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Keywords:

  • Cutaneous lymphocyte antigen;
  • B lymphocytes;
  • Skin;
  • Mucosa;
  • Secondary immunization

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunizationwith Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA+ cells was 13–21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA+, while oral and rectal immunizations were followed by only 1% of CLA+ ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34–48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9–14% were CLA+. In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA+ cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders.

Abbreviations:
CLA:

Cutaneous lymphocyte antigen

ISC:

Immunoglobulin-secreting cell

ASC:

Antibody-secreting cell

PLN:

Peripheral lymph node

HR:

Homing receptor

ELISPOT:

Enzyme-linked immunospot assay

TT:

Tetanus toxoid

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The targeting of immune mechanisms, both protective and pathological, in the body depends on an elaborate system of differential homing of lymphocytes in the body 17. Conventional virgin lymphocytes are known to recirculate relatively homogeneously through secondary lymphoid tissues [Peyer's patches, peripheral lymph nodes (PLN), tonsils, spleen etc.], while being able to extravasate only poorly at extralymphoid sites such as the skin 17. Virgin-to-effector cell transition of lymphocytes has been found to change the migratory behavior of lymphocytes in two main respects: the cells gain tissue-selective homing properties and some populations obtain the ability to efficiently localize in extralymphoid tissues, too.

The tissue selectivity of homing is regulated in large part at the level of lymphocytes recognizing post-capillary venular endothelial cells (EC) via the interaction of differentially expressed lymphocyte homing receptors (HR) and their EC ligands 17. Moreover, adhesion molecules mediating specific retention of lymphocytes in particular tissue microenvironments may also contribute to the tissue selectivity of immune mechanisms 79. Lymphocyte HR/EC adhesion molecule pairs regarded to participate in tissue-selective homing of lymphocytes in humans include the PLN HR, L-selectin and PLN addressin (PNAd) 1012; the gut HR, α4β7 integrin and mucosal vascular cell addressin (MAdCAM)-1 1315; and the skin-selective HR, cutaneous lymphocyte antigen (CLA) and E-selectin 16, 17].

CLA is defined by the rat monoclonal antibody (mAb) HECA-452 and is regarded to serve as the major T cell ligand for the vascular adhesion molecule E-selectin 16, 17. The CLA/E-selectin interaction is required for efficient T cell location in skin. As compared to peripheral blood, memory T cells expressing CLA have been shown to be highly enriched in both resting and inflamed skin, but not at other lymphoid or extralymphoid sites 1822. Antigen-specific T cells responsible for skin-associated immune reactions are preferentially found in the CLA+ fraction of memory cells 23, 24, and CLA is expressed by skin-homing neoplastic cells of the mycosis fungoides and adult T cell leukemia, but not extracutaneous T cell malignancies 25, 26. In the great majority of studies, the expression of CLA has been investigated on T cells, whereas research on its expression on B cells is sparse.

To our knowledge, there are no in vivo data on the regulation of CLA expression on effector B cells. To this end, we set forth to study the expression of CLA on circulating antigen-specific antibody-secreting cells (ASC) induced by antigen encounter at different sites of the body. The expression of CLA was found to depend on the site of antigen encounter and, interestingly, to be up-regulated upon intestinal antigen re-encounter.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Outlining the results

In the present study the CLA expression was studied on both total immunoglobulin-secreting cells (ISC) and antigen-specific ASC. The ISC at a given moment are regarded to consist of numerous populations of recently activated ASC, as a given ASC population is known to be found in the circulation only transiently 2730. Hence, in the present study, the examination of HR on ISC reveals the general profile of circulating effector B cells of the individual, whereas investigation of specific ASC allows the analysis of the influence of various factors (site of antigen encounter, nature of the antigen, primary vs. secondary encounter) on CLA expression of effector B cells.

2.2 General characteristics of the ASC responses

After both oral and rectal primary vaccinations with Salmonella typhi Ty21a, the responses were in most cases dominated by IgA, consistent with our previous studies 2730. After parenteral primary immunization with Ty21a, in the majority of the cases, IgM dominated the response, while in the tetanus toxoid (TT) group the responses were in all cases dominated by IgG. Consistent with our previous studies with the same Ty21a vaccines 27, 28, the responses to the oral and to the rectal vaccines were of higher magnitude than those to the parenteral vaccine (p<0.05 and p<0.01, respectively; Fig. 1). The geometric mean of Ty21a-specific ASC in the parenterally immunized group was 14/106 PBMC for IgA-ASC, 5/106 cells for IgG-ASC and 15/106 cells for IgM-ASC. In the orally immunized group the respective figures were 75, 18 and 19 ASC/106 cells and in the rectally immunized group, 62/106, 10/106 and 17/106 cells. In the TT group the geometric mean of the TT-specific ASC/106 cells was 23 for IgA-ASC, 1031 for IgG and 8 for IgM. Nine volunteers in the parenterally and nine in the orally immunized group were not available for ASC analysis after primary immunization.

After secondary Ty21a immunization, either oral or parenteral, the number of ASC did not differ statistically significantly from that after primary immunization through the same immunization route (data not shown). This probably reflects the timing of the immunization: our previous studies have shown that the number of ASC elicited by secondary immunization depends on the interval between priming and boosting 31, 32. In the present study a booster effect is revealed by the qualitative changes of CLA expression after secondary immunization (see below).

In the following, the data are presented based on the sum of IgA-, IgG- and IgM-ISC or -ASC found, followed by a separate comparison between the three isotypes. In order to get reliable statistics for the proportions of cells expressing a given marker, we set an exclusion limit of 20 ISC/ASC that needed to be identified among the cells studied.

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Figure 1. Numbers of circulating antigen-specific ASC. The figures represent the sum of IgA-, IgG- and IgM-ASC 7 days after parenteral, oral or rectal immunization with S. typhi Ty21a. The ASC values are given as geometric means ± SEM. The number of individuals in each group is indicated under the column.

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2.3 General profile of HR expression on ISC

CLA was expressed on 13–21% of ISC in the different vaccination groups. When comparing the CLA expression to that of the other HR on ISC, CLA was found to be expressed significantly less frequently than L-selectin (p<0.001) or α4β7 (p<0.001) in all study groups (e.g. in the oral group 13% of ISC were CLA+vs. 62% L-selectin+ or 45% α4β7+); this parallels with our previous results on HR expression on ISC of unimmunized healthy volunteers 33.

2.4 Expression of HR on ASC

2.4.1 Numbers of volunteers providing data for HR analyses

After primary vaccination with Ty21a, the inclusion limit of 20 ASC was exceeded in 14/15 parenterally, in 15/17 orally and in 6/8 rectally immunized volunteers. After booster vaccination with Ty21a, the limit was exceeded in 15/16 parenterally and in 15/18 orally boosted volunteers. The limit was exceeded in all five volunteers receiving the parenteral TT vaccine. The results of HR expression in this study are based on those volunteers that exceeded the inclusion limit.

2.4.2 Expression of CLA

After primary immunization, the CLA HR was expressed on 13%, 1% and 1% of ASC in the parenterally, orally and rectally immunized Ty21a groups, respectively (Fig. 2). The higher expression in the parenterally immunized group was statistically significant (p<0.001 for oral and p<0.01 for rectal immunization groups). The expression of CLA on ASC in the parenterally immunized groups did not differ from that on ISC of the same volunteers (Fig. 3a), while in the orally (Fig. 3b) and in the rectally (Fig. 3c) immunized groups the proportions of CLA+ cells were significantly lower among ASC than among ISC of the same volunteers (p<0.001 and p<0.01 for the respective groups).

After secondary immunization, by contrast, CLA was found to be up-regulated also after intestinal antigen encounter: after oral re-immunization CLA was expressed on 48% and 35% of ASC in the orally and in the parenterally primed groups, respectively (Fig. 4). This proportion of CLA+ ASC was significantly higher than that found after primary immunization either through oral (p<0.001) or parenteral (p<0.05) route. Parenteral re-immunization was followed by 15% and 9% of CLA+ ASC after oral and parenteral priming, respectively (Fig. 4). These proportions were higher than that after oral primary immunization (p<0.001), but similar to that after parenteral primary immunization.

In the TT group, CLA was expressed on 15% of ASC. This proportion was significantly higher than that after oral (p<0.001) or rectal (p<0.01) primary immunization with Ty21a, but similar to that in the parenteral Ty21a groups, regardless of the occasion of the immunization (primary or secondary). These data, suggesting that the nature of the antigen does not have influence on CLA expression, are consistent with previous findings with other HR 34.

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Figure 2.  Expression of HR on circulating specific ASC after parenteral, oral or rectal immunization with S. typhi Ty21a. The bars indicate arithmetic means (± SD) of percentages of specific ASC expressing the skin HR, CLA, the PLN HR, L-selectin, or the gut HR, α4β7. CLA expression is less frequent than that of the other HR and it depends on the immunization route. The number of individuals is indicated under each column. *;*;*;p<0.001; *;*;0.001<p<0.01; *;0.01<p<0.05 by Student's t-test.

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Figure 3. Expression of the skin HR, CLA, on circulating specific ASC and total ISC of the same volunteers 7 days after parenteral (a), oral (b) or rectal (c) vaccination with S. typhi Ty21a. The bars indicate arithmetic means (± SD) of percentages of cells expressing CLA among specific IgA-, IgG- or IgM-ASC and -ISC. The number of individuals from whom the data were pooled is indicated under each column.

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Figure 4. Expression of CLA on circulating antigen-specific ASC after primary and secondary immunization with S. typhi Ty21a through parenteral or oral routes. The bars indicate arithmetic means (± SD) of percentages of cells expressing CLA. Even though oral primary immunization was not able to induce cells with CLA, re-immunization through the same route resulted in significant numbers of CLA+ cells.

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2.4.3 Expression of α4β7

The α4β7 HR was expressed on 44% of ASC after parenteral Ty21a vaccination (Fig. 2). Consistent with our previous studies 28, 35, 36, the expression of α4β7 on ASC induced by oral or rectal immunization (both 98%) was significantly higher than that in the parenterally immunized group.

2.4.4 Expression of L-selectin

L-Selectin was expressed on 78% of ASC after parenteral Ty21a vaccination (Fig. 2). Consistent with our previous studies 28, 35, 36, the expression of L-selectin on ASC induced by oral (29%) or rectal (35%) Ty21a immunization was significantly lower than that on parenterally induced ASC.

2.5 Contribution of the various isotypes to the differences found in HR expression

Separate examination of the three isotypes among ISC (Fig. 3) in the orally and the rectally immunized volunteers revealed that CLA was expressed less frequently by IgA-ISC than by IgG-ISC (p<0.001), consistent with our previous findings 33. Moreover, a comparison between the three typhoid study groups revealed that IgA-ISC in the orally immunized volunteers expressed CLA less frequently than those in the parenterally immunized group (p<0.05), which was probably a reflection of the significant population of typhoid-specific CLA IgA-ASC in the orally immunized volunteers.

The comparisons between the ASC isotypes after vaccinations could not be carried out in all cases, because the ASC responses within a given isotype did not always exceed the exclusion limit of 20 detected ASC. The proportions of CLA+ cells among ASC did not differ between the isotypes in any of the immunization groups (Fig. 3). Likewise, for α4β7 and L-selectin expression on ASC, no differences were found between the isotypes (data not shown).

3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Antigen encounter at the skin is followed by a transportation of the antigen to local lymph nodes, where specific small lymphocytes undergo virgin-to-effector cell transition, then exit the lymph nodes and enter the blood stream via lymphatics. These cells leave the circulation in post-capillary venules of their target tissues with help of the tissue-selective HR on their surface. Homing to cutaneous sites is regarded to be largely mediated by CLA. Consistently, the expression of CLA has been shown to have clinical significance in various skin diseases such as leukemic cutaneous T cell lymphoma 25, cutaneous anaplastic large cell lymphoma 37, or various cutaneous inflammations such as milk-induced eczema 38, allergic contact dermatitis or atopic dermatitis 39.

So far, the great majority of studies on CLA expression on lymphocytes deal with T cells, while documentation on the expression of CLA on human B cells has been sparse. We have previously shown that CLA is expressed by a proportion of circulating ISC 33, and Yoshino et al. 40 and Rott et al. 41 have shown CLA expression on memory B cells. The tiny, but important, populations of final specific effector B cells, the antigen-specific ASC, can currently be investigated only by combining cell sorting with functional analysis of the resulting populations, yet this cell population is expected to provide us front-line information on the regulation of CLA expression in vivo. To our knowledge, the present study is the first to investigate CLA expression on these final effector B cell populations of known specificity. The study describes a differential expression of CLA depending on the site of the primary antigen encounter and demonstrates that re-immunization may change the HR profile of the activated lymphocytes.

Firstly, the general level of CLA expression on circulating activated B cells (ISC) was evaluated. Previous studies on T cells have shown that CLA is expressed on 15% of T cells in the human peripheral blood 18, on 78% of T cells in clinically normally appearing skin 21, 22, 42, on 85% of T cells in cutaneous chronicinflammation and on <5% of T cells in extracutaneous tissues 18. In the present study, about 15% of circulating effector B cells were found to express CLA. Closer examination on the two currently studied cell populations among ISC, antigen-specific ASC induced by mucosal vs. parenteral immunization, reveals that circulating ISC are not a homogeneous populationof cells with respect to their CLA expression, but consist of smaller populations some of which are CLA and some CLA+.

The microenvironment where the antigen encounter takes place was found to have a profound influence on the expression of CLA on B cells. According to the existing dogma of lymphocyte homing, cells tend to home back to sites where the primary contact with their antigen took place 17. The present study is consistent with this dogma: while cells activated at the intestinal mucosa (oral or rectal immunization) were practically all CLA, cutaneous encounter with the antigen (parenteral immunization) resulted in significant numbers of CLA+ cells, indicating a potential of these cells to home to cutaneous sites.

It is noteworthy that even after parenteral immunization not all of the cells seemed to be targeted to the skin, as CLA was expressed on only 10–25% of the specific cells in the parenterally immunized volunteers. Hence, cutaneous sites seem to differ from the intestine as inductive sites: all B cells activated at the intestinal mucosa exhibit homing potentials back to the gut (100% of cells express α4β7) 28, 35, 36, 43, whereas the majority of cells activated at cutaneous tissues seem tobe targeted to extracutaneous sites. This seems to serve the purpose with respect to the different numbers of cells needed for immune defense at these sites as related to their surface area: the intestinal mucosa has been estimated to represent a surface area of 400 m244, while the skin exhibits an area of less than 2 m2. Obviously, excessive accumulationof cells to cutaneous sites could turn out to be harmful.

The intestinal exposure to an antigen elicits circulating specific B cells without a potential to home to the cutaneous tissues, which suggests that the intestinal mucosa is a microenvironmentlacking the conditions required for up-regulation of CLA. Interestingly, however, it appears that antigen encounter at the intestinal mucosa does not always result in total absence of CLA on the activated cells: oral re-immunization was found to result in ASC with a significant proportion of CLA+ cells, even though primary immunization through the same route was unable to do this. This finding is exciting when trying to understand both its causes and its consequences. The principal difference between the secondary and primary immune response is that the effector cells arise from memory cells instead of naive cells. It is plausible to think that this is reflected in the homing profile of the resulting effector B cells. Furthermore, it is possible that the secondary encounter with the antigen in the intestine takes place in conditions that would influence the anatomical site of antigen presentation and consequently the HR expression of the activated cells: these conditions include the presence of antigen-specific effector cells in the lamina propria and secretory antibodies in the mucosa. At this point, the causes of CLA up-regulation after oral re-immunization will need further investigation.

The up-regulation of CLA found after oral re-immunization might have significance in the pathogenesis of cutaneous disorders where an orally encountered antigen is regarded to play a central role: an oral re-encounter with the antigen could result in formation of excessive numbers of specific lymphocytes with homing potentials to the skin, and, consequently, to harmful accumulation of effector cells in the cutaneous tissues. Especially in food allergy, there exists a close association between skin and intestine with skin symptoms predominating in many food allergies 45. Consistently, milk-induced eczema has been shown to be associated with the expansion of CLA+ T cells 38. Accordingly, the induction of CLA on memory B cells may be important for the symptoms of food allergy, routing allergen-specific B cells to close proximity of this target organ. On the other hand, the finding of up-regulation of CLA after intestinal antigen re-encounter might have also therapeutical applications with respect to the possibility of focused targeting of lymphocytes to cutaneous tissues.

The up-regulation of CLA after oral re-immunization is in line with our recent study on homing potentials of ASC after re-immunization (Kantele et al., manuscript in preparation). It demonstrates that oral re-immunization is followed by appearance of ASC with unique HR profile: these orally induced cells expressed both α4β7 and L-selectin in high proportions, while those induced by parenteral re-immunization did not differ from those after primary immunization. The significant up-regulation of L-selectin after oral re-immunization appears concomitant with the up-regulation of CLA demonstrated in the present study. In the present study a simultaneous up-regulation of CLA and L-selectin was also found after parenteral primary immunization. These results are consistent with studies on T cells revealing that there exists a physiological co-regulation of CLA and L-selectin. In fact, the vast majority of CLA+ memory T cells in both blood andskin have been found to be also L-selectin+19, 46.

In conclusion, the present study shows that the expression of CLA on human effector B cells depends on the site of initial antigen encounter. Intestinal mucosa represents a microenvironment lacking the conditions required for up-regulation of CLA expression during virgin-to-effector cell transition of B cells. The subcutaneous tissue, by contrast, seems to favor an up-regulation of CLA expression. After antigen re-encounter, on the other hand, up-regulation of CLA can also take place at the intestinal mucosal, an event with possible significance in the pathogenesis of certain cutaneous disorders. These differential homing potentials achieved by the choice of immunization route offer more accurate grounds for evaluating the effect of the immunization routes used. Moreover, increasing knowledge on the regulation of CLA expression on lymphocytes may permit novel approaches to the diagnosis and therapy of cutaneous and inflammatory disorders.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Outlining the study plan

Examination of HR expression on the minor but important population of the active effector cells among all B cells requires a combination of cell sorting with assessment of the effector function, the Ig secretion, with an enzyme-linked immunospot assay (ELISPOT): investigation of the cells actively secreting Ig let alone antibodies of certain specificity has not been possible with sole flow cytometry.

In the present study the expression of CLA was studied on peripheral blood ISC in general and on circulating specific ASC induced either by oral, parenteral or rectal primary immunization, or by oral or parenteral secondary immunization with a whole-cell vaccine, S. typhi Ty21a, or, for comparison, by parenteral immunization with a protein vaccine, TT. Moreover, after primary immunizations with Ty21a, the expression of CLA was compared to that of other HR (L-selectin, α4β7). The expression all three HR was examined by combining immunomagnetic cellsorting with the ELISPOT assay for all ISC or for S. typhi- or TT-specific ASC.

4.2 Vaccinations and collection of samples

Sixty-three healthy volunteers (age 18–51 years) participated in this study. None of the vaccinees had a history of typhoid fever and none of them had received a typhoid vaccine previously. All of the vaccinees had received TT intramuscularly (i.m.) 5–10 years earlier. Informed consent was received from all participants. The study protocol was approved by the Ethics Committees of the National Public Health Institute, the Hospital for Children and Adolescents, University of Helsinki, and the University of Jyväskylä.

The typhoid vaccine was given to 24 volunteers parenterally, 26 orally and 8 rectally as primary immunization. In the parenterally primed group, a booster vaccination was given to eight volunteers parenterally and to nine volunteers orally, and in the orally primed group, to eight volunteers parenterally and to nine orally. Booster vaccinations were given 3–30 months after the primary immunization. The five volunteers not receiving typhoid vaccines were vaccinated with TT parenterally.

The parenteral Ty21a vaccine was prepared in the Swiss Serum and Vaccine Institute (Bern, Switzerland) according to a previously described protocol 27. The vaccinees were given in the left arm, both as primary and secondary vaccinations, an i.m. injection (0.5 ml) of this vaccine, each dose consisting of 0.5×109 formalin-killed bacteria. As the oral and the rectal vaccine, the live, attenuated S. typhi Ty21a vaccine (Vivotif Berna, Swiss Serum and Vaccine Institute) consisting of at least 2×109–6×109 bacteria perdose was used. The oral vaccine was administered according to the manufacturer's instructions, while the rectal one was recovered from the enteric-coated capsule, suspended in 0.9% NaCl and administered intrarectally with help of a syringe as previously described in detail 28. The oral and rectal immunizations were carried out by giving the volunteers, both as primary and secondary vaccination, three vaccine doses 2 days apart. Both the parenteral and the enteric-coated Ty21a vaccines were kindly provided by Dr. C. Herzog and Dr. E. Fürer (Swiss Serum and Vaccine Institute). The vaccine containing TT and diphtheria toxoid (DT) (Tetanus-D-vaccine) was prepared in the Vaccine Department of the National Public Health Institute, Helsinki. It consisted of 5 Lf TT and 2 Lf DT (immunizing efficacy 40 IU for TT and 4 IU for DT) per 0.5 ml; each of the five vaccinees received 0.5 ml of this vaccine i.m. in the left arm. Samples of heparinized blood were collected from the volunteers 7 days after vaccinations.

4.3 Isolation of mononuclear cells

The mononuclear cells were isolated from the heparinized venous blood by Ficoll-Paque density gradient centrifugation as previously described 29.

4.4 Separation of the receptor-negative and -positive cell populations

The separation of the cells into receptor-negative and -positive populations has been described previously in detail 33, 35, 36. Briefly, the cells were incubated in the first stage with the mAb: anti-CLA (HECA-452; kindly provided by Dr. Eugene C. Butcher, Stanford, CA), anti-α4β7 (ACT-1; kindly provided by Millennium Pharmaceuticals, Cambridge, MA) or anti-L-selectin (Leu8; Becton-Dickinson, San Jose, CA) for 30 min on ice. The cells were then washed twice with 1% FCS-PBS and incubated on ice for 30 min with Dynal® M-450 magnetic beads coated with sheep anti-mouse IgG (Dynal, Oslo, Norway) cross-reactive with rat Ig. The beads with the attached cells were then separated from the suspension by applying a magnet outside the test tubes, and the supernatants with the receptor-negative cells were collected. Next, the beads were washed, the magnetic separation repeated, and the supernatant pooled with the initial population of negatively selected cells. The receptor-positive cells attached to the beads were then suspended in medium. Both the receptor-positive and -negative cell populations were immediately analyzed with ELISPOT for numbers of ISC and for vaccine antigen-specific ASC. The efficiency of the cell separations has been controlled in earlier experiments as previously described 33, 35, 36, 47.

4.5 Assay of specific antibody-secreting cells (ELISPOT)

The receptor-positive and -negative cell populations were each assayed for specific ASC with ELISPOT; the assay of specific ASC has been described in detail previously 29. In brief, 96-well microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) were coated with a whole-cell preparation of formalin-killed Salmonella strain SL2404 (108 bacteria/ml PBS) sharing with the vaccine strain the O-9,12 antigen 29, or with a preparation of purified TT (kindly provided by Rose-Marie Ölander, National Public Health Institute; 0.5 Lf/ml carbonate buffer, pH 9.6). The unspecific binding sites were blocked with 1% BSA in PBS. Then the cells were incubated in the wells for 3 h, and antibodies secreted during this time were detected with alkaline-phosphatase-conjugated swine anti-human IgA, IgM (diluted 1:100; Orion Diagnostica, Helsinki, Finland) or goat anti-human IgG (diluted 1:500; Sigma, St. Louis, MO) antisera followed by application of substrate (5-bromo-4-chloro-3-indolylphosphate; Sigma) in melted agarose. For each Ig isotype surface marker combination, 0.8×106–4.8×106 cells were screened. Specific ASC were enumerated by counting the spots in the wells in a light microscope.

4.6 Assay of immunoglobulin-secreting cells

ISC were enumerated as described in detail previously 29. Microtiter plate wells were coated with human IgA-, IgG- or IgM-specific antisera. The next steps were carried out as described above for specific ASC.

4.7 Statistics

The proportions of the receptor-positive ASC or ISC were calculated as follows: % of receptor-positive cells among ASC or ISC = (100 × the number of ASC or ISC in receptor-positive population) / (sum of the number of ASC or ISC in receptor-positive and receptor-negative populations).

Percentages of cells expressing the different receptors were determined as arithmetic means of the proportions of ASC or ISC expressing the given cell surface marker. Statistical comparisons were carried out using Student's t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The authors thank Professor Sirpa Jalkanen and Professor Heikki Arvilommi for constructive criticism of the manuscript, Dr. C. Herzog and Dr. E. Fürer for providing the typhoid vaccines, Professor Eugene C. Butcher for providing the HECA-452 mAb, Dr. Michael Briskin for providing the ACT-1 mAb, and Dr. Rose-Marie Ölander for providing the preparation of purified TT. This work was supported by the Sigrid Jusélius Foundation and the European Union Contract QLK2-CT-1999–00228.

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