Human CD34+ blood cells induce T-cell unresponsiveness to specific alloantigens only under costimulatory blockade  Mario Arpinati, Carolina Terragna, Gabriella Chirumbolo, Simonetta Rizzi, Benedetta Urbini, Francesca Re, Sante Tura, Michele Baccarani, Damiano Rondelli  Experimental Hematology  Volume 31, Issue 1, Pages 31-38 (January 2003) DOI: 10.1016/S0301-472X(02)01018-4

Figure 1 Anti-CD40L mAb and CTLA4-Ig inhibit T-cell alloreactivity to CD34+ cells. Freshly isolated CD34+ blood cells were irradiated and mixed with 5 × 104 HLA-mismatched MNC at 1:2 S/R ratio in the presence of different doses of anti-CD40L mAb (A, left), or CTLA4-Ig (A, right). Each molecule was used separately, or in combination, in further MLC experiments where T-cell alloreactivity to CD34+ cells in the presence of costimulatory blockade was measured by 3H-thymidine incorporation assay after 6 days of culture (B). Inhibition of allogeneic T-cell response by anti-CD40L mAb plus CTLA4-Ig was greater than by anti-CD40L mAb (p = 0.008), or CTLA4-Ig alone (p = 0.05). Results are given as mean ± SD of four separate experiments. Experimental Hematology 2003 31, 31-38DOI: (10.1016/S0301-472X(02)01018-4)

Figure 2 Induction of CTL responses by CD34+ cells is principally mediated by B7:CD28 signal. After priming allogeneic MNC with irradiated CD34+ blood cells, with or without anti-CD40L mAb and/or CTLA4-Ig, the cytotoxic effect against alloantigen-specific PHA blasts was assessed in a standard 51Cr release assay. CTL generation was greatly reduced by the presence of CTLA4-Ig, whereas little effect was elicited by anti-CD40L mAb. Autologous control effectors always showed less than 10% lytic activity. This result is representative of three separate experiments. Experimental Hematology 2003 31, 31-38DOI: (10.1016/S0301-472X(02)01018-4)

Figure 3 Effect of anti-CD40L mAb and/or CTLA4-Ig added to irradiated CD34+ cells in primary MLC on T-cell response in secondary MLC. Allogeneic MNC were stimulated in primary MLC by HLA-mismatched irradiated CD34+ cells (S/R ratio of 1:2) with or without anti-CD40L mAb and/or CTLA4-Ig. After 6 days the cells were harvested and rested for 2 days. The cells then were restimulated with allogeneic MNC (from the same donor as in primary MLC) in the absence (−IL-2) or in the presence of 50 UI/mL IL-2 (+IL-2). Alloantigen-specific T-cell secondary proliferative response was measured by 3H-thymidine assay after 3 days. Results are given as stimulation index ± SD. Experimental Hematology 2003 31, 31-38DOI: (10.1016/S0301-472X(02)01018-4)

Figure 4 Unresponsiveness induced by CD34+ cell stimulation and costimulatory blockade is antigen specific. Allogeneic MNC were stimulated in primary MLC by HLA-mismatched irradiated CD34+ cells (S/R ratio of 1:2) without (black bars) or with (white bars) anti-CD40L mAb and CTLA4-Ig. After 6 days the cells were harvested and rested for 2 days. The cells then were restimulated with allogeneic MNC from the same donor as in primary MLC (alloantigen) for 3 days or from a third party donor (3rd party) for 6 days or with tetanus toxoid (TT) for 6 days, or with PHA for 3 days. Proliferative response was assessed by 3H-thymidine incorporation assay. Responder cells that had been activated in the presence of costimulatory blockade were unresponsive to the same alloantigen previously presented by CD34+ cells, which showed proliferative responses against third party, or tetanus toxoid antigen, or to PHA, comparable to control cells. Results are given as stimulation index ± SD. Experimental Hematology 2003 31, 31-38DOI: (10.1016/S0301-472X(02)01018-4)

Figure 5 Effect of costimulatory blockade in primary MLC on cytokine production upon alloantigen rechallenge. Allogeneic MNC were stimulated in primary MLC by HLA-mismatched irradiated CD34+ cells (at 1:2 S/R ratio) without (white bars) or with (black bars) anti-CD40L mAb and CTLA4-Ig. After 6 days the cells were harvested and rested for 2 days. The cells then were restimulated with allogeneic MNC from the same donor as in primary MLC for 24 hours. Culture supernatants were collected and IFN-γ, TNFα, IL-2, IL-10, IL-5, and IL-4 were measured by flow cytometry using the CBA kit (Becton-Dickinson). Costimulatory blockade induced suppression of IFN-γ and resulted in an increase of IL-10 production. IL-2 and IL-4 results were always under the limit of detection for this assay, which is 12 pg/mL for all the cytokines. Cytokines production is given as mean ± SD, and results are representative of three separate experiments. Experimental Hematology 2003 31, 31-38DOI: (10.1016/S0301-472X(02)01018-4)

Figure 6 Increased IL-10 expression in anergic responder cells upon alloantigen rechallenge. Allogeneic MNC were stimulated in primary MLC by HLA-mismatched irradiated CD34+ cells (S/R ratio of 1:2) without (white bars) or with (black bars) anti-CD40L mAb and CTLA4-Ig. After 6 days the cells were harvested and rested for 2 days. The cells then were restimulated with allogeneic MNC from the same donor as in primary MLC for 6 hours (left) or 24 hours (right). The cells were collected and quantitative mRNA expression of IFN-γ, IL-2, IL-10, and IL-4 analyzed by real-time PCR (see Methods). As early as 6 hours after antigen rechallenge in the cells that had been activated by CD34+ cells and costimulatory blockade, higher expression of IL-10, reduced expression of IL-2, and no IFN-γ were observed compared to control cultures. Upon prolonging the culture to 24 hours the pattern of cytokine expression was maintained. Results are given as the ratio of ΔΔCt in antigen-stimulated/unstimulated cells. Experimental Hematology 2003 31, 31-38DOI: (10.1016/S0301-472X(02)01018-4)