Role of CD2 cross-linking in cytoplasmic calcium responses and T cell activation.
The relationship between the increase of intracellular free Ca2+ concentration ([Ca2+]i) in resting T cells after stimulation with monoclonal antibody (mAb) to CD2 (E rosette receptor) and the subsequent proliferation response was investigated. Although the combination of 9.6 plus 9-1 mAb to CD2 was both mitogenic and induced an increase in [Ca2+]i, cross-linking of an individual CD2 mAb on the cell surface induced an increase in [Ca2+]i without directly stimulating T cell proliferation. The [Ca2+]i response from cross-linking an individual mAb was not epitope dependent, since 21 of 21 mAb to CD2 were effective when cross-linked with a polyclonal goat anti-mouse Ig second step. The kinetics of calcium mobilization was highly dependent upon the procedure for cross-linking, since the binding of biotin-conjugated 9.6 mAb followed by avidin gave a large and rapid response, whereas cross-linking of 9.6 with an anti-kappa mAb, 187.1, caused a minimal response, and the cross-linking of 9.6 followed by a polyclonal goat anti-mouse Ig gave an intermediate response. In addition, ligation of CD2 by rosetting with sheep red blood cells alone was sufficient to cause increased [Ca2+]i. In functional studies only the procedures associated with minimal CD2 cross-linking induced proliferation of resting T cells in combination with interleukin (IL)2. The proliferation also required IL 1 or accessory cells. Cross-linking 9.6 on the cell surface also enhanced proliferation in the presence of phorbol myristate acetate or a CD28 mAb, 9.3, under conditions that were accessory cell independent. In contrast to the proliferation following stimulation with 9.6 plus 9-1, the combination of 9.6 plus 9-1 F(ab')2 fragments lost mitogenic activity. The 9.6 plus 9-1 F(ab')2 combination was similar to 9.6 cross-linking in that either could induce responsiveness to recombinant IL2 or CD28 mAb 9.3. The combination of 9.6 plus 9-1 F(ab')2 fragments was still able to increase [Ca2+]i in T cells with slow kinetics. Together these results suggest that the binding of mAb to CD2 under conditions that cause a slow rather than a rapid increase in [Ca2+]i is associated with T cell activation. Furthermore, they suggest that in studies of T cell activation, sheep erythrocyte rosette formation should not be used to isolate T cells since rosetting may effect [Ca2+]i.
Ledbetter
J A
JA
ONCOGEN, Seattle, Washington 98121.
Rabinovitch
P S
PS
Hellström
I
I
Hellström
K E
KE
Grosmaire
L S
LS
June
C H
CH
D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology*; D018801:CD2 Antigens; D018106:CD28 Antigens; D002118:Calcium / Q000502:physiology*; D000939:Epitopes; D007375:Interleukin-1 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D008163:Luminescent Measurements; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface / Q000502:physiology*; D011961:Receptors, Fc / Q000502:physiology; D011971:Receptors, Immunologic / Q000502:physiology*; D012397:Rosette Formation; D013601:T-Lymphocytes / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D018106:CD28 Antigens; D000939:Epitopes; D007375:Interleukin-1; D007376:Interleukin-2; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011961:Receptors, Fc; D011971:Receptors, Immunologic; D002118:Calcium