Antigen presenting, contrasuppressor human T cells

Immunology Today, vol. 7, No. 3, 1986

reviewsAntigen presenting,contrasuppressor humanT cells

Two principal human immunoregulatory T cells have been defined. T helper/inducer (Th/i) lymphocytes are recognized by monoclonal antibodies to CD4 (T4 or Leu 3) antigen and T suppressor (Ts) and cytotoxic (Tc) lymphocytes by monoclonal antibodies to CD8 (T8 or Leu 2) antigen• Antigenspecific T h cells and factors are essential for an effective B-cell response in humans 1-s and rhesus monkeys 6,7. Antigen-specific T~ cells and factors have also been described in humans 8 lo and rhesus monkeys 7,11. In most of these investigations helper and suppressor cells and their factors were identified separately. However, helper and suppressor cell activities coexist during primary in-vitro sensitization of human lymphocytes 12 and primary invivo and secondary in-vitro sensitization of human ~° and rhesus monkey lymphocytes 13. The concept of contrasuppression, proposed by Gershon and his colleagues14was first identified in a human context as the best interpretation of a set of observed T4 + and T8 + cell interactions 1° and followed up by a series of cell depletion and reconstitution experiments is 2o• The existence of human T8 + contrasuppressor cells has also been suggested in experiments showing that T8 + cells suppress pokeweed-mitogen-driven immunoglobulin production but amplify it when irradiated T8 + cells are used21. These T8 + augmenting cells may be contrasuppressor cells22. In-vitro studies of helper and suppressor functions To study human T-cell helper and suppressor activities in parallel, T cells are stimulated by antigen in Marbrook-Diener flasks and supernatants are tested for helper activity on direct antibody forming cells, using unprimed mouse (CBA/Ca or B10.BR) spleen cells and dinitrophenylated (DNP) antigen ~°. It is a requirement of this technique that primate helper and suppressor factors cross the species barrier3,~l. To assay supernatant suppressor factor activity, murine antigen-specific helper cells are generated and then cultured with the supernatant from human T cells, murine B cells and antigen 1°. Although several antigens have been used 3'6'11, extensive studies were pursued only with a streptococcal antigen (SA). SA has a molecular weight of 185000, is predominantly protein and rather hydrophobic in nature and has two major determinants SAI/I123. Mononuclear cells from donors lacking DRw6 expression (HLA-DRw6~ released helper activity with 1000 ng m1-1 of SA, whereas suppressor activity was elicited by 1, 10, 100 or 10000 ng m1-1 Department of Oral Immunology and Microbiology, United Medical and Dental Schools of Guy's and St Thomas" Hospitals, London SE1 9RT, UK © 1986, Elsevier Science Publishers B.V., Amsterdam

0167

4919/86/$02.00

T. Lehner SA1° (Fig. 1). Conversely, DRw6 + mononuclear cells generated helper activity with 1 ng ml -~ SA and suppressor activity with 10, 100, 1000 or 10000 ng m1-1 SA. Rather surprisingly, autoradiography ls'16 has shown that binding of 12Sl-SA to DRw6- T cells is maximal with 1000 ng ml -~ SA. This concentration generated helper but not suppressor activity (Fig. 1). DRw6 + T cells showed optimal binding at 1 ng m1-1 SA which generated helper but not suppressor activity. The assay thus established that helper and suppressor functions coexisted, appeared to be antigen dose-dependent and the concentration of SA binding to T cells was the same as that required to elicit helper activity. When T4 + and T8 + subpopulations were separated by negative and positive selection 1 0 ,1 5 ,1 6 , • ' 10 T8-depleted cells lost both suppressor activity and antigen binding capacity 15'16 but retained the helper activity which was elicited by all concentrations of SA (Fig. 1). Removal of T8 + suppressor cells thus enabled T4 + cells to provide help over the range of SA concentrations tested• In contrast, depletion of T4 + cells resulted in loss of helper activityI 0 and the remaining cells had an ability to produce suppressor activity over the entire dose-range of SA used (Fig, 1). The hypothesis postulated to account for the discrete antigen dose-dependent helper activity (1000 ng m1-1 SA) was that T8 +, antigen-binding cells function as contrasuppressor cells which enable T4 + helper cells to break through the dominant T8 + suppressor activity1°'16 To examine these events further unseparated DRw6- mononuclear cells were treated with 1000 ng ml -~ SA and monoclonal anti-SA and complement to remove antigen-binding cells. This resulted in loss of helper activity. However, in the remaining cells, suppressor activity was induced not only by 1 ng ml-', as with unseparated cells, but also by 1000 ng SA. The treatment seemed therefore to have removed T8 + antigen-binding cells which presented antigen to T4 + helper cells and to have led to the loss of helper function. Monocyte depletion also resulted in the loss of helper activity but suppressor activity was induced only by the same dose of SA that induced suppressor activity by the intact population. These findings are consistent with the notion that depletion of T8 + SA-adherent cells not only removes a subset of T8 + SA-binding and presenting cells but also contrasuppressor cells. Two T8 + subsets

These cell depletion studies suggest that there are two subsets of T8 + antigen-binding cells: one

87

Immunology Today, vol. 7, No. 3, 1986

-r'#,1/l#,14/,$ T4+T8

T4

binds and presents anti¢ HeJp gen to T4 + helper cells ._~ 100 80and induces helper activity, the other has a con3 trasuppressor function, for the cells react with ~ Suppre~slon T4 + helper cells to prevent the T8 + suppressor ~ i" ~b'- I ~o~o'"1 subset (a third T8 + subI 100 10,000 I 100 10,000 set) from inhibiting the helper activity. To test this hypothesis the lectin V i c i a v i l l o s a (VV) was selected as this binds ~ Suppress;on murine contrasuppressor .~_ cells 2s. Panning with VV separated T8 + cells into a . 60 T8+VV + (VV-adherent) o 40 subset (19.7 + 3.2%) 2 which bound 12Sl-labelled 2 SA or tetanus toxoid o I~ I lo~o I t I lb I 10'0o I I00 I0,000 1 100 10,000 readily and a T8+VV (W-non-adherent) subset SA (ng per ml) SA (ng per ml) (48.1 ___ 11.4%) of cells Fig. 1. Effect ot killing T cells (DRw5 ) with monoclonal anti-T4 or anti-T8 antibodies and complement, on which bound little antithe helper -- :, suppressor ~ and antigen binding ~ functions to streptococcal antigen gen 19. Furthermore, (5A). The T-ceil functions indicated on the left as 'T4 + T8" are those with untreated mononudear cells. The separation of T or T8 cells functions on the right indicate T8-depleted ('T4') or T4-depleted ('TS') cells. The vertical bars show 1251C,_A binding as the mean grain count per 500 cells. into T8+SA + (adherent; 3.3 + 0.4%) and T8+SA (non-adherent) cells revealed that 12SI-SA binding tively as monocytes 19. No helper activity was obwas confined mostly to the T8+SA + cells 26. served with T8+VV + cells alone or when T4 + helper These separation methods with VV or SA percells were reconstituted with T8+VV cells (Fig. 2). mitted examination of antigen presentation by cell T8+SA + cells could also present SA as effectively as subpopulatjons. Separated or reconstituted ceils monocytes to induce helper activity 2° (Fig. 2). (total of 5 x 1 0 6 cells) were cultured for 1 day with However, fewer T8 + SA + cells (10%) than T8 VV + DNP-SA and the supernatant factors generated cells (20%) were required for antigen presentation, were assayed with mouse spleen cells as described presumably because the former have a greater above. T4 + cells were not induced by SA alone to proportion of SA-specific cells. generate helper activity but require the antigen Both T8 + SA- (Ref. 20) and T8 + VV- (Ref. 19) presenting function of monocytes (Fig. 2). How cells suppressed the helper activity of T4 t cells and ever, T8*VV + cells induced helper activity as effecmonocytes induced by SA (Fig. 3; T8 + VV- data not shown). The T8 +, SA and T8 + VV- cell factors also suppressed mouse helper cells, suggesting that the 400] suppressor cells are induced by SA in the absence of accessory cells, as was found by others 27'28. Indeed, reconstitution of T8 + SA cells with monocytes or T8 t SA + cells had little effect on the suppression generated by T8 + SA cells 2°. This is also important evidence against the possibility that T8 + SA + cells prevent suppression by a direct effect on T8 t SAililiiiii cells. Although T8 + SA cells suppressed the helper VV + SA + V V - S A - ' *Mo** * V V + * * S A + W V - S A activity elicited by SA with T4 + cells and monocytes, T8 + T8 + ,T4 + + T8 + T8 + substitution of T8 + SA + cells (or T8 + VV + cells) for T4 + + + Ratio of monocytes converted the suppressor to a predomiT4:Mo/T8+_.4:1,**4.5:0. 5 T4+ T4 + nantly helper activity (Fig. 3). Contrasuppressor activity thus seemed to reside in the SA t and VV t Fig. 2. Antigen presenting function induced by T8+ VV+ or T8+ SA+ cells, subsets of T8 + cells. Although the same subsets of reconstituted with T4+ cells and SA; the positive controls are Mo with T4+ cells T8 + cells are involved in antigen presentation and and negative controls are T8+ VV- or T8+ SA cells with T4+ cells. T4 + cells were contrasuppression, these functions may be expressreconstituted with T8+ VV+ or T8 + VV or monocytes at a ra#io of 4:1 cells. T4+ ed by different subpopulations of these cells. cells were reconstituted with T8+ SA + or 7-8+ SA or Mo at a ratio of 4.5:0.5. The Cross-over reconstitution experiments between antigen presenting function is expressed as the helper activity tested by the mouse spleen B ceil antibody forming assay. T8 + SA + and T8 + VV + cells on the one hand and

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immunology Today, vol. 7, No. 3, 1985

T8 + SA- and T8 + VV cells on the other 26 suggested that T8 + VV + cells contain antigen binding, presenting and contrasuppressor ceils and that the T8 + SA + cells might be an antigen-specific subset of this population.

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The target for antigen presenting and contrasuppressor cells

The term contrasuppression is sometimes misinterpreted to suggest that the contrasuppressor ceils directly inhibit suppressor cells. This is not found on reconstitution of T8 + SA + with T8 + SA- cells, for the suppression exerted by T8 + SA- cells remains unchanged. However, adding T8 + SA + cells to T8 ÷ SA- cells in the presence of T4 + cells results in helper and little suppressor activity (Fig. 3.), whereas reconstitution of T8 + SA with T4 + cells in the presence or absence of monocytes exerts predominantly suppression. It seems that the target for both the antigen presenting and contrasuppressor functions of T8 + SA+ cells is the T4 + helper cell (Fig. 4). In the mouse the target for contrasuppressor cell is also the Lyt 1+ helper cell 14'2s'29'3°. Nevertheless, the possibility that two different subsets of T4 + cells might act as the targets for T8 + antigen presenting and contrasuppressor cells cannot be excluded. It is pertinent to note that in mice, the Lyt 2 + I-J + cell functions as a contrasuppressor inducer cell which acts via a transducer cell on a Lyt 1+, L3T4 +, I-J + contrasuppressor effector cell 14'25,29. The closest analogy with the human system suggests that the T8 + I-J + cell (see below) might correspond to the Lyt 2 + I-J + contrasuppressor inducer cell and that a T4 + contrasuppressor effector cell might be required for the mouse analogy to be maintained. The target for suppressor activity of the T8 ÷ SA-/VV- cell is also the T4 + helper cell (Fig. 4). The final common pathway of action of T8 + SA+/VV + , T8 + SA /VVcells and monocytes appears to be the T4 + cells but the possibility that B cells might be an additional target has not yet been explored. T8 + cell function specificity

The specificities of antigen binding T8 + cells (Table I(a)), helper or suppressor factor (Table I(b)) and helper, suppressor and contrasuppressor functions (Table I(c)), have been documented elsewhere 15'16'~9'2°'31, and are presented here with reference to SA, keyhole limpet haemocyanin (KLH) or tetanus toxoid (TT). A criticism sometimes raised has been that the technique uses first human T cells which, on activation with antigen, release helper or suppressor factor and these factors are then directed to mouse spleen B cells for antibody formation and Th cells for suppression. In order to counter this we have developed a new assay, using autologous human T cells and B cells (R. Brines and T. Lehner, unpublished). This has confirmed the concept based on the xenogeneic assay that T8 + cells bind and pre-

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Fig.3. Contrasuppressorfunction induced by T8+ SA+ cells is presented as the parallel helper and suppressor activities generated by reconstituted T4+ and T8+ SA- cells with T8+ SA+ cells but not with Mo. T4+ cells were reconstituted with T8+ SA- and either T8+ SA+ cells or monocytes at a ratio.of3:1.5:0.5; the concentration of SA added was 1000 ng ml -~. The helper and suppressor activities were tested by the mouse spleen B ceil antibody forming assay. sent antigen to T4 ÷ cells and may also exert contrasuppressor activity. The rationale for culturing human T cells for only 24 h was based on the assumption that the T cells were sensitized to the SA naturally in vivo, so that only a short antigenic pulse is required in v i t r o to generate the specific helper or suppressor factor 1°. This assumption has now been confirmed in rhesus

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Fig.4. A model for interactions between Mo (monocytes) T8+ AP/CSC(antigen presenting, contrasuppressorcells), T8~ SC(suppressorcells) and T4+ HC (helper cells) on the antibody response of B cells. The antigen binding and classII binding sites are probably part of one molecule (the T-ceilreceptor).

89

Immunology Today, vol. 7, No. 3, 1986

Table I. The specificitiesof: (a) antigenbindingT8+ cellstested by autoradiography:(b) helperand s,Jppressorfactors tested by in~munoadsorption: (c) helperand suppressorfunctions assayedby anti-DNPantibodyforming cells Streptococcalantigen (a) Inhibition of antigenbinding Net grain count-+SEM

Without 70.1+13.7

(b) Immunoadsorptionof: Helperfactor (AFCa_+SEM) Suppressorfactor (AFC_+SEM)

Bound 97.5_+7.2 28.3_+5.2

(c) Helperorsuppressorfunction on cell reconstitutionb T4+ + T8+ W +c T4+ + T8+ W -c T4+ + T8+ + T8+ W +

With 2.4_+2.4

Keyholelimpet haemocyanin Percent 98.3+1.7

Without 62.5_+6.9

Not bound 22.3+_7.4 208.7_+10.1

Bound 38.2+7.3 216.7_+3.8

With 56.0+7.2

Percent 10.2+4.4 Not bound 191.5_+6.8 34.8_+3.2

TNP-tetanustoxoid Help (%) Suppression(%) 25 7 0 4 0 4

DNP-streptococcalantigen Help (%) Suppression(%) 324 0 127 97 309 10

a Antibody

forming cells b Streptococcal antigen was used in the priming culture and DNP-streptococcalantigen or TNP-tetanus to×old in the cooperative culture c Vicia villosa adherent (VV+) or non-adherent (VV-) cells monkeys, for only monkeys immunized in vivo with SA will generate in v i t r o SA specific helper and suppressor factors within 24 h 13. Phenotype of contrasuppressor cells

Significantly more T8 ÷ cells than T4 + cells react with monoclonal antibodies to la13 (DA6.231) (8.8 _ 1.8% and 1.8 + 0.7%, respectively) 32. This proportion is increased in T8 ÷ VV + cells (11.3 _+ 1.4%) and T8 ÷ SA + cells (27.4%) but decreased in T8 + V V - cells (3.4 +__ 0.1%) 19 and T8 + SA- cells (0.35%) 2o. 12sl-SA binding to T8 + cells was inhibited by monoclonal anti-lal3 but not control anti-lao~ antibodies. Furthermore, treatment of T8 + VV + cells with monoclonal anti-lal3 antibody and complement, followed by reconstitution with untreated T4 ÷ helper cells, with or without T8 ÷ V V suppressor cells showed significant inhibition of helper and contrasuppressor activity (R. Brines and T. Lehner, unpublished). Thus, T8 + VV +, antigen binding, presenting and contrasuppressor cells express on their cell surface la13 determinants which are probably DRI3 molecules because anti-DP antibodies did not inhibit antigen binding to T8 ÷ cells. However, DQ molecules might also be involved, Fig. 5. The Proportionof cellsreacting with antH-J [] or anti-la~ [] monodonal antibodies; Inhibition of T2S/. streptococcal antigen binding with antil-J or anti-lae antibodies; inhibition of helper activity in SA presentation and contrasuppression, when T8+ VV+ (Mo or T8+ W-) cells were treated with antH-J or anti-la~ and complement.

because antigen binding to T8 + cells was inhibited by anti-DQ antibodies 32. T8 + VV ÷ cells may also express surface determinants which cross-react with the murine t-J molecules 17 (Fig. 5). Anti-I-J antibodies react with a significantly greater proportion of T8 (or T8 ÷ VV ÷) cells than T4 cells or macrophages and they inhibit binding of I251-SA to T8 cells. Both the antigen presenting and contrasuppressor functions of T8 ÷ VV + ceils, unlike those of monocytes or T8 ÷ VV suppressor cells, are inhibited by anti-I-J antibodies and complement but not control anti-lac~ antibodies ~7. It is important to differentiate the T8 ÷ antigen binding, presenting and contrasuppressor cell subset from monocytes (Table II). There are clear phenotypic differences but it might be argued that a very small number of accessory cells in the T8 ÷ population could account for antigen binding and presentation. However, this is unlikely to explain the inhibition of 1251-SA binding to T8 + cells but not monocytes by monoclonal antibodies to T8 and I-J determinants or conventional antibodies to suppressor factor ~6. Furthermore, complement dependent killing of T8 ÷ cells with monoclonal antibody

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Immunology Today, vol. 7, No. 3, 1986

Table U. DifferentiationbetweenT8+ cellsand monocytes T8+ cells

Monocytes

T cellphenotypes

T8+, T5+, T3+, T4- Lessthan 5%

Esterasea

Punctate

Diffuse

Phagocytosisof latex

No

Yes

BindsSAat:

4°C

37°C

Inhibitionof ~s I-SAbinding with monoclonalantibodyto: T8 Yes I-J Yes antiserumto suppressor factor Yes

No No No

Inhibitionof helperactivityby killingwith complementand monoclonal antibodyto: T8 I-J

Yes Yes

No No

Contrasuppressorfunction Yes

No

aAcidnaphthylacetateesterase to T8 or I-J significantly decreased the helper function when reconstituted with T4 + cells 17'19. Similar treatment of monocytes had no effect (Table II). T8 + cells can bind antigen specifically, as recently demonstrated independently 33 with antiidiotypic antibodies to tetanus toxoid which react with T8 + but not T4 + cells. Although all our evidence, so far, suggests that the antigen binding, presenting and contrasuppressor functions reside in one or two subsets of T8 + VV + cells, the proof of this assertion is dependent on cloning the T cells.

A model for T8 + antigen binding, presenting and contrasuppressor cell subset The studies reviewed here suggest that a subset of T8 +, T3 +, T4-, la +, J-like + , VV and antigen binding cells play a part in immunoregulation. The inhibition of antigen binding by monoclonal anti-I-J raises the possibility that antigen binding might be associated with the 'J-like' molecule (Fig. 4). The T8 + VV + cell can interact with T4 + cells by means of the la-antigen (or la-J-antigen) complex and corresponding receptor on the T4 + cells. The existence of class II receptors on T4 + cells34"3s would be consistent with the T8 + W + cells presenting antigen to T4 + helper cells and inducing helper activity in the absence of accessory cells. Hence, the J-like molecule might function either in facilitating binding of the foreign antigen with the T cell receptor, as the I-J may function as the anti-self receptor induced by self-la36; or in antigen presentation, after the antigen is re-expressed as la-antigen or la-J-antigen complex 36,37. This may enable the T8 + VV + cells to engage preferentially the receptors on T4 + cells, as

in addition to the foreign and la antigens binding the T-cell receptor structure, the J-like molecule may act as an additional binding site (anti-self receptor36). This may prevent similar T8 + VV- (suppressor cell) determinants from reacting with corresponding T4 + receptors. Although the most economical hypothesis suggests that the same population of T8 + cells is responsible for antigen binding, presentation and contrasuppression, preliminary evidence of cloned T8 + cells suggests that two or more sub-populations of cells might be involved.

Functional aspects The potential role of antigen-binding and antigen-presenting T cells is in modulating the immunological cell circuit. Helper activity might be induced in a microenvironment in the absence of accessory cells and this might apply particularly to the intestinal and respiratory tracts. If the same cell subset which induces antigen presentation is also responsible for contrasuppression then a considerable degree of cellular economy is preserved. Contrasuppression might account for covert Ts-cell activity in Crohn's disease in which suppression is opposed by another lymphocyte population 38. In rheumatoid arthritis synovial tissue lymphocytes generate a decreased suppressor cell activity but similar helper activity as that found in normal peripheral blood lymphocytes 39. However, the authors emphasized that the disease can develop in the face of potent suppressor activity. The augmenting synovial T cells are T8 + la + (Ref. 40) and might represent contrasuppressor cells which prevent detection of suppressor ceils. This concept might have important implications in autoimmunity, as la + antigen presenting epithelial cells have been implicated 41. These la + cells may not only present antigen but also prevent the effect of suppressor cells, thereby inducing an autoimmune response in the face of suppressor activity 42. Contrasuppression might therefore account for autoimmunity at a local level (e.g., the thyroid gland), despite systemic suppression. The immunological balance could be tipped towards decreased immunity or to tolerance by decreasing contrasuppression and this might be achieved by the removal of contrasuppressor cells. As depletion of contrasuppressor cells may enable natural T-cell suppressor mechanisms to operate more effectively, this approach might be useful in the treatment of autoimmune diseases and possibly in organ transplantation. I should like to express my gratitude to the late Dr Richard K. Gershon for the valuable discussions and encouragement in pursuing the early stages of these investigations. I should also like to thank Dr Douglas R. Green and my collaborators Tina Jones, Janet Avery and Robert Brines for the exchange of ideas and experimental work contributing to this review. Many of the investigations carried out in this review were supported by the Medical Research Council project grants.

91

computerinterface References

I Dosch, H.M. and Gelfand, E.W. (1977)J. Immunol. 118, 302 2 Geha, R.S., Mudawwar, R. and Schneeberger, E. (t977)J. Exp. Med. 145, 1436 3 Zvaifler, N.J., Feldmann, M., Howie, S. etaL (1979) Clin. Exp. ImmunoL 37, 328 4 Heijnen, C.J., Uytdehaag, F., Dollekamp, I. etaL (1979)in Antibody production in man: in vitro synthesis and clinical implications (Fauci, A.S. and Ballieux R.E. eds) p. 231 5 Lehner, T., Lamb, J.R., Welsh, K.l. etal. (1981) Nature (London) 292, 770 6 Lamb, J.R., Kontiainen, S. and Lehner, T. (1980)1 ImmunoL 124, 2384 7 Lehner, T., Lamb, J.R. and Kontiainen, S. (t982) Clin. Exp. Immunol. 47, 706 8 Uytdehaag, F., Heijnen, C.J. and Ballieux, R.E.(1978)Nature (London) 271,556 9 Kontiainen, S., Woody, J.N., Rees,A. etal. (1981) Clin. Exp. ImmunoL 43, 517 10 Lehner, T. (1982)J. ImmunoL 129, 1936 11 Lamb, J.R., Kontiainen, S. and Lehner, T. (1979) Infect. Immun. 26, 903 12 Morimoto, C., Reinherz,E.L. and Schlossman, S.F.(1981)1 ImmunoL 127, 69 13 Lehner, T., Caldwell, J. and Avery, J. (1984) Eur. J. Immunol. 14,814 14 Gershon, R.K., Eardley, D.D., Durum, S. etal. (1981)1 Exp. Med. 153, 1533 15 Lehner, T. (1983) Eur. J. ImmunoL 13,370 16 Lehner, T. and Jones, T. (1984) Immunology 53, 215 17 Lehner, T., Brines, R., Jones, T. etal. (1984) Clin. Exp. Immunol. 58, 410 18 Lehner, T., Avery, J. and Smith, R. (1985) Immunology. 54, 701 19 Lehner,T., Avery, J. and Jones, T. (1985) Immunology 54,713 20 Lehner, T., Avery, J. and Jones, T. (1985) Clin. Exp. ImmunoL 61 ,.203

A collection of reprints or references is often difficult and tedious to use and maintain. Here John Weiler reviews BIBLIOFILE and REFERENCEMANAGER, two sophisticated software packages which help the user to manage a reprint file. BIBLIOFILEis command oriented (you have to know the commands to run the program) and REFERENCEMANAGER is menu oriented (choicesare presented on the screen),

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Immunology Today, voL 7, No. 3, 1986

21 Thomas, Y., Rogozinski, L., Rabbani Le Roy etaL (1984)J. MoL Cell, ImmunoL 1,103 22 Green, D. R. (1984)1 Mol. Cell. ImmunoL 1, 111 23 Russell,M.W., Bergmeier, L.A., Zanders, E.D. etaL (1980) Infect. Immun. 28, 486 25 Green, D. R., Eardley, D.D., Kimura, A. etaL (1981)Eur. J. Immunol. 11,973 26 Lehner, T., Jones, T. and Avery, J. (1986) Clin. Exp. ImmunoL 63,312 27 Feldmann, M. and Kontiainen, S. (1976) Eur. J. Immunol. 6, 302 28 Pierres, M. and Germain, R.N. (1978) J. Immunol. 121, 1306 29 Ptak, W., Green, D.R., Durum, S.K. etal(19~1~)~Eur.J. Immunol. 11,980 30 Iverson, M., Ptak, W., Green, D.R. etal. (1983)J. Exp. Med. 158, 982 31 Lehner, T. (1983) Immunology 48, 695 32 Lehner, T. and Jones, T. (1984) Clin. Exp. ImmunoL 56, 683 33 Geha, R.S.(1984)J. Immunol. 133, 1846 34 Biddison, W.E,, Rao, P.E.,Talle, M.A. etaL (1982) J, Exp. Med. 156, 1065 35 Meuer, S.C., Fitzgerald, K.A., Hussey, R.E.etaL (1983)1 Exp. Med. 157, 705 36 Uracz, W., Asano, Y., Abe, R. etaL (1985) Nature (London) 316, 741 37 Flood, P., Yamauchi, K. and Gershon, R.K. (t 982)J. Exp. Med. 155, 361 311 Elson,C.O., Graeff, A.S., James, S.P.etaL (1981) Gastroenterology 80, 1513 39 Chattopadhyay, C., Chattopadhyay, H., Natvig, J.B: e#al. (1979) Stand. J. ImmunoL 1O, 309 40 Romain, P.L., Burmester, G.R., Enlow, R.W. etal. (1982) Rheumatol. Int. 2, 121 41 Bottazzo, G.F., PujoI-Borrell, R., Hanafusa, T. etaL (1983) Lancet ii, 1115 42 Lehner, T. (1984)ImmunoL Today. 5, 339

The computer and the reprint file fromJohnM. Weiler

them. The user must have his own editor which writes ASCII text files and not all users have one. I used the editor supplied with my C compiler but could also have used 'SIDEKICK'. BIBLIOFILE needs a text editor for users who do not have one. References must be entered into a BIBLIOFILE database using an editor and cannot be entered automatically BIBLIOFILE is a sophisticated package after a database search (e.g. by of programs that allows references 'Medline' or Bibliographic Retrieval to be stored (REAP), displayed (RE- Service search). Entries must conVIEW), searched (RESEARCH), ex- form exactly to BIBLIOFILE rules for tracted from the large database that entry; for example, it will only accept may be created and used in manu- lower case letters. The BIBLIOFILE scripts (RECITE, RECITE2, RECITE3), user enters: authors, year, title, jouralphabetized (RESORT), combined nal or book or source, volume and from more than one bibliography pages, and various codes; the user is (REMERGE) and formatted and allowed entries of up to 2000 characters. BIBLIOFILE is WordStarprinted (REFORM). BIBLIOFILE offers a large number oriented and some of the printing of options to manipulate reprints, commands will not work with other but not the ability to enter or edit word processors.

BIBLIOFILE also lacks menus. The user must know the commands, which at times are confusing because they may have no intrinsic meaning (e.g. REAP, RECITE, REVIEW, REFORM). This becomes less of a disadvantage once the user becomes familiar with BIBLIOFILE and may speed up operation because he can go to exactly the command he wishes to enter but learning is made more difficult, particularly for computer novices. The manual is poor: it was written for the CP/M operating system whereas the diskette I received is for PC-DOS. The manual is necessary because, without menus, BIBLIOFILE initially is not particularly 'friendly'. The manual refers, often tantalizingly, to 'planned' additions. I cannot recommend BIBLIOFILEto computer novices; it requires extensive use of the disk operating system

© 1986, ElsevierScience Publishers&V., Amsterdam 0167-4919/86/$02.00