Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics.

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

B cells promote CD8 T cell primary and memory responses to subunit vaccines.

The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called "helper" cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Anti-CD8 monoclonal antibody-mediated depletion alters the phenotype and behavior of surviving CD8+ T cells.

It is common practice for researchers to use antibodies to remove a specific cell type to infer its function. However, it is difficult to completely eliminate a cell type and there is often limited or no information as to how the cells which survive depletion are affected. This is particularly important for CD8+ T cells for two reasons. First, they are more resistant to mAb-mediated depletion than other lymphocytes. Second, targeting either the CD8α or CD8ß chain could induce differential effects. We show here that two commonly used mAbs, against either the CD8α or CD8ß subunit, can differentially affect cellular metabolism. Further, in vivo treatment leaves behind a population of CD8+ T cells with different phenotypic and functional attributes relative to each other or control CD8+ T cells. The impact of anti-CD8 antibodies on CD8+ T cell phenotype and function indicates the need to carefully consider the use of these, and possibly other "depleting" antibodies, as they could significantly complicate the interpretation of results or change the outcome of an experiment. These observations could impact how immunotherapy and modulation of CD8+ T cell activation is pursued.

Antigen-inexperienced memory CD8+ T cells: where they come from and why we need them.

Memory-phenotype CD8+ T cells exist in substantial numbers within hosts that have not been exposed to either foreign antigen or overt lymphopenia. These antigen-inexperienced memory-phenotype T cells can be divided into two major subsets: 'innate memory' T cells and 'virtual memory' T cells. Although these two subsets are nearly indistinguishable by surface markers alone, notable developmental and functional differences exist between the two subsets, which suggests that they represent distinct populations. In this Opinion article, we review the available literature on each subset, highlighting the key differences between these populations. Furthermore, we suggest a unifying model for the categorization of antigen-inexperienced memory-phenotype CD8+ T cells.

Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner.

Virtual memory cells (VM) are an antigen-specific, memory phenotype CD8 T-cell subset found in lymphoreplete, unchallenged mice. Previous studies indicated that VM cells were the result of homeostatic proliferation (HP) resembling the proliferation observed in a lymphopenic environment. Here we demonstrate that HP is ongoing in lymphoreplete mice, the degree of which is dictated by the number of naive CD8 T cells with a sufficiently high affinity for self-antigen interacting with peripheral IL-15. VM cell transcriptional profiles suggest a capacity to mediate protective immunity via antigen non-specific bystander killing, a function we show is dependent on IL-15. Finally, we show a VM-like population of human cells that accumulate with age and traffic to the liver, displaying phenotypic and functional attributes consistent with the bystander protective functions of VM cells identified in the mouse. These data identify developmental and functional attributes of VM cells, including their likely role in protective immunity.

CD8α+ dendritic cell trans presentation of IL-15 to naive CD8+ T cells produces antigen-inexperienced T cells in the periphery with memory phenotype and function.

Various populations of memory phenotype CD8(+) T cells have been described over the last 15-20 y, all of which possess elevated effector functions relative to naive phenotype cells. Using a technique for isolating Ag-specific cells from unprimed hosts, we recently identified a new subset of cells, specific for nominal Ag, but phenotypically and functionally similar to memory cells arising as a result of homeostatic proliferation. We show in this study that these virtual memory (VM) cells are independent of previously identified innate memory cells, arising as a result of their response to IL-15 trans presentation by lymphoid tissue-resident CD8α(+) dendritic cells in the periphery. The absence of IL-15, CD8(+) T cell expression of either CD122 or eomesodermin or of CD8a(+) dendritic cells all lead to the loss of VM cells in the host. Our results show that CD8(+) T cell homeostatic expansion is an active process within the nonlymphopenic environment, is mediated by IL-15, and produces Ag-inexperienced memory cells that retain the capacity to respond to nominal Ag with memory-like function. Preferential engagement of these VM T cells into a vaccine response could dramatically enhance the rate by which immune protection develops.

Maternal impairment of transposon regulation in Drosophila melanogaster by mutations in the genes aubergine, piwi and Suppressor of variegation 205.

TP5, a P element inserted in the telomere-associated sequences of the X chromosome, represses the excision of other P elements in the germ line through a combination of maternal and zygotic effects. The maternal component of this repression is impaired by heterozygous mutations in the aubergine and Suppressor of variegation 205 genes; one mutation in the piwi gene also appears to impair repression. In the female germ line, the level of TP5 mRNA is increased by these impairing mutations. The impairing aubergine and piwi mutations also increase the level of germ-line mRNA from CP, a transgene that encodes the P-element transposase; however, the Suppressor of variegation 205 mutation does not. These findings are discussed in terms of a model of P-element regulation that involves post-transcriptional and chromatin re-organizing events mediated by maternally transmitted small RNAs derived from the telomeric P element.

Does RNA interference influence meiotic crossing over in Drosophila melanogaster?

Mutations in the RNA interference (RNAi) genes aubergine (aub), homeless and piwi were tested for effects on the frequency, distribution and coincidence of meiotic crossovers in the long arm of the X chromosome. Some increases in crossover frequency were seen in these tests, but they may have been due to a maternal effect of the balancer chromosomes that were used to maintain the RNAi mutations in stocks rather than to the RNAi mutations themselves. These same balancers produced strong zygotic interchromosomal effects when tested separately. Mutations in aub and piwi did not affect the frequency of crossing over in the centric heterochromatin of chromosome II; nor did a balancer chromosome III.