Immunological memory represents the ability of specific immune cells to recall an encounter with a cognate pathogen. This results in an immediate response upon a second exposure to the same infectious agent. Thus, conventional memory T cells are generated after encountering specific foreign antigens. In contrast, a group of innate memory T cells develops without initial exposure to foreign antigen that specifically binds to a cognate T cell receptor.1 These naturally occurring memory-like T cells, also known as “virtual” memory T cells, are generated under steady-state conditions. They accumulate during aging in both humans and mice.2 By comparing the TCR repertoire of young and old mice, an early study identified age-dependent clonal expansion and age-related changes in CD8+ but not CD4+ T cells.3 Interestingly, more than 50% of CD62LhiCD44hi central memory T cells in the peripheral blood of aged naive mice have been suggested to represent virtual memory T cells based on their lower expression levels of CD49d.4 Infection studies in mice employing Listeria monocytogenes showed that both virtual memory T cells and conventional antigen-specific memory T cells were able to provide protection.5 However, in contrast to virtual memory T cells, conventional memory T cells produced fivefold higher levels of IFN-γ upon short-term in vitro TCR stimulation.5
The migration properties of different types of naive and memory immune cells have been studied for decades in animals using intravenous adoptive cell transfer. Although this approach is suited to studying the homing of immune cells from blood via specialized high endothelial vessels to lymph nodes (LNs), it does not provide any information regarding LN homing of cells that arrive via afferent lymphatics. To study the mechanisms permitting entry of lymph-derived cells and their subsequent intranodal positioning, we established an intralymphatic (IL) cell transfer technique in mice. With the help of microinjectors, immune cells are placed into the lymphatic vessels draining from the foot paw toward the popliteal LN.6 This approach revealed that in noninflamed LNs, lymph-derived naive CD4 T cells enter the paracortical T cell zone (TCZ) from the medullary sinuses, whereas activated T cells and dendritic cells enter the TCZ through the subcapsular sinus (SCS) floor at interfollicular areas (IFA). Both routes of homing rely on chemokine receptors, with CCR7 playing a prominent role.7 In addition, the atypical chemokine receptor ACKR4 is expressed on endothelial cells of the ceiling but not of the floor of the SCS. By scavenging CCR7 ligands, ACKR4 actively shapes chemokine gradients pointing from the SCS lumen toward the LN parenchyma that facilitate homing of cells into the TCZ.8
In the present study, we investigated how virtual memory CD8 T cells behave once they arrive at the SCS following IL transfer. CD8 T cells from LNs and spleen of naive aged mice were MACS-purified and, based on the expression of CD3, CD8, CD62L, and CD44, subsequently sorted into virtual central memory (Tcm; CD3+CD8+CD44+CD62L+) and virtual effector memory (Tem; CD3+CD8+CD44+CD62L−) populations. Tcm and Tem populations were stained with eFluor450 (red) or eFluor670 (green), respectively, or vice versa. Subsequently, they were then IL injected at a 1:1 ratio into lymphatic vessels draining toward the popliteal LN of mice. The recipients received either 106 plaque-forming units of mouse cytomegalovirus (MCMV) or PBS 3 days earlier by footpad injection. Three hours after IL administration, the positioning of transferred cells in the SCS, the IFA, the TCZ and the medulla of the popliteal LN was determined histologically. In noninflamed LNs, approximately half of the Tcm population migrated deeply into the TCZ, while the other cells were found in the IFA and the medulla but were hardly present in the SCS (Fig. 1a). In contrast, under steady-state conditions, Tem cells were primarily found in the medulla and to some degree in the IFA and the SCS but were not detected in the TCZ (Fig. 1a). Interestingly, IL transfer to virus-inflamed LNs showed a different picture. In this case, neither Tcm nor Tem cells made it into the TCZ, and both populations were detected in the medulla and the SCS (Fig. 1a). In particular, Tem cells showed a strong accumulation in the SCS. Immunohistology also revealed a strong accumulation of CD11b+ and CD11c+ cells in the IFA, most likely reflecting monocytes and DCs, respectively (Fig. 1b).
Altogether, the data from the present study indicate that Tcm cells, but not Tem cells, arriving via afferent lymphatics in noninflamed LNs can enter the TCZ to potentially scan DCs for presented cognate antigens. In contrast, Tem cells in particular and, to a lower degree, Tcm cells are retained in the SCS in inflamed LNs to potentially either directly combat virus-infected cells in that area or become reactivated in the IFA. Such behavior would also be in line with the role of other immune cells positioned in direct proximity to the SCS floor that have been shown to play an essential role in mounting adaptive immune responses. For instance, LN SCS macrophages clear lymph-borne viruses and present them to antiviral B cells.9 In addition, innate-like lymphocytes located close to the SCS actually migrate back and forth to the SCS lumen and rapidly produce IL-17 upon bacterial and fungal challenge.10
Collectively, our findings provide new insights into the mechanisms that drive homing and intranodal migration of lymph-derived memory CD8 T cells under steady-state and inflamed conditions.
Jameson, S. C., Lee, Y. J. & Hogquist, K. A. Innate memory T cells. Adv. Immunol. 126, 173–213 (2015).
Nikolich-Zugich, J. Ageing and life-long maintenance of T-cell subsets in the face of latent persistent infections. Nat. Rev. Immunol. 8, 512–522 (2008).
Callahan, J. E., Kappler, J. W. & Marrack, P. Unexpected expansions of CD8-bearing cells in old mice. J. Immunol. 151, 6657–6669 (1993).
Chiu, B. C., Martin, B. E., Stolberg, V. R. & Chensue, S. W. Cutting edge: Central memory CD8 T cells in aged mice are virtual memory cells. J. Immunol. 191, 5793–5796 (2013).
Lee, J. Y., Hamilton, S. E., Akue, A. D., Hogquist, K. A. & Jameson, S. C. Virtual memory CD8 T cells display unique functional properties. Proc. Natl Acad. Sci. USA 110, 13498–13503 (2013).
Braun, A. et al. Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration. Nat. Immunol. 12, 879–887 (2011).
Martens, R. et al. Efficient homing of T cells via afferent lymphatics requires mechanical arrest and integrin-supported chemokine guidance. Nat. Commun. 11, 1114 (2020).
Ulvmar, M. H. et al. The atypical chemokine receptor CCRL1 shapes functional CCL21 gradients in lymph nodes. Nat. Immunol. 15, 623–630 (2014).
Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007).
Zhang, Y. et al. Migratory and adhesive cues controlling innate-like lymphocyte surveillance of the pathogen-exposed surface of the lymph node. eLife 5, e18156 https://doi.org/10.7554/eLife.18156 (2016).
This work was supported by Deutsche Forschungsgemeinschaft, (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2155 “RESIST” – Project ID 39087428, DFG grants SFB900-B1 and FOR2830-P006 to RF and Deutsche Akademische Austauschdienst grant GSSP2014-57034101-91557342 to GN
The authors declare no competing interests.
About this article
Cite this article
Nikolova, G., Weiss, S., Bosnjak, B. et al. Differential retention of lymph-borne CD8 memory T cell subsets in the subcapsular sinus of resting and inflamed lymph nodes. Cell Mol Immunol (2020). https://doi.org/10.1038/s41423-020-0451-6