Migration
Inhibitory
Factor
(MIF)
and
Proliferative
Responses
by Human Lymphocyte Subpopulations Separated by Sheep Erythrocyte Rosette Formation ’
Human pcripheral l~lootl lymphocytes wet-c sqaratctl hy a conlhinatim of roscttc formation \vith sheep erythrocytcs ant1 tliffcreutial density ccntrifugation into suhpopulations of rosette positive (T-enriched) cells and rosette nrgative (T tlcplctctl) cells. These cvere then tcstctl i,l rrilrrj for the lx-otluction of niacrophage migration inhibitory factor ( RIIF) ant1 for incorlmration of “H-thymitlinc in response to specific antigens. Hot17 T enriched and T depleted cell populatibus protluccd ,\4 II; but only T enrichctl cells exhihitcrl significant antigcmintlucctl “H-thymitlinr incorporation. These findings using a T ccl1 surface marker as the hasis for cell separation, a technique which should not alter the I! ccl1 surface, coutirm an earlirr report in \vhich human cells \vcre scl)aratctl on the basis r>f 5urfacc itnniu~lo~loh~~lin. a I: cell marker.
INTROl)UCTION The production of nmqhnge migrdion inliihtory factor ( RI1 F ) ant1 the prolifcrati\-e response of humnn peripheral l~lootl lyni~hocytcs to specific mtigen have long been thought to he iu Sty0 correlates of tlelnyetl hypersensitivity and, therefore, exclusively T lymphocyte functions. Recently. this assumption has l,een s110wt1, at least in part, to be incorrect. I!sing affinity chromatograpl~y to obtain highly purifietl populntions of 11um~tn T and I3 I\-tnphocytes, Chess rlf 01. tlenmstrntetl that botlt T and 1: hum;tn lymphocytes respond to mitogens l)ut onlv T Ijmphocytes proliferate in response to specific antigenic stimulntion ( 1, 2,): In contrast, hlI1; ~2’35 pro(lucetl l)\: 1~0th ‘I‘ ant1 13 lynil)liocyte5 in response to specific antigen (3 ).
1 Supported in part by LSPHS grants 111-11720. i\I-07685, anti ;\~I~)5577 an(l 3 Kr;lnt from the New England Peahotly Homr Foundation. ” Reprint rcqucsts should he directed to l)r. J!ruce H. I.ittman, Rob-t Ii. I3righam Hos[)ital, 115 Parker Hill Ave.. Boston, hfasaachusetts 02120; recipient of ;L Cancer rkscarch I;cllo\,ship Award from the r1merican Cancer Society, klassachusctts I)ivision, 111~. :1 Recipient of a Research Career Ikvelo~rmcnt :\\varcl, National Itlstituteh ,,f Ijealth, #s K 04 .4 I -707V6.
242
LITTMAK,
DA~JD
AND
KOCKLIN
altered the B cell in some way so that it would now respond to antigen by producing AlIF whereas normally it did not. This possibility was further suggested by the observations of Mackler rf nl. (3) who found that EAC rosetted B lymphocytes spontaneously produced monocyte chemotactic factor and mitogenic factor while B cells dissociated from EAC produced much less (5). To test whether this indeed had occurred, human peripheral blood lymphocytes were separated by a method which should not alter the B lymphocytes. This was accomplished by the specific binding of T lymphocytes to sheep erythrocytes (6) and separating these rosetted cells by differential density centrifugation from the non-rosetted B lymphocytes, monocytes and “null” cells. These cell populations were tested for MIF production and proliferation in response to specific antigenic stimulation. The results of these studies indicate that T depleted lymphocytes do not proliferate but still produce MIF in responseto specific antigen even when separated by a technique which does not affect their surfaces. T enriched cells (> 91% T lymphocytes) produce MIF in responseto the same stimulus. MATERIALS
AND METHODS
Five hundred ml of heparinized blood was sedimented with.70,000 MW dextran (final concentration = 0.6 g s). Mononuclear cells were obtained by differential density centrifugation utilizing a FicollHypaque mixture (LSM solution, Litton Bionetics, Kensington, Md.) as previously described (7). This cell population, called the unseparated population, was mixed with 0.5% sheep erythrocyte (E) suspensionin Hank’s balanced salt solution (HBSS) in the ratio of 10 ml of lymphocytes at 5 X 10R/ml : 10 ml of 0.5y0 E. This mixture was warmed at 37°C for 5 min and centrifuged for 10 min at 1809. Nineteen milliliters of supernatant was removed and 0.1 ml AB serum, previously absorbed twice with E at O”C, was added to each tube and then incubated in an icewater bath for 60 min. The cells were gently resuspended with a Pasteur pipette and layered over a Ficoll-Hypaque mixture for a second separation. Cells remaining at the Ficoll-HBSS interface were mainly monocytes and rosette-negative lymphocytes (referred to as the T depleted cell population) and cells at the bottom of the tubes were rosetted lymphocytes (referred to as the T enriched cell population). All three cell populations were subjected to a hypotonic ammonium chloride solution to lyse red blood cells as previously described (8). In the experiments reported, 5 x lo4 autologous adherent cells per 10” lymphocytes were added to each cell population. These adherent cells were obtained from well washed monolayers of Ficoll separated mononuclear cells. They were scraped from petri dishes with a rubber policeman while in a cold 10m4&Z EDTA-saline solution. This adherent cell population was previously foutid to be 98% phagocytic cells by latex ingestion. A fourth cell population composed of a mixture of the T enriched and T depleted cell populations in the original proportions (referred to as the recombined population) was also prepared when enough cells were obtained. Proliferation assq. Two X 10” lymphocytes in TC 199 (Microbiological Associates, Bethesda, Md.) containing 15% AB serum with penicillin (100 U/ml) and streptomycin (100 pg/ml) was cultured in quadruplicate microtiter wells in 0.2 ml volumes with or without antigen for 6 days at 37°C in 5% CO, and air. One &i of 3H-thymidine (specific activity 6.7 Ci/mM, New England Nuclear, Boston, Mass.) was added for the last 20 hr of incubation and incorporation of Preparation
of lyq?lzocyte
szrbpopdations.
.\I II:
I;Ho\l
llI’Il.\S
I.Ylll’rlot‘Y’l‘R
243
SI~1~1’01’1’1..\‘I‘I0NS
ll1i5 l)r(wir5or ilrtc, cvlluhr I )N,A \v;ts (lctcrlllitlt~(l 1)~. licpiicl scintill;iticui coriiltillg ;15 coulith I)c’r minute ilicot-l)or;ltic,11 of ,‘t~i-tliymitlillc ( 0 ). Kehulth are es~msstxl ;in(l as a stimulation intles ( ratio antip ~tiniulatetl C~ml/cp~ll \vitliout antigen I. .Intigens usetl were lmrifietl I)rotein tleriv:lti\~e (I’L’I) ) (kintllj~ Im~vitlctl 1)~ tlrc k:.S.-Jnpiil Coolm-ntive Science I’rcqyani, (Leographic i\le(liciiie I:r:wch. Nl 1 I, Iktlicstln. LItI. ) at 1 pg/ml and 2;tre~~tokin;~sr-stre~~to~lor~l:~se ( 5X-I) ) at 50 Ilnits,/ml ( 1xtlerle IAmxtorics. t’earl Riwr, N.1’. ). .111/: tr.s.srry. 10; lyni~~lioc\-tes iii 2 ml bw-e cdturetl iii ~Ietlium TC-l(Y) containing I.;‘/, -4 I: sermi Lvitli jxnicillin ant1 strq)tomycin \vith or \vithout antigen. After 4s lir the sulmnatants bvere liarvested antI antigen \v;is atltlctl to the control sulwr\vcrc‘ tlicn nssaycvl for ;\I117 activity natant. Tlic su~~ernatants as ~mviousl~ clescrihetl ( 10 ). liesdts are eslmssctl as tlicb Iwrceiit inliihiticm of migration as calculatetl ironi the follmving foriiiuln :
I)c,tcrrlrilztrtio11 of 7‘ mti 13 cdl purity. Fc~llo~ving cell sqmation, ;tll three ~~o~~ul:~tions bvcrc roscttetl bvitli l< to tleteriiiiiic the percentage of rosette positive and negative cells ( 1 I ‘) f’reliminary experiments tlemonstratetl that N H,CI treatment of rosetted cells to Iyse slicel) erythrocytes ( E ) does no alter tlic ;J)ilitv of these cell5 to he rosettetl with E a second time. Rosettetl cells \vere counted in turn groul)s : those lvitli l-2 I3 rbc
A
IJnsep T enriched T depleted (B)
B
[Jnsep T enriched T depleted (B)
c
48 75 3
Total
9% Nonrosetted lymphocytes
1-2 rbc 20 22 3
68 97 6
32 3 94
49 78 5
18 13 5
67 91 10
33 9 90
Unsep T enriched T depleted (B)
71 86 13
14 12 1
85 98 14
1.5 2 86
1)
Unsep T enriched T depleted (B)
73 8.5 4
8 12 3
81 97 7
19 3 93
E
Unsep T enriched T depleted (B)
70 82 3
5 11 8
75 93 11
25 7 89
F
Unsep T enriched T depleted (B)
67 82 2
19 11 1
86 93 3
14 7 97
been counted as lymphocytes. The T cell populations contained only < l-1170 Ig bearing lymphocytes (see Table 2). Lynzphocyte proliferation and MIF production. The comparative results of the proliferation and MIF assays for the various cell populations are listed in Table 3. Specific antigen uniformly produced significant proliferative responses in the unseparated, T enriched and recombined populations. The T depleted population generally had higher baseline levels of ‘H-thymidine incorporation but exhibited minimal increased incorporation of “H-thymidine in the presence of antigen compared to the response of the unseparated or T enriched cell populations from the same donor. For example, the stimulation indices of the T depleted populations to SK-SD were only l-l 1% of the responseof the unseparated cell populations. Antigen specificity of the proliferative response by the T enriched cells was demonstrated using a tuberculin negative, SK-SD positive donor (see Table 4). A nonspecific mitogenic effect of PPD on the T depleted cells (12) was observed using 1 pg PPD. No proliferative response to PPD by this donor’s T enriched cells was noted while T depleted cells responded minimally in dose response fashion to PPD. Using a closeof PPD which was minimally stimulatory in a nonspecific fashion, the T depleted cell responsein a tuberculin positive donor was very small compared to that of the T enriched or unseparated populations. It should be noted that unseparated cells and T enriched cells of both donors responded well to SK-D, while the T depleted populations did not respond at all.
‘,C Inhibition of m;tcrophagc* migration -.~___-_-._--__
I~ynlphoc)‘t~
transformatiorl c[xll'L
E\[’
9'8
N?at
1:2
1:1
1, l[nwp
17
7
1h
1-I
22
21
2.z
10
I‘ cwrichctl
I‘ tlrplwtl
licxoml)inc~tl
E\I,
Non,. SKSI
(I( I
)
2,070 1.674
(‘I‘ + I{)
I I,/
I~llSCll
11.3 1.3-U Nonc~
SK-SI liccc,ml,inv(l
__--
(‘I’ +
)
3.722 3,wo
1.3.1
I4
7
11.0
1f,
0
5
I.1
.31
21
22
2-r
0
- 2
-13
I()
----.-.--tri :‘H-th>mitlinc.
11Counts per minute incorpcxation ‘8 Stimulation In(lc*s = cpm antigen prcsent/cpm I
1’1’1) SK-SI
IO ;’ 17’;,
)
25’;. 20’ ‘I( ‘1‘ dq~letcd 1’1’1 ) SK-SI)
-4’ -5’;
;
- 1 .i 1, ; 231,;’
11c; 17’{,
0’ ( 7’;
-- 10’ ; 25(.;’
- 16’ ; X’ ;
(Ii 1 cdl, xc,;
221,;’
18’ ;’ 1SC’;;
-5’
I
28’ ;”
10 (, ;,’ 2 7 ‘,/( ’
248
LITTM.~N,
D.4vm
AND
KOCKLIN
results obtained by Rocklin et al. where affinity chromatography was used as a means of cell separation (3). The method employed in the present study for cell separation has the advantage of not interacting with the B cell surface but sacrifices absolute cell purity. Although we cannot rule out that the small percentage of T cells present in the T depleted population produced the MIF, this is unlikely since more MIF was produced by the T depleted cell population. Further, the T depleted population thus obtained did not proliferate significantly when compared to the unseparated or T enriched l>opulations. Thus, cell separation based on a techniclue which should not affect the B cell yielded results similar to previous studies using affinity chromatography (3). T and B cell MIF was previously shown to elute in the 23,000 MW range by Sephadex chromatography and is probably the same. Human B and T cell chemotactic factors have also been compared and foulid to be identical by both gel filtration chromatography and isoelectric focusing techniques (5). Dilution studies demonstrated that more MIF activity was produced by the T depleted cells than by the unseparated or T enriched populations. If B and T cell MIF are identical, then B cells must make quantitatively more MIF than T cells, Other investigators (13, 14) have found that mediator production by B lymphocytes in the guinea pig could not be induced by specific antigens but could be nonspecifically induced by PPD. In the present study when cells from a SK-SD positive, tuberculin negative blood donor were tested, SK-SD stimulated MIF production but PPD did not. Furthermore, SK-SD was capable of inducing MIF production by T depleted lymphocytes as well as by T enriched lymphocytes. T depleted cells from a donor positive to both PPD and SK-SD produced MIF in response to both antigens. As noted previously, we found that PPD nonspecifically stimulated 3H-thymidine incorporation by the T depleted cell population. This presumably represents a B cell mitogenic effect of PPD. T enriched cells from the same donor failed to demonstrate any increased thymidine incorporation (see Table 4). These observations differ from some previous reports (2) but agree with those of other investigators (12). Some possible explanations include differences in the PPD preparation used or a differing method of cell separation. Contaminating T cells in the T depleted cell population are an unlikely explanation since the T enriched cells from the PPD negative donor did not show increased 3H-thymidine incorporation. It is of interest that in some experiments not presented here the T enriched lymphocyte population functioned poorly in both proliferation and MIF assays. For instance, in one experiment, the T enriched cell response to SK-SD was only 6.6 times control, the T depleted cell response was 8.0 times control while the unseparated cells showed 97.4 times the control amount of 3H-tI~ymidine incorporation. On recombining T enriched plus T depleted cells in the same proportion as in the unseparated population, a stimulation index of 98.5 was obtained. Since the method of separation of T lymphocytes depletes this cell population of monocytes, 5 x 10’ adherent cells per 10G lymphocytes were added to each culture. In all subsequent experiments where this was done the T enriched cell population functioned u,eIl in both assays. This confirmed several previous reports which indicated a monocyte requirement for specific antigenic stimulation of T cells obtained from guinea pigs and man (14-16).
we IKIIY contirtuetl l~wvious rc’l)orts (l-3) that ‘I‘ cells (our 1 II ccmclu&m, 7’ enriclxtl lqulatioii j proliferate and prwluce 5111; ill respoiix to a slvxific antigciiic stimulus while I< cells (‘I’ tleldetetl ccl15 in this work ) do not lxwlifcratc Iut tit) lmduce MI 1; when sul~j~ctetl to the s:mw stimulus. 11.~2 have also confirmetl that 13 cdls lxduce greater LlI 1; acti\-it\, than cclual niunhcrs of T cells from the saiiie donor. This, using tbvo tlifferelit lecliiiicluw, one of which sl~oultl not dtm- 13 cells, simihr results wre ol)tainetl. r\ltliou~li the in z*ifr-0 hl I I; test has Iwn shm31 to corrrlate kvitli tlelayetl hylxnensitivity ii2 CUJ ( lo), it tloes not csclusively measure 1‘ cell function. However. since these cell lqnilations arc not 100%
I)Ilrv
-1‘
Or
13 CdlS
if fewer
ccJntamin;rting
I)iJSSihlC
that
(this
iS
lmh1dy
illll)cJSSihlv
to
:IChie\e
hy
Xl)-
lll~~thOd
),
T cells ;m rccluiretl to facilitate I\11 IT prductioii than to Stll)lXJrt 13 Cdl ~~rlJ~ifer~ltiOl1, tlicn T cell clelwiitlent I( cell XIII; lmduction inay ;~c~n~~it for these results. This is tliswssctl in detail elscwlierc (3 ). It is, of course, \vitli tliytnus,
the
ddity
tliynitis
Of
1:
Cdh
to
puhiCe
111
r’
dCpldS
U]Wll
501116:
illteraCti~l1
lmtlwt or T Iynil)hocytes \vliicli occurs at ;I tinle ln-itrr to cdl sep;ll-atloll i,l Z~izV. This cellular coolmation does occur during sensitkhm to :ui antigeli. Tlic ul)servation that patieiits with wrtain inlililmodeficieiicy diseases have i~ormal IZ cell function as nssessetl 1)~ aritilwtl~ production hut lack cellular imniunc iutlction ant1 do not product 3111; (20 ) gives sulq~~-t to tlw a-guiliellt that at soi11t’ lmint 1%cell 91 IF is tliymris tlelxn~lcnt (21, 22).
Inhibitory
Factor
(MIF)
and
Proliferative
Responses
by Human Lymphocyte Subpopulations Separated by Sheep Erythrocyte Rosette Formation ’
Human pcripheral l~lootl lymphocytes wet-c sqaratctl hy a conlhinatim of roscttc formation \vith sheep erythrocytcs ant1 tliffcreutial density ccntrifugation into suhpopulations of rosette positive (T-enriched) cells and rosette nrgative (T tlcplctctl) cells. These cvere then tcstctl i,l rrilrrj for the lx-otluction of niacrophage migration inhibitory factor ( RIIF) ant1 for incorlmration of “H-thymitlinc in response to specific antigens. Hot17 T enriched and T depleted cell populatibus protluccd ,\4 II; but only T enrichctl cells exhihitcrl significant antigcmintlucctl “H-thymitlinr incorporation. These findings using a T ccl1 surface marker as the hasis for cell separation, a technique which should not alter the I! ccl1 surface, coutirm an earlirr report in \vhich human cells \vcre scl)aratctl on the basis r>f 5urfacc itnniu~lo~loh~~lin. a I: cell marker.
INTROl)UCTION The production of nmqhnge migrdion inliihtory factor ( RI1 F ) ant1 the prolifcrati\-e response of humnn peripheral l~lootl lyni~hocytcs to specific mtigen have long been thought to he iu Sty0 correlates of tlelnyetl hypersensitivity and, therefore, exclusively T lymphocyte functions. Recently. this assumption has l,een s110wt1, at least in part, to be incorrect. I!sing affinity chromatograpl~y to obtain highly purifietl populntions of 11um~tn T and I3 I\-tnphocytes, Chess rlf 01. tlenmstrntetl that botlt T and 1: hum;tn lymphocytes respond to mitogens l)ut onlv T Ijmphocytes proliferate in response to specific antigenic stimulntion ( 1, 2,): In contrast, hlI1; ~2’35 pro(lucetl l)\: 1~0th ‘I‘ ant1 13 lynil)liocyte5 in response to specific antigen (3 ).
1 Supported in part by LSPHS grants 111-11720. i\I-07685, anti ;\~I~)5577 an(l 3 Kr;lnt from the New England Peahotly Homr Foundation. ” Reprint rcqucsts should he directed to l)r. J!ruce H. I.ittman, Rob-t Ii. I3righam Hos[)ital, 115 Parker Hill Ave.. Boston, hfasaachusetts 02120; recipient of ;L Cancer rkscarch I;cllo\,ship Award from the r1merican Cancer Society, klassachusctts I)ivision, 111~. :1 Recipient of a Research Career Ikvelo~rmcnt :\\varcl, National Itlstituteh ,,f Ijealth, #s K 04 .4 I -707V6.
242
LITTMAK,
DA~JD
AND
KOCKLIN
altered the B cell in some way so that it would now respond to antigen by producing AlIF whereas normally it did not. This possibility was further suggested by the observations of Mackler rf nl. (3) who found that EAC rosetted B lymphocytes spontaneously produced monocyte chemotactic factor and mitogenic factor while B cells dissociated from EAC produced much less (5). To test whether this indeed had occurred, human peripheral blood lymphocytes were separated by a method which should not alter the B lymphocytes. This was accomplished by the specific binding of T lymphocytes to sheep erythrocytes (6) and separating these rosetted cells by differential density centrifugation from the non-rosetted B lymphocytes, monocytes and “null” cells. These cell populations were tested for MIF production and proliferation in response to specific antigenic stimulation. The results of these studies indicate that T depleted lymphocytes do not proliferate but still produce MIF in responseto specific antigen even when separated by a technique which does not affect their surfaces. T enriched cells (> 91% T lymphocytes) produce MIF in responseto the same stimulus. MATERIALS
AND METHODS
Five hundred ml of heparinized blood was sedimented with.70,000 MW dextran (final concentration = 0.6 g s). Mononuclear cells were obtained by differential density centrifugation utilizing a FicollHypaque mixture (LSM solution, Litton Bionetics, Kensington, Md.) as previously described (7). This cell population, called the unseparated population, was mixed with 0.5% sheep erythrocyte (E) suspensionin Hank’s balanced salt solution (HBSS) in the ratio of 10 ml of lymphocytes at 5 X 10R/ml : 10 ml of 0.5y0 E. This mixture was warmed at 37°C for 5 min and centrifuged for 10 min at 1809. Nineteen milliliters of supernatant was removed and 0.1 ml AB serum, previously absorbed twice with E at O”C, was added to each tube and then incubated in an icewater bath for 60 min. The cells were gently resuspended with a Pasteur pipette and layered over a Ficoll-Hypaque mixture for a second separation. Cells remaining at the Ficoll-HBSS interface were mainly monocytes and rosette-negative lymphocytes (referred to as the T depleted cell population) and cells at the bottom of the tubes were rosetted lymphocytes (referred to as the T enriched cell population). All three cell populations were subjected to a hypotonic ammonium chloride solution to lyse red blood cells as previously described (8). In the experiments reported, 5 x lo4 autologous adherent cells per 10” lymphocytes were added to each cell population. These adherent cells were obtained from well washed monolayers of Ficoll separated mononuclear cells. They were scraped from petri dishes with a rubber policeman while in a cold 10m4&Z EDTA-saline solution. This adherent cell population was previously foutid to be 98% phagocytic cells by latex ingestion. A fourth cell population composed of a mixture of the T enriched and T depleted cell populations in the original proportions (referred to as the recombined population) was also prepared when enough cells were obtained. Proliferation assq. Two X 10” lymphocytes in TC 199 (Microbiological Associates, Bethesda, Md.) containing 15% AB serum with penicillin (100 U/ml) and streptomycin (100 pg/ml) was cultured in quadruplicate microtiter wells in 0.2 ml volumes with or without antigen for 6 days at 37°C in 5% CO, and air. One &i of 3H-thymidine (specific activity 6.7 Ci/mM, New England Nuclear, Boston, Mass.) was added for the last 20 hr of incubation and incorporation of Preparation
of lyq?lzocyte
szrbpopdations.
.\I II:
I;Ho\l
llI’Il.\S
I.Ylll’rlot‘Y’l‘R
243
SI~1~1’01’1’1..\‘I‘I0NS
ll1i5 l)r(wir5or ilrtc, cvlluhr I )N,A \v;ts (lctcrlllitlt~(l 1)~. licpiicl scintill;iticui coriiltillg ;15 coulith I)c’r minute ilicot-l)or;ltic,11 of ,‘t~i-tliymitlillc ( 0 ). Kehulth are es~msstxl ;in(l as a stimulation intles ( ratio antip ~tiniulatetl C~ml/cp~ll \vitliout antigen I. .Intigens usetl were lmrifietl I)rotein tleriv:lti\~e (I’L’I) ) (kintllj~ Im~vitlctl 1)~ tlrc k:.S.-Jnpiil Coolm-ntive Science I’rcqyani, (Leographic i\le(liciiie I:r:wch. Nl 1 I, Iktlicstln. LItI. ) at 1 pg/ml and 2;tre~~tokin;~sr-stre~~to~lor~l:~se ( 5X-I) ) at 50 Ilnits,/ml ( 1xtlerle IAmxtorics. t’earl Riwr, N.1’. ). .111/: tr.s.srry. 10; lyni~~lioc\-tes iii 2 ml bw-e cdturetl iii ~Ietlium TC-l(Y) containing I.;‘/, -4 I: sermi Lvitli jxnicillin ant1 strq)tomycin \vith or \vithout antigen. After 4s lir the sulmnatants bvere liarvested antI antigen \v;is atltlctl to the control sulwr\vcrc‘ tlicn nssaycvl for ;\I117 activity natant. Tlic su~~ernatants as ~mviousl~ clescrihetl ( 10 ). liesdts are eslmssctl as tlicb Iwrceiit inliihiticm of migration as calculatetl ironi the follmving foriiiuln :
I)c,tcrrlrilztrtio11 of 7‘ mti 13 cdl purity. Fc~llo~ving cell sqmation, ;tll three ~~o~~ul:~tions bvcrc roscttetl bvitli l< to tleteriiiiiic the percentage of rosette positive and negative cells ( 1 I ‘) f’reliminary experiments tlemonstratetl that N H,CI treatment of rosetted cells to Iyse slicel) erythrocytes ( E ) does no alter tlic ;J)ilitv of these cell5 to he rosettetl with E a second time. Rosettetl cells \vere counted in turn groul)s : those lvitli l-2 I3 rbc
A
IJnsep T enriched T depleted (B)
B
[Jnsep T enriched T depleted (B)
c
48 75 3
Total
9% Nonrosetted lymphocytes
1-2 rbc 20 22 3
68 97 6
32 3 94
49 78 5
18 13 5
67 91 10
33 9 90
Unsep T enriched T depleted (B)
71 86 13
14 12 1
85 98 14
1.5 2 86
1)
Unsep T enriched T depleted (B)
73 8.5 4
8 12 3
81 97 7
19 3 93
E
Unsep T enriched T depleted (B)
70 82 3
5 11 8
75 93 11
25 7 89
F
Unsep T enriched T depleted (B)
67 82 2
19 11 1
86 93 3
14 7 97
been counted as lymphocytes. The T cell populations contained only < l-1170 Ig bearing lymphocytes (see Table 2). Lynzphocyte proliferation and MIF production. The comparative results of the proliferation and MIF assays for the various cell populations are listed in Table 3. Specific antigen uniformly produced significant proliferative responses in the unseparated, T enriched and recombined populations. The T depleted population generally had higher baseline levels of ‘H-thymidine incorporation but exhibited minimal increased incorporation of “H-thymidine in the presence of antigen compared to the response of the unseparated or T enriched cell populations from the same donor. For example, the stimulation indices of the T depleted populations to SK-SD were only l-l 1% of the responseof the unseparated cell populations. Antigen specificity of the proliferative response by the T enriched cells was demonstrated using a tuberculin negative, SK-SD positive donor (see Table 4). A nonspecific mitogenic effect of PPD on the T depleted cells (12) was observed using 1 pg PPD. No proliferative response to PPD by this donor’s T enriched cells was noted while T depleted cells responded minimally in dose response fashion to PPD. Using a closeof PPD which was minimally stimulatory in a nonspecific fashion, the T depleted cell responsein a tuberculin positive donor was very small compared to that of the T enriched or unseparated populations. It should be noted that unseparated cells and T enriched cells of both donors responded well to SK-D, while the T depleted populations did not respond at all.
‘,C Inhibition of m;tcrophagc* migration -.~___-_-._--__
I~ynlphoc)‘t~
transformatiorl c[xll'L
E\[’
9'8
N?at
1:2
1:1
1, l[nwp
17
7
1h
1-I
22
21
2.z
10
I‘ cwrichctl
I‘ tlrplwtl
licxoml)inc~tl
E\I,
Non,. SKSI
(I( I
)
2,070 1.674
(‘I‘ + I{)
I I,/
I~llSCll
11.3 1.3-U Nonc~
SK-SI liccc,ml,inv(l
__--
(‘I’ +
)
3.722 3,wo
1.3.1
I4
7
11.0
1f,
0
5
I.1
.31
21
22
2-r
0
- 2
-13
I()
----.-.--tri :‘H-th>mitlinc.
11Counts per minute incorpcxation ‘8 Stimulation In(lc*s = cpm antigen prcsent/cpm I
1’1’1) SK-SI
IO ;’ 17’;,
)
25’;. 20’ ‘I( ‘1‘ dq~letcd 1’1’1 ) SK-SI)
-4’ -5’;
;
- 1 .i 1, ; 231,;’
11c; 17’{,
0’ ( 7’;
-- 10’ ; 25(.;’
- 16’ ; X’ ;
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results obtained by Rocklin et al. where affinity chromatography was used as a means of cell separation (3). The method employed in the present study for cell separation has the advantage of not interacting with the B cell surface but sacrifices absolute cell purity. Although we cannot rule out that the small percentage of T cells present in the T depleted population produced the MIF, this is unlikely since more MIF was produced by the T depleted cell population. Further, the T depleted population thus obtained did not proliferate significantly when compared to the unseparated or T enriched l>opulations. Thus, cell separation based on a techniclue which should not affect the B cell yielded results similar to previous studies using affinity chromatography (3). T and B cell MIF was previously shown to elute in the 23,000 MW range by Sephadex chromatography and is probably the same. Human B and T cell chemotactic factors have also been compared and foulid to be identical by both gel filtration chromatography and isoelectric focusing techniques (5). Dilution studies demonstrated that more MIF activity was produced by the T depleted cells than by the unseparated or T enriched populations. If B and T cell MIF are identical, then B cells must make quantitatively more MIF than T cells, Other investigators (13, 14) have found that mediator production by B lymphocytes in the guinea pig could not be induced by specific antigens but could be nonspecifically induced by PPD. In the present study when cells from a SK-SD positive, tuberculin negative blood donor were tested, SK-SD stimulated MIF production but PPD did not. Furthermore, SK-SD was capable of inducing MIF production by T depleted lymphocytes as well as by T enriched lymphocytes. T depleted cells from a donor positive to both PPD and SK-SD produced MIF in response to both antigens. As noted previously, we found that PPD nonspecifically stimulated 3H-thymidine incorporation by the T depleted cell population. This presumably represents a B cell mitogenic effect of PPD. T enriched cells from the same donor failed to demonstrate any increased thymidine incorporation (see Table 4). These observations differ from some previous reports (2) but agree with those of other investigators (12). Some possible explanations include differences in the PPD preparation used or a differing method of cell separation. Contaminating T cells in the T depleted cell population are an unlikely explanation since the T enriched cells from the PPD negative donor did not show increased 3H-thymidine incorporation. It is of interest that in some experiments not presented here the T enriched lymphocyte population functioned poorly in both proliferation and MIF assays. For instance, in one experiment, the T enriched cell response to SK-SD was only 6.6 times control, the T depleted cell response was 8.0 times control while the unseparated cells showed 97.4 times the control amount of 3H-tI~ymidine incorporation. On recombining T enriched plus T depleted cells in the same proportion as in the unseparated population, a stimulation index of 98.5 was obtained. Since the method of separation of T lymphocytes depletes this cell population of monocytes, 5 x 10’ adherent cells per 10G lymphocytes were added to each culture. In all subsequent experiments where this was done the T enriched cell population functioned u,eIl in both assays. This confirmed several previous reports which indicated a monocyte requirement for specific antigenic stimulation of T cells obtained from guinea pigs and man (14-16).
we IKIIY contirtuetl l~wvious rc’l)orts (l-3) that ‘I‘ cells (our 1 II ccmclu&m, 7’ enriclxtl lqulatioii j proliferate and prwluce 5111; ill respoiix to a slvxific antigciiic stimulus while I< cells (‘I’ tleldetetl ccl15 in this work ) do not lxwlifcratc Iut tit) lmduce MI 1; when sul~j~ctetl to the s:mw stimulus. 11.~2 have also confirmetl that 13 cdls lxduce greater LlI 1; acti\-it\, than cclual niunhcrs of T cells from the saiiie donor. This, using tbvo tlifferelit lecliiiicluw, one of which sl~oultl not dtm- 13 cells, simihr results wre ol)tainetl. r\ltliou~li the in z*ifr-0 hl I I; test has Iwn shm31 to corrrlate kvitli tlelayetl hylxnensitivity ii2 CUJ ( lo), it tloes not csclusively measure 1‘ cell function. However. since these cell lqnilations arc not 100%
I)Ilrv
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Or
13 CdlS
if fewer
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that
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to
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hy
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T cells ;m rccluiretl to facilitate I\11 IT prductioii than to Stll)lXJrt 13 Cdl ~~rlJ~ifer~ltiOl1, tlicn T cell clelwiitlent I( cell XIII; lmduction inay ;~c~n~~it for these results. This is tliswssctl in detail elscwlierc (3 ). It is, of course, \vitli tliytnus,
the
ddity
tliynitis
Of
1:
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111
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lmtlwt or T Iynil)hocytes \vliicli occurs at ;I tinle ln-itrr to cdl sep;ll-atloll i,l Z~izV. This cellular coolmation does occur during sensitkhm to :ui antigeli. Tlic ul)servation that patieiits with wrtain inlililmodeficieiicy diseases have i~ormal IZ cell function as nssessetl 1)~ aritilwtl~ production hut lack cellular imniunc iutlction ant1 do not product 3111; (20 ) gives sulq~~-t to tlw a-guiliellt that at soi11t’ lmint 1%cell 91 IF is tliymris tlelxn~lcnt (21, 22).
