Ageing is a multifactorial process [24, 25] integrating general metabolic changes and immune specific alterations such as progressive decrease in thymic export [21–23]. An expected consequence of declining T cell production is the development of progressive T cell lymphopenia. The incidence of T cell lymphopenia during human ageing is extensively reported in blood samples [11–13] but a more controversial picture emerges from lymphoid organs in murine studies. T cell numbers recovered from spleen or pooled secondary lymphoid organs are reported to be unchanged [24, 39] or reduced [14, 15] depending notably on the organs considered. In that respect, our current analysis in C57BL/6 mice reconciles this apparent contradiction regarding cell numbers recovered from secondary lymphoid organs: mesenteric lymph nodes and spleen being essentially preserved, whereas superficial lymph nodes studied exhibited significant T cell depletion affecting primarily CD4 T cell in middle-aged animals but also CD8 T cells at older ages. A second common observation regarding immune ageing is the differential behaviour of CD4 and CD8 T cell compartments when ageing developed . Evidence of T cell lymphopenia differently affecting CD4 and CD8 T cell compartments is also described in bone marrow transplantation protocols or in a model of genetic thymectomy [15, 34, 35]. These differential behaviours of CD4 and CD8 T cells are also substantiated by phenotypic and molecular analyses [28, 36, 44]. Numerous publications attribute such differential behaviour to specific age related oligoclonal expansion of CD8 T cells both in mice and humans [45–48]. Accordingly, we observed that CD4 T cell numbers recovered from secondary lymphoid organs were rapidly affected: a significant decay was detected in middle-aged animals, whereas CD8 T cells were essentially preserved. However, we demonstrated here that differential behaviour of CD4 and CD8 T cells also arise among their naïve T cell compartments. The size of the naïve T cell compartment theoretically results from the combined effect of thymic production, homeostatic control and peripheral activation leading to recruitment of T cells from the naïve pool. Because loss of thymic production observed during ageing affects equally single positive CD4 or CD8 thymocytes production , our results suggest that naïve CD8 T cells are preserved due to poor turn over, whereas naïve CD4 T cell pool appears highly stimulated and directly impacted by partial reduction in thymic production. This faster consumption rate of CD4 T cells may reflect the more pleiotropic function of CD4 T cells compared to CD8 T cells, CD4 T cells being central partners of innate, humoral and cytotoxic responses.
A third classical feature of T cell ageing is a shift from naïve towards effector/memory T cell predominance observed in blood and lymphoid organs . This shift may rely on either naïve T cell decay and/or increase of the effector/memory fraction. By providing naïve and effector/memory absolute numbers, we demonstrated that a different balance between naïve decay and increase in effector/memory cells seems to develop among CD4 and CD8 T cells. In middle-aged animals, naïve CD4 T cell decay was prominent whereas effector/memory CD4 T cell numbers were, albeit significantly, only mildly increased. To note, we excluded regulatory T cells, a particular subset of CD4 T cells exhibiting suppressive activity, from naïve and effector/memory CD4 T cell pool analysis. In contrast, CD8 T cell numbers were essentially preserved in middle-aged animals: no significant difference was detected when considering total, naïve or effector/memory CD8 T cell numbers separately. In old animals, a different picture emerged: a prominent increase in effector/memory CD8 but not CD4 T cells was detected. Our data suggest that changes in activation profile is essentially related to decrease in naïve T cell numbers when considering CD4 T cell compartment, whereas oligoclonal CD8 expansion occurring in older mice also contributes to naïve towards effector/memory shift among CD8 T cell compartment. Collectively, we demonstrated the existence of an unparalleled impact of ageing on naïve CD4 and effector/memory CD8 T cells.
More importantly, we provided in this report the first evaluation of the impact of ageing on CD4 and CD8 T cells residing in secondary and tertiary lymphoid organs and in non-lymphoid tissues. In striking contrast to secondary lymphoid organs, we detected a progressive accumulation of CD4 T cells in all gut associated lymphoid tissues (GALT) considered: Peyer’s patches, lamina propria from the small intestine and the colon. Such increase was not detected for CD8 T cells in Peyer’s patches but was observed at a lesser amplitude in lamina propria. CD4 T cell numbers recovered from lamina propria were consistently up to 6-fold higher than CD8 T cell numbers recovered. Thus, although both CD4 and CD8 T cells appear to accumulate in the lamina propria with age, such effect may be particularly crucial for the CD4 T cell compartment. As a control, we next analysed CD4 and CD8 T cell accumulation in lungs and liver. CD8 but not CD4 T cell accumulation was detected in the lungs mucosa associated lymphoid tissues suggesting CD4 T cell accumulation in the intestine was a specific feature of the GALT. Analyses of T cell recovery in the liver revealed essentially preserved T cell numbers although later time points may be required to fully ascertain the impact of ageing on liver resident T cells. This observation is in accordance with previous publications demonstrating a high increase of DN T cell rather than CD4 or CD8 T cells in aged liver [40, 41]. Thus, CD4 and CD8 T cell distribution/accumulation during ageing appears highly differing depending on the tissues considered. The mechanisms responsible for such predominant age dependent accumulation of CD4 T cells in the GALT remain to be further investigated. Two main hypotheses can be formulated: CD4 T cell accumulation in the intestine may reflect age related skewing of CD4 T cell distribution from secondary lymphoid organs to the GALT or specific proliferative activity of CD4 T cells residing in the gut ensuring local CD4 T cell production. Evaluating Ki67 expression in GALT CD4 T cells provided insight showing that local proliferation is not significantly increased but even reduced during ageing. This observation suggests that change in T cell numbers in the gut are predominantly induced by recruitment rather than local proliferation. Accordingly, preserved proportion of FOXP3+ regulatory T cells suggests that CD4 T cell accumulation applies to both conventional and regulatory T cells. Additionally, we detected an association between the severity of T cell lymphopenia at lymphoid sites and CD4 T cell accumulation in colonic lamina propria (cLP). The absence of association with small intestine lamina propria (siLP) may reflect either the lower fold of CD4 T cell accumulation in siLP although different mechanisms of accumulation developing in cLP and siLP respectively cannot be excluded. Increasing numbers of reports demonstrated the crucial role of microbial colonization of mucosal site and mucosal immunity on immune responses [50, 51]. It is tempting to speculate that mucosal immunity may directly influence the severity of lymphoid T cell lymphopenia. However, we cannot strictly discriminate in our experimental settings the causal or consequential link between lymphoid T cell lymphopenia and mucosal accumulation. This question will require further investigations.
Collectively, analyses of T cell recovery in multiple sites indicate that T cell lymphopenia is not a consistent feature of ageing: T cell lymphopenia was essentially restricted to the CD4 subset in some secondary lymphoid organs. Such heterogeneity questions the exact incidence of T cell lymphopenia developing during ageing. One may consider that redistribution of CD4 T cells rather than T cell lymphopenia is the crucial phenomena occurring during ageing.
Finally, these observations are also highly relevant in context of accelerated ageing. For instance, HIV infection is frequently defined as accelerated ageing, due to persistent T cell lymphopenia and skewed naïve to effector/memory phenotype. However, HIV infection is associated to drastic CD4 T cell depletion in the gut prior to the progressive depletion detected in the blood . Such observation constitutes a major drawback to the characterization of HIV infection as an accelerated ageing process. One may question the long-term thread that local CD4 T cell depletion in the gut may induce in old HIV infected patients.