Donna L. Farber

Cutting Edge: Tissue-Retentive Lung Memory CD4 T Cells Mediate Optimal Protection to Respiratory Virus Infection

We identify in this article a new class of lung tissue-resident memory CD4 T cells that exhibit tissue tropism and retention independent of Ag or inflammation. Tissue-resident memory CD4 T cells in the lung did not circulate or emigrate from the lung in parabiosis experiments, were protected from in vivo Ab labeling, and expressed elevated levels of CD69 and CD11a compared with those of circulating memory populations. Importantly, influenza-specific lung-resident memory CD4 T cells served as in situ protectors to respiratory viral challenge, mediating enhanced viral clearance and survival to lethal influenza infection. By contrast, memory CD4 T cells isolated from spleen recirculated among multiple tissues without retention and failed to mediate protection to influenza infection, despite their ability to expand and migrate to the lung. Our results reveal tissue compartmentalization as a major determining factor for immune-mediated protection in a key mucosal site, important for targeting local protective responses in vaccines and immunotherapies.

Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62Lhigh) and effector memory (TEM; CD62Llow) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-γ production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-γ production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly TEM in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on TEM responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of TEM cells in autoimmunity and chronic disease.

The FcRγ Subunit and Syk Kinase Replace the CD3ζ-Chain and ZAP-70 Kinase in the TCR Signaling Complex of Human Effector CD4 T Cells

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3ζ-related FcRγ signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3ε, but not CD3ζ. The TCR/CD3/FcRγ complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.

Heterogeneity of the Memory CD4 T Cell Response: Persisting Effectors and Resting Memory T Cells

Defining the cellular composition of the memory T cell pool has been complicated by an inability to distinguish effector and memory T cells. We present here an activation profile assay, using anti-CD3 and antigenic stimuli, that clearly distinguishes effector and memory CD4 T cells and defines subsets of long-lived memory CD4 T cells based on CD62 ligand (CD62L) expression. The CD62Llow memory subset functionally resembles effector cells, exhibiting hyper-responsiveness to antigenic and anti-CD3 mediated stimuli, high proliferative capacity, and rapid activation kinetics. The CD62Lhigh memory subset functionally resembles resting memory cells, exhibiting hyporesponsiveness to anti-CD3 stimuli, lower proliferative capacity, and slower activation kinetics. Our results indicate that the memory CD4 T cell pool is heterogeneous, consisting of persisting effectors and resting memory T cells.

Memory T Cells in Transplantation: Generation, Function, and Potential Role in Rejection

The adaptive immune system is endowed with long‐lived memory to recall previous antigen encounters and respond more effectively to them. Memory immune responses are mediated by antigen‐specific memory T lymphocytes that exhibit enhanced function compared with naïve T cells that have never encountered antigen. While the generation of memory T cells specific for pathogens is beneficial in providing protective immunity, memory T cells specific for alloantigens can be deleterious to the recipient of a transplanted organ. In graft rejection, memory T cells mediate accelerated, ‘second‐set’ rejection and their presence has been associated with increased propensity for early rejection. Recent findings have demonstrated that alloreactive memory T cells can be generated via exposure to alloantigens, as well as stimuli that are cross‐reactive with alloantigens, and are therefore likely present in ‘naïve’ individuals. This review focuses on the characteristics of memory T cells which make them of special interest to the transplant community, including differential activation requirements, broad homing properties, and resistance to tolerance induction. The multiple ways in which memory T cells can contribute to early and late graft rejection are discussed, as well as potential targets for combating alloreactive memory to be considered in the future design of tolerance induction strategies.

Prolonged Antigen Presentation Is Required for Optimal CD8+ T Cell Responses against Malaria Liver Stage Parasites

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization—a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

CTLA4 Expression Is an Indicator and Regulator of Steady-State CD4+FoxP3+ T Cell Homeostasis

The presence of FoxP3+ regulatory T cells (Tregs) is necessary for control of deleterious immune responses in the steady state; however, mechanisms for maintaining the frequency and quality of endogenous Tregs are not well defined. In this study, we used in vivo modulators of the CD28 and CTLA4 pathways administered to intact mice to reveal mechanisms controlling the homeostasis and phenotype of endogenous Tregs. We demonstrate that expression of the negative costimulatory regulator CTLA4 on FoxP3+ Tregs in vivo is a direct consequence of their rapid, perpetual homeostasis. Up-regulation of CTLA4 expression occurs only on FoxP3+ Tregs undergoing extensive proliferation and can be abrogated by inhibiting the CD28 pathway, coinciding with a reduction in FoxP3+ Treg proliferation and frequency. We further demonstrate that CTLA4 negatively regulates steady-state Treg homeostasis, given that inhibiting CTLA4 signaling with an anti-CTLA4 blocking Ab greatly enhances Treg proliferation and overall Treg frequency. Our findings provide new insight into the origin and role of CTLA4 expression on natural FoxP3+ Tregs and reveal opposing effects of costimulation modulators on the steady-state level and quality of Tregs, with implications regarding their effects on endogenous Tregs in patients receiving immunotherapy.

Regulation of CD103 Expression by CD8 + T Cells Responding to Renal Allografts

CD103 is an integrin with specificity for the epithelial cell-specific ligand, E-cadherin. Recent studies indicate that CD103 expression endows peripheral CD8 cells with a unique capacity to access the epithelial compartments of organ allografts. In the present study we used a nonvascularized mouse renal allograft model to 1) define the mechanisms regulating CD103 expression by graft-infiltrating CD8 effector populations, and 2) identify the cellular compartments in which this occurs. We report that CD8 cells responding to donor alloantigens in host lymphoid compartments do not initially express CD103, but dramatically up-regulate CD103 expression to high levels subsequent to migration to the graft site. CD103+CD8+ cells that infiltrated renal allografts exhibited a classic effector phenotype and were selectively localized to the graft site. CD8 cells expressing low levels of CD103 were also present in lymphoid compartments, but three-color analyses revealed that these are almost exclusively of naive phenotype. Adoptive transfer studies using TCR-transgenic CD8 cells demonstrated that donor-specific CD8 cells rapidly and uniformly up-regulate CD103 expression following entry into the graft site. Donor-specific CD8 cells expressing a dominant negative TGF-β receptor were highly deficient in CD103 expression following migration to the graft, thereby implicating TGF-β activity as a dominant controlling factor. The relevance of these data to conventional (vascularized) renal transplantation is confirmed. These data support a model in which TGF-β activity present locally at the graft site plays a critical role in regulating CD103 expression, and hence the epitheliotropism, of CD8 effector populations that infiltrate renal allografts.

Differential SLP-76 Expression and TCR-Mediated Signaling in Effector and Memory CD4 T Cells

We present in this study novel findings on TCR-mediated signaling in naive, effector, and memory CD4 T cells that identify critical biochemical markers to distinguish these subsets. We demonstrate that relative to naive CD4 T cells, memory CD4 T cells exhibit a profound decrease in expression of the linker/adapter molecule SLP-76, while effector T cells express normal to elevated levels of SLP-76. The reduced level of SLP-76 is memory CD4 T cells is coincident with reduced phosphorylation overall, yet the residual SLP-76 couples to a subset of TCR-associated linker molecules, leading to downstream mitogen-activated protein (MAP) kinase activation. By contrast, effector CD4 T cells strongly phosphorylate SLP-76, linker for activation of T cells, and additional Grb2-coupled proteins, exhibit increased associations of SLP-76 to phosphorylated linkers, and hyperphosphorylate downstream Erk1/2 MAP kinases. Our results suggest distinct coupling of signaling intermediates to the TCR in naive, effector, and memory CD4 T cells. Whereas effector CD4 T cells amplify existing TCR signaling events accounting for rapid effector responses, memory T cells engage fewer signaling intermediates to efficiently link TCR triggering directly to downstream MAP kinase activation.

T Cell Rewiring in Differentiation and Disease

The TCR-mediated signals leading to IL-2 production by T cell lines and naive T cells have been well characterized; however, the biochemical signaling mechanisms leading to disparate functions such as differentiation, effector cytokine production, death, and dysfunction due to immune pathologies are