 2).

Figure 1.

0.7 ± 0.27, Sp °2 0.89 ± 0.45. *Statistically significant decrease in splenectomized individuals as compared to the controls (non-parametric one-way analysis of variance by Kruskal-Wallace analysis: %CD4+, P = 0.002; %CD4+CD45RA+, P = 0.005; Mann-Whitney U-test, splenectomized individuals versus healthy adult volunteers: %CD4+, P = 0.0006; %CD4+CD45RA+, P = 0.005).

Figure 2.

Fig 2. Analysis of the level of CD4+CD45RA+ cells versus time since splenectomy, age at examination and age at splenectomy. Levels of CD4+CD45RA+ cells were examined in the peripheral blood of splenectomized individuals by flow cytometry as described in Materials and Methods. No statistically significant correlation was found between levels of CD4+CD45RA+ cells and time (months) since splenectomy (upper panel), age at examination (middle panel) or age at splenectomy (lower panel), as calculated using Spearman analysis of correlation.

T-cell response to mitogens, CD3 stimulation, recall antigen or primary antigen after post-traumatic splenectomy

The results presented in 2 Table II show that lymphoproliferative responses of purified mononuclear cells stimulated with plant mitogens (PHA, ConA, PWM) or monoclonal antibody against CD3 (OKT3) employed at optimal concentrations were slightly lower in splenectomized individuals as compared to the controls, although the difference did not reach statistical significance. In addition, no statistically significant difference between splenectomized individuals and controls could be found when IFN-gamma production following stimulation with mitogens or CD3-mAb was examined ( Table II). Furthermore, both T-cell proliferation and IFN-gamma production in response to optimal concentrations of a purified protein recall antigen (tetanus toxoid) or a bacterial recall antigen (heat-inactivated E. coli) were comparable in splenectomized and control subjects ( Table II; all study individuals tested had a history of previous tetanus toxoid immunization). However, the capacity to mount a primary T-cell response to a neoantigen in vitro was significantly impaired following splenectomy. The results presented in Fig 3 show that T cells isolated from splenectomized individuals show a significantly lower proliferative response to stimulation with keyhole limpet haemocyanin (KLH) employed at an optimal concentration. This could be demonstrated in two different, previously described, experimental systems, using either mitomycin-C-treated peripheral blood mononuclear cells containing blood-derived dendritic cells ( Plebanski et al, 1992 ) or monocyte-derived dendritic cells generated in the presence of cytokines such as GM-CSF and IL-4 ( Jonuleit et al, 1997 ) as antigen-presenting cells, and purified T cells or the isolated CD4+ T-cell subset as responder cells, respectively (Fig 3).

Table 2. Table II. Lymphoproliferative responses following stimulation with mitogens (PHA, ConA, PWM), CD3 monoclonal antibody or recall antigens (E. coli, tetanus toxoid). Freshly isolated peripheral blood mononuclear cells (MNC) were stimulated with CD3 monoclonal antibody (OKT3, 10 ng/ml), PHA (1:1250) or ConA (12 μg/ml) for 3 d and PWM (1:1000), heat-inactivated E. coli (5 × 106 bacteria/ml) or tetanus toxoid (10 Lf/ml) for 7 d. As a control, parallel cultures were incubated in the presence of medium alone. Lymphoproliferative responses were then assessed by measuring 3H-thymidine incorporation. For examination of IFN-gamma release, parallel cultures of MNC were stimulated for 1, 3 or 7 d with antigen or mitogen, as indicated, and cytokine release was then determined in cell-free supernatants by ELISA. Values represent mean ± SEM. No statistically significant difference could be found between splenectomized and control individuals. Thumbnail image of
Figure 3.

21 ± 14809, P = 0.147).

Decreased antibody response to primary immunization with a viral antigen, hepatitis A vaccine, after post-traumatic splenectomy

To further investigate whether the impairment in primary immune responsiveness observed in vitro could have functional consequences for a primary immune response in vivo, the capacity to respond after immunization with a T-cell-dependent neoantigen, commercially available hepatitis A vaccine, was examined in hepatitis A seronegative splenectomized individuals (n = 8) and controls (n = 6). Hepatitis A serum antibody levels were measured before and after a first and a second immunization. The results shown in Fig 4 demonstrate that absence of the spleen was associated with an impairment in primary antibody responses to this clinically relevant T-cell-dependent viral antigen. As compared to the controls vaccinated and tested in parallel with the splenectomized individuals, significantly lower levels of serum antibodies against hepatitis A were found in splenectomized study subjects after the first and after the second hepatitis A vaccination. In addition, a statistically significant correlation was found between the percentage of peripheral blood CD4+CD45RA+ T cells and the level of serum antibodies after the second hepatitis A vaccination when both splenectomized and control individuals were analysed together (P < 0.05, Fig 5).

Figure 4.

5RA+ cells within the splenectomy group (27%).

Figure 5.

).

In contrast to the observed impairment in responsiveness to primary immunization with hepatitis A virus antigen, the antibody response of splenectomized individuals following booster vaccination with a bacterial protein antigen appeared to be normal. All study subjects had a history of vaccination against tetanus toxoid, with booster immunizations as recommended (i.e. every 5–10 years) before the study. Serum levels of antibodies against tetanus toxoid were comparable between splenectomized individuals and controls, indicating that if primary immunization occurred very early in life, well before splenectomy, antibody responses to booster immunization are most probably unaffected by the absence of the spleen (antibody levels against tetanus toxoid (IE/ml, ELISA, mean ± SEM, min–max [n]): controls, 2.66 ± 0.58, 0.22–7.2 [13]; surgery controls, 2.81 ± 0.51, 0.9–5.6 [11]; splenectomized individuals, 2.25 ± 0.42, 0.1–5.10 [12]).

DISCUSSION

Expression of different isoforms of the common leucocyte antigen CD45 characterizes functionally different subsets of CD4+ T cells. Expression of the higher molecular weight isoform, CD45RA, identifies mainly CD4+ T cells that have been released from the thymus into the periphery but have not yet encountered antigen, so-called naive CD4+ T cells, whereas the low molecular weight isoform, CD45RO, is expressed on memory CD4+ T-cells, i.e. T cells previously activated by antigen. Several functional characteristics distinguish CD45RA+, functionally naive, unprimed CD4+ T cells from CD45RO+ antigen-primed CD4+ T cells: for example, CD45RA+ CD4+ T cells show a higher proliferative capacity in response to mitogenic stimulation and are the CD4+ T-cell subset responding in an in vitro primary T-cell stimulation, e.g. with KLH ( Plebanski et al, 1992 ; Mehta-Damani et al, 1995 ; Young et al, 1997 ); in contrast, CD45RO+ CD4+ T cells produce a wider variety and increased amounts of T-cell cytokines such as IFN-gamma, IL-4, IL-5 and are the predominant CD4+ T-cell subset responding to recall antigens (e.g. Tet Tox) in vitro ( Sanders et al, 1988 ; Akbar et al, 1991 ).

The present study confirms previous reports in children splenectomized for haematological disorders ( Wang et al, 1988 ), in children ( Kreuzfelder et al, 1991 ) or adults ( Müller et al, 1988 ; Ferrante et al, 1987 ; Tsai et al, 1991 ) splenectomized for severe abdominal trauma and in children with congenital asplenia ( Wang & Hsieh, 1991) showing that absence of the spleen leads to a reduction in the percentage of CD4+ T cells. In addition, the present findings demonstrated for the first time that this reduction in CD4+ T cells following splenectomy was due to a selective decrease in the CD4+CD45RA+ T-cell subset, while the distribution of the reciprocal subset of CD4+ T cells, characterized by the expression of CD45RO, is unchanged. As has been previously described ( Wang et al, 1988 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991; Müller et al, 1988 ; Ferrante et al, 1987 ), a clear trend towards lymphocytosis was observed in the splenectomized group, which could best be explained by the absence of the spleen as a reservoir for circulating lymphocytes, and this increase in absolute lymphocyte counts partially compensated for the relative reduction in CD45RA+CD4+ T cells.

The exact mechanism(s) leading to the observed selective and long-term reduction in CD4+ CD45RA+ T cells following splenectomy remain(s) to be investigated. However, several circumstances of the present study indicate that this finding is related only to the absence of the spleen. The study was carried out in otherwise healthy adults splenectomized because of severe abdominal trauma, so that an influence of mechanisms affected by underlying disease or immunosuppressive therapy could be ruled out. Furthermore, the observed reduction of CD4+CD45RA+ cells was a long-term consequence of the absence of the spleen and not related to the acute circumstances that accompanied splenectomy, since it was found many years after post-traumatic splenectomy on two independent occasions more than a year apart, and was evident as compared both to healthy volunteers and to a control group that underwent abdominal surgery with spleen-conserving measures in the same time period before the study.

The spleen is a normal haemopoietic organ during fetal life, and the potential for splenic haemopoiesis remains and occurs postnatally under certain circumstances (e.g. thalassaemia, osteopetrosis). However, the role of the spleen in human T-cell development is largely unknown. A selective decrease of the CD45RA+ subset of CD4+ T cells has previously been described in other patient populations ( Mackall et al, 1995 ; Roederer et al, 1995 ; Bonyhadi et al, 1993 ; Watanabe et al, 1997 ), and has been ascribed to dysfunction of the thymus, known to be an important source of CD45RA+ CD4+ T cells ( Fuji et al, 1992 ; Pilarski et al, 1989 ). Following intensive chemotherapy in patients with neoplastic disorders, regeneration of CD4+ T cells is brought about by the thymus-dependent reappearance of the CD45RA+ CD4+ T-cell subset ( Mackall et al, 1995 ). In HIV-infected individuals a progressive loss of naive T cells of both the CD4 and CD8 lineage has been found, which could be due to HIV-induced destruction of the thymus, thereby removing a potentially important source of naive T-cell regeneration ( Roederer et al, 1995 ; Bonyhadi et al, 1993 ). A severe and long-term impairment in the regeneration of naive CD4+ and CD8+ T cells has recently been demonstrated in patients treated for Hodgkin's disease ( Watanabe et al, 1997 ) and has been explained by destruction of the thymus following mediastinal irradiation. However, the majority of patients in this study also received more extensive irradiation above and below the diaphragm, which also includes splenic irradiation known to lead to long-term functional hyposplenia ( Coleman et al, 1982 ). Furthermore, it was not stated how many of the patients underwent splenectomy, so that participation of a dysfunctional or absent spleen in the mechanisms leading to the observed long-term reduction of naive T cells in patients treated for Hodgkin's disease cannot be ruled out completely.

A recent study showed that in the mouse the spleen is greatly enriched for early T-cell precursors capable of repopulating the thymus and the intestine of lethally irradiated mice ( Hamad et al, 1995 ). Therefore it is feasible to speculate that the observed long-term decrease in CD45RA+CD4+ cells is related to a possible role of the spleen in the generation and/or differentiation of naive unprimed T cells or their precursors. However, other explanations cannot be excluded. A shortened half-life and/or accelerated destruction rate of CD4+CD45RA+ cells in the absence of the spleen would suggest a role for the spleen in the maintenance and/or regeneration of appropriate levels of this CD4 subset. Furthermore, recent evidence indicates that antigen-primed T cells can revert back to a CD45RA+ phenotype of quiescent, slow-responding (i.e. functionally naive), long-living memory T cells (reviewed in Bell et al, 1998 ); in the absence of the spleen, the rate of reversion to this memory T-cell phenotype could be decreased, pointing towards a role for the spleen in the maintenance of normal levels of long-living memory T cells. Alternatively, decreased levels of CD45RA+CD4+ cells following splenectomy could be due to an accelerated differentiation of naive into memory CD4+ T cells. Several findings of the present study do not support this hypothesis. The percentage of activated (HLA-DR positive) T cells was normal, thus giving no indication for an increased rate of differentiation from CD45RA+ naive to CD45RO+ primed T cells. Furthermore, the level of CD45RO+CD4+ cells was unchanged, and the reduction in CD4+CD45RA+ cells was accompanied by a decrease in the percentage of CD4-positive and CD3-positive cells, thus suggesting a true reduction in the CD45RA+CD4+ cell subset rather than a mere interchange between the CD4+ cell subpopulations. Our findings also make a decrease in the surface expression of the CD45RA molecule unlikely as the only explanation for the observed decrease in CD45RA+CD4+ cells. Flow cytometric analysis using Leu-18 as the CD45RA-specific monoclonal antibody showed a relatively marked distinction between CD45RA-positive and CD45RA-negative cells in patients and controls, without indication for a down-regulation of CD45RA expression (i.e. decreased fluorescence intensity of Leu-18 staining) in the splenectomized individuals (Fig 1, first part). Furthermore, a change in the distribution of the CD4+ subsets between different body compartments seems unlikely to be the only explanation for the observed decrease in the percentage of peripheral blood CD45RA+CD4+ cells. Naive unprimed lymphocytes emigrate to the periphery from primary lymphoid tissues (bone marrow, thymus) and home to the different secondary lymphoid tissues such as peripheral lymph nodes, Peyer's patches or the spleen, but do not migrate efficiently to nonlymphoid tissue (reviewed in Mackay, 1992; Butcher & Picker, 1996). Thus an increase in CD45RA+CD4+ cells in the peripheral blood rather than a decrease would be expected in the absence of the spleen, analogous to the increase in absolute lymphocyte numbers observed by us and by others ( Wang et al, 1988 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991; Müller et al, 1988 ; Ferrante et al, 1987 ) due to the lack of the organ's reservoir function for circulating lymphocytes.

A previous study suggested a more pronounced effect of splenectomy on cell-mediated immunity in the first 5 years after removal of the spleen ( Melamed et al, 1982 ). In the present study the splenectomized individuals were studied a relatively long time after splenectomy (minimum 4 years and 1 month), and no correlation was found between decreased CD4+CD45RA+ cells and time since splenectomy. Furthermore, no statistically significant correlation could be found between age at splenectomy or age at examination and percentage of CD4+CD45RA+ lymphocytes (Fig 2); however, the decrease in CD4+CD45RA+ lymphocytes seemed to be more pronounced in individuals splenectomized at the age of 23 or older (n = 3, % CD4+CD45RA+ lymphocytes, mean ± SEM, 9.7 ± 1.2), as compared to those splenectomized between the age of 12 and 22 (n = 9, 15 ± 1.8, P = 0.047, Mann-Whitney U-test). Although the present study did not not include individuals splenectomized very early in life, these findings could indicate that the loss of the CD4+CD45RA+ subset following splenectomy is more pronounced in older individuals whose thymus has largely become atrophic. An age-dependent effect of splenectomy on CD4+ subset distribution is further supported by a previous study reporting a different effect of splenectomy on CD4+ subset distribution if removal of the spleen occurred early in childhood, leading to a more pronounced decrease of the CD4+CD29+ (i.e. essentially the CD45RA) subset ( Kreuzfelder et al, 1991 ). Age-related variations of the effect of splenic loss on lymphocyte subpopulations could also explain the discordant results obtained in some of the previous studies reporting normal absolute and relative numbers of lymphocyte subpopulations in otherwise healthy adults splenectomized after abdominal trauma ( Balsalobre & Carbonell-Tatay, 1991; Passlick et al, 1991 ); however, final clarification of this point awaits further investigation of the pathophysiologic mechanisms involved.

Functional irregularities in cell-mediated immunity, such as an impaired lymphoproliferative response to mitogenic stimulation, have previously been reported in splenectomized or asplenic patient populations ( Downey et al, 1987 ; Kreuzfelder et al, 1991 ; Wang & Hsieh, 1991). The present study shows only a slight, statistically not significant, decrease in lymphoproliferative responses following mitogenic stimulation with PHA, ConA, PWM or anti-CD3 in the splenectomized population; however, as these stimuli were used at optimal concentrations, a decreased response under suboptimal concentrations cannot be excluded. As compared to the reciprocal CD45RO-expressing CD4+ subset, CD4+CD45RA+ cells are known to mount a more pronounced proliferative response to activation with mitogenic stimuli ( Sanders et al, 1988 ; Akbar et al, 1991 ). A decrease in this subset could therefore play a role in the decreased mitogen-induced lymphoproliferative responses observed as a trend in this study or as a statistically significant effect in other populations with anatomical asplenia ( Downey et al, 1987 ). Previous studies also showed defective cutaneous DTH reaction to recall antigens ( Balsalobre & Carbonell-Tatay, 1991) and decreased levels of CD4+CD29+ T cells known to respond to recall antigens ( Kreuzfelder et al, 1991 ) in children after post-traumatic splenectomy. In the present study, functional tests of cell-mediated immunity that are known to involve mainly the CD4+CD45RO+ memory T-cell subset, such as lymphoproliferative responses or IFN-gamma production to recall antigens (e.g. tetanus toxoid, E. coli bacterial antigen) were comparable between splenectomized and control individuals, as were levels of CD4+CD45RO+ cells. The apparent discrepancy between these results and previous findings by others could be due to different marker combinantions used to define the CD4+ memory T-cell subset, due to different read out systems applied to examine T-cell responses to recall antigen (cutaneous DTH reaction versus in vitro lymphoproliferative responses) or due to the different age groups studied; furthermore, it cannot be excluded that a decreased response to recall antigens could become evident if suboptimal concentrations were employed.

In a recent study the importance of the spleen for the development of a primary CD4+ T-cell response was demonstrated in a mouse model of respiratory infection with influenza virus ( Tripp et al, 1997 ). Although in this model the infection is localized to the mucosal surface and the immune response mainly takes place in the regional lymph nodes, the spleen probably plays a role in the blood-borne dissemination of antigen and/or antigen-loaded APCs. Removal of the spleen and disruption of the normal localization of the primary immune response to the draining lymph nodes at the level of CD62L, the lymph node homing receptor expressed on naive T cells, allowed the bone marrow to function as a site of primary immune responses. Interestingly, this mainly compensated for the development of a CD8+ CTL response, while the total number of antigen-specific CD4+ T-helper precursors was still decreased. The present study provides further evidence for a possible role of the spleen in primary immune responses to T-cell dependent antigens. As a functional correlate for the observed decrease in CD4+CD45RA+ cells, the CD4+ T-cell subset responding in an in vitro primary T-cell stimulation ( Plebanski et al, 1992 ; Mehta-Damani et al, 1995 ; Young et al, 1997 ), splenectomized individuals showed an impaired primary T-cell response in vitro to a novel antigen, keyhole limpet haemocyanin (KLH). Two different experimental systems employing different types of APCs, mitomycin-C-treated peripheral blood mononuclear cells ( Plebanski et al, 1992 ) or monocyte-derived dendritic cells generated in the presence of cytokines such as GM-CSF and IL-4 ( Jonuleit et al, 1997 ), gave essentially the same results. Furthermore, the capacity to present recall antigen (tetanus toxoid) and the expression of MHC class II or co-stimulatory molecules such as CD80, CD83 and CD86 (preliminary results, data not shown) was comparable in monocyte-derived dendritic cells from splenectomized individuals and controls, thus indicating a defect at the level of the responding CD4+ T-cell subset rather than an impairment at the level of the antigen-presenting cell.

IgM is the main immunoglobulin isotype produced during primary encounter of the immune system with a new antigen, and decreased levels of serum IgM have been reported following splenectomy ( Eibl, 1985), which could reflect an impairment in the capacity to respond to novel antigens. A decreased primary antibody response to a T-cell-dependent antigen has been previously observed in splenectomized individuals ( Sullivan et al, 1978 ), but an experimental antigen (bacteriophage φX174) was used and applied intravenously, which is known to affect antibody responses after splenectomy ( Hosea et al, 1981a ). The present study extends these previous observations by showing an impairment in primary antibody responses in splenectomized individuals using a clinically relevant T-cell-dependent antigen, inactivated hepatitis A virus vaccine, which was applied by intramuscular injection, thus providing evidence for a possible functional in vivo relevance of the observed abnormalities in CD4 subset distribution in asplenic individuals. Hepatitis A vaccine was choosen as a model antigen for primary immunization in the present study, because it is a clinically relevant viral antigen and vaccination had a clear potential benefit for the study individuals. Although it cannot be ruled out completely that the decreased antibody response observed following primary immunization is due to impaired B-cell function, the concomitant observation of an impaired primary T-cell response in vitro makes a defect at the T-cell level more likely; furthermore, IgG antibody responses to a T-independent bacterial antigen, pneumococcal polysaccharide (data not shown), and IgG-antibody titres to tetanus toxoid examined after repeated immunizations at regular intervals were comparable in splenectomized and control individuals, indicating that the observed impairment in IgG antibody production was limited to primary T-dependent immune responses. A statistically significant correlation between decreased relative levels of CD4+CD45RA+ T cells and reduced HAV antibody responses was found when splenectomized and control individuals were analysed together (Fig 5), whereas absolute numbers of CD4+CD45RA+ cells did not correlate with HAV antibody levels (rho = 0.3077, P > 0.05, data not shown). A trend towards lymphocytosis and a slight, statistically not significant, increase in the absolute number of other lymphocyte subsets (e.g. CD8-, CD19- or CD56-positive cells, Table I) was observed following splenectomy, and could theoretically be involved in the observed impairment in primary immune responsiveness. Although this possibility cannot be totally excluded, a significant impairment in T-cell responsiveness to the neoantigen KLH could be confirmed using isolated CD4+ T cells (Fig 3, lower panel), which can best be explained by a relative reduction of CD4+CD45RA+ cells within the CD4 population, as compared to the unchanged percentage of the reciprocal memory CD4+ cell subset. The observed statistical correlation between reduced relative levels of CD4+ CD45RA+ and reduced antibody titres following immunization with a primary antigen could thus reflect a disturbance in the relation between memory and naive CD4+ T-cell subsets as present in the secondary lymphoid tissues, the principal site of induction of an immune response. Even though our findings could point towards a more general association between levels of CD45RA+CD4+ T cells and primary immune responsiveness, further studies are required to confirm this observation. In addition, further studies have to be performed to investigate a possible relevance of the present findings for the duration and magnitude of immune responses and protection following hepatitis A vaccination in splenectomized individuals. Although antibody levels after the first and second vaccination were lower in splenectomized individuals as compared to the controls, they increased after the second immunization, indicating that the decreased antibody levels observed following primary immunization in the absence of the spleen could be normalized by repeated booster immunizations.

Acknowledgements

The authors wish to thank Smith Kline-Beecham Biologicals, Rixensart, Belgium, Pasteur Mérieux Connaught Austria GmbH, Vienna, Austria, and Chiron-Behring GmbH & Co., Vienna, Austria, for the generous provision of vaccines for immunization studies in immunocompromised populations. The authors are grateful to Andreas Eder and Bernhard Höcher for assistance with the flow cytometric analysis and to Milada Miricka and Eleonore Gschaider for expert technical assistance.

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