You are walking out of your advisor\'s office after an hour long meeting where D

Question

You are walking out of your advisor's office after an hour long meeting where Dr Feeble kept trying to convince you that the B cell response to your newly discovered peanut allergen "nutz" is T-independent. You are convinced he is wrong and want to prove once and for all the immune response to nutz is truly T-dependent (and you are the superior immunologist). You look around the lab and find monoclonal antibodies to CD40L, B7, CD 19, IgM and IgE (appropriate for flow cytometry, immunofluorescence, or western blotting). You also have CFSE (which can be incorporated to monitor proliferating cells) and ATP-gamma-s (which is nonhydrolyzable and shuts down any ATP-dependent reaction including kinases). You also have unlimited access to spleen cells from mice and unlimited amounts of nutz antigen and all the equipment in the department. You grin wryly and begin to describe your plan of experimentation on the pieces of recycled notebook paper available in the lab to prove your scientific superiority by demonstrating the immune response to nutz is truly T-dependent. HA-HA! (draw out your approach and list the experiment(s) needed to refute one of the hypotheses) Your less bitter labmate leans over the bench to see what you are working on and says "Cool idea! .. but, if you're correct, then w hat are your expected results from that experiment?"

Explanation / Answer

a. My approach would be to prove that nutz allergen immune response is T cell dependent. The best way to show this is to shut off the T cell pathway and show that B-cell activation alone is not sufficient for the activation of the immune response. CD40-CD40L interaction is one of important occurs during the T cell-dependent immune response. Inject the mice with anti-CD40L antibody to block this interaction in vivo. Then immunize the mice with nutz allergen, isolate the spleen cells and culture then in vitro. Then check for the T cell immune response using the specific antibodies by flow cytometry.

Another approach would be isolate and sort the T cell from mouse spleen. Culture them with anti-CD40L antibody in vitro and label them with CFSE dye. In the presence B cells, co-culture these T cells and stimulate with the nutz allergen. Monitor the rate of proliferation of T cells under the inactivated state.

b. If the immune response is T cell dependent, in both the cases you will not find any immune response as you have totally shut down the T cell activation. If the immune response is only B cell dependent you can see an increase the IgM and IgE antibody production without the need of T cell activation.

Usually, T cells expressing CD40L provides signal for the B cell activation and proliferation.

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