Figure 3. CD28 co-activation remarkably enhanced TCR/CD3 co-clustering or interaction with CD4 on the cell membrane of the anti-CD3 Ab-activated CD4 T cells.

(A) The NSOM dual color images of one representative of the T-cells co-stimulated with anti-CD3 Ab and anti-CD28 Ab. The CD28 co-stimulation led to marked increases in numbers of CD3-CD4 co-clustering nano-domains or micro-domains (upper panel) as well as in the density and size (middle panel) of co-localized CD3 and CD4 nano-domains or micro-domains compared to the anti-CD3 Ab alone (Fig. 2A). Lower panels show the T cell topography and topography-fluorescence overlay images. Note that complete co-clustering of CD3 and CD4 took place in the central areas of nano-domains or micro-domains, leaving only small numbers of CD3-only or CD4-only clusters in the peripheral area. CD28 co-stimulation led to increases in secretion of IL-2 by CD3 Ab-activated T cells (data not shown here). (B) The NSOM dual-color images of one representative of the anti-CD28 Ab alone-stimulated T-cells. Note that anti-CD28 Ab stimulation led to the formation of few independent CD3 or CD4 nano-domains, but there was no apparent co-clustering or co-localization of CD3 and CD4 in the nano-domains on the membrane. Lower panels show the T cell topography and topography-fluorescence overlay images. (C) Molecular-density analysis shows the molecular density of CD3 or CD4 molecules on the membrane of un-stimulated T cells, T cells stimulated with anti-CD28 Ab only, anti-CD3 Ab only and T cells co-stimulated with anti-CD3 Ab and anti-CD28 Ab. Anti-CD3 Ab-stimulated T cells exhibited significantly-greater molecular density of CD3 or CD4 compared to unstimulated T-cell, PMA/Ionomycin-stimulated T-cells or anti-CD28 Ab only-stimulated T cells (p<0.05); Anti-CD28/anti-CD3 Ab co-stimulation significantly increases density of co-localized CD3–CD4 nano- or micro-domains (p<0.05) compared to anti-CD3 Ab stimulation only. Similar results were observed regardless of whether CD3 and CD4 were stained with QD605 or QD655. Up to 10 cells were analyzed. (D) Molecular-number analysis shows the percentages of total CD3 or CD4 molecules that arrayed to form nano- or micro-domains on the membrane of un-stimulated T cells, T cells stimulated with anti-CD28 Ab only, anti-CD3 Ab only and T cells co-stimulated with anti-CD3 Ab and anti-CD28 Ab. Anti-CD3 Ab-stimulated T cells exhibited significantly higher numbers of CD3 or CD4 nano-domains and co-localized CD3–CD4 nanodomains compared to unstimulated T cells, PMA/Ionomycin-stimulated T-cells or anti-CD28 Ab only-stimulated T cells (p<0.05); Anti-CD28/anti-CD3 Ab co-stimulation significantly increases numbers of co-localized CD3–CD4 nano- or micro-domains (p<0.05) compared to anti-CD3 Ab stimulation only. Up to 10 cells were analyzed.