The effect of age on the cognate function of CD4+ T cells

Abstract

With increasing age, the ability to produce protective antibodies in response to immunization declines, resulting in reduced efficacy of vaccination. We have examined how reductions in CD4(+) T-cell function contribute to reduced humoral responses, using a model that allows us to compare identical numbers of antigen-specific naive T cells from young and aged T-cell receptor transgenic mice. Naive cells from aged mice exhibit reduced responses, both in vitro and in vivo. In vitro, responses of aged T cells can be enhanced by addition of interleukin (IL)-2. In vivo, using an adoptive transfer model with young hosts, naive cells from aged mice exhibit significant reductions in cognate helper function, leading to reduced B-cell expansion and differentiation. These age-related defects could be overcome by prior in vitro T helper 2 effector generation with aged T cells. This improvement in cognate function of the aged effectors may be related to the enhancement of CD154 expression, which occurs on aged T cells in the presence of exogenous IL-2. We also found no difference in B-cell expansion and differentiation when young cells were transferred to young or aged hosts. Our results indicate that age-related reductions in humoral responses are mainly due to defects in the cognate helper function of naive CD4(+) T cells from aged individuals.

Fig. 1. Model for how defects in naive CD4⁺ T-cell function occur with increasing age

In young animals, thymic output is high, leading to a rapid turnover of T cells in the periphery. As animals age and thymic involution turnover of T cells in the periphery of aged animals must decline. This model would result in T cells in the periphery of aged animals being chronologically older than those in younger animals.

Fig. 2. In vitro responses of naive CD4⁺ T cells from aged mice can be enhanced by the addition of exogenous interleukin (IL)-2

Purified populations of transgenic (Tg) CD4⁺ T cells from young (black) and aged (red) mice were stimulated with peptide antigen and antigen-presenting cell (APC) (DCEK—ICAM fibroblasts) with or without exogenous IL-2 (80 U/ml) for 4 days. (A) Fold expansion of each effector population was calculated after 4 days of culture. (B) Equal numbers of day 4 effectors were restimulated with Ag/APC alone. Supernatants were recovered after 24 h and assayed for the presence of IL-2. (C) Young (open) and aged (shaded) day 4 effectors were stained for CD4, Vβ3, and the indicated cell-surface markers. Flow cytometry histograms are gated on CD4⁺Vβ3⁺ cells. Dotted lines represent isotype-control staining. (D) Young and aged naive Tg CD4⁺ T cells were cultured under Th1 polarizing conditions for 4 days. Equal numbers of effectors were restimulated with Ag/APC alone. Supernatants were recovered after 24 h and assayed for the presence of IL-2 and interferon γ. (E) Young and aged naive Tg CD4⁺ T cells were cultured under Th2 polarizing conditions for 4 days. Equal numbers of effectors were restimulated with Ag/APC alone. Supernatants were recovered after 24 h and assayed for the presence of IL-4 and IL-5. For all graphs *P < 0.05 by Student’s t-test.

Fig. 3. Naive CD4⁺ T cells from aged mice exhibit reduced in vivo expansion and cognate helper function

Purified populations of transgenic (Tg) CD4⁺ T cells from young (black) and aged (red) mice were transferred i.v. to CD4KO hosts (10⁶ per host). Hosts were immunized with 200 μg of 4-hydroxy-3-nitrophenyl acetyl conjugated to PCC (NP-PCC)/alum i.p. on the same day. (A) On day 6, the recovery of donor T cells was determined by flow cytometry. Donor T cells were identified by CD4 Vβ3 staining. (B) On day 6, cells (spleen and lymph node) from the hosts were stimulated ex vivo with phorbol myristate acetate and ionomycin for 4 h and intracellular staining for interleukin (IL)-2 was performed. The percent of each donor CD4⁺ Vβ3 population staining positive for IL-2 was determined by flow cytometry. (C) On day 14, the number of NP-specific host B cells was determined by staining with NP conjugated to the fluorochrome allophycocyanin (NP-APC). The number of NP⁺ germinal center (GC) phenotype B cells (CD38^lo PNA^hi) was also determined by flow cytometry. (D) On day 14, serum was collected from each host and assayed for the presence of NP-specific immunoglobulin G1 (IgG1) antibodies. For all graphs *P < 0.05 by Student’s t-test.

Fig. 4. In vitro-generated Th2 effectors generated from aged naive cells exhibit good cognate helper function

Th2 effectors were generated from young (black) and aged (red) CD4⁺ T cells and then transferred i.v. into CD4KO hosts(10⁷ per host). Hosts were immunized with 200 μg of 4-hydroxy-3-nitrophenyl acetyl conjugated to PCC (NPPCC)/alum i.p. on the same day. (A) On day 14, the number of NP-specific host B cells was determined by staining with NP-AC. The number of NP⁺ germinal center (GC) phenotype B cells (CD38^lo PNA^hi) was also determined by flow cytometry. (B) On day 14, serum was collected from each host and assayed for the presence of NP-specific immunoglobulin G1 (IgG1) antibodies.

Fig. 5. CD154 expression and NFκB binding of aged CD4⁺ T cells can be enhanced by culturing with exogenous interleukin (IL)-2

Purified populations of transgenic (Tg) CD4⁺ T cells from young (black) and aged (red) mice were stimulated with peptide antigen and antigen-presenting cell (DCEK—intercellular adhesion molecule fibroblasts) with or without exogenous IL-2 (80 U/ml). (A) At the indicated time points, the expression of CD154 was determined on the CD4⁺Vβ3⁺ populations by flow cytometry. (B) After 4 days of culture, equal numbers of each effector population were stimulated with anti-Vβ3/anti-CD28 for 4 h and nuclear extracts were then prepared. Electrophoretic mobility shift assays were performed using [³²P]dCTP-labeled probe for NFκB.

Fig. 6. Young donor CD4⁺ T cells exhibit good cognate function in young and aged hosts

Purified populations of transgenic (Tg) CD4⁺ T cells from young mice were transferred i.v. to young (black) or aged (red) CD4KO hosts (10⁶ per host). Hosts were immunized with 200 μg of 4-hydroxy-3-nitrophenyl acetyl conjugated to PCC (NP-PCC)/alum i.p. on the same day. (A) On day 14, the number of NP-specific host B cells was determined by staining with NP-APC. The number of NP⁺ germinal center (GC) phenotype B cells (CD38^lo PNA^hi) was also determined by flow cytometry. (B) On day 14, serum was collected from each host and assayed for the presence of NP-specific immunoglobulin G1 (IgG1) antibodies.