Susanne Heinzel

Antigen affinity, costimulation, and cytokine inputs sum linearly to amplify T cell expansion

T cell responses are initiated by antigen and promoted by a range of costimulatory signals. Understanding how T cells integrate alternative signal combinations and make decisions affecting immune response strength or tolerance poses a considerable theoretical challenge. Here, we report that T cell receptor (TCR) and costimulatory signals imprint an early, cell-intrinsic, division fate, whereby cells effectively count through generations before returning automatically to a quiescent state. This autonomous program can be extended by cytokines. Signals from the TCR, costimulatory receptors, and cytokines add together using a linear division calculus, allowing the strength of a T cell response to be predicted from the sum of the underlying signal components. These data resolve a long-standing costimulation paradox and provide a quantitative paradigm for therapeutically manipulating immune response strength. T cells follow a linear calculation when integrating costimulatory and cytokine signals. Stimulatory signals add up for T cells T cell activation is a dynamic process. T cells encounter multiple input signals such as antigens, costimulatory molecules, and cytokines at different times and anatomical locations during an infection. But how do T cells integrate this information to determine the extent to which they divide? To find out, Marchingo et al. stimulated mouse T cells in culture with different combinations of inputs and also tracked antigen-specific T cell responses in mice infected with influenza virus. They found that T cells linearly sum the various stimulatory inputs they receive to determine their “division destiny.” Science, this issue p. 1123

Peptide HER2(776-788) represents a naturally processed broad MHC class II-restricted T cell epitope.

HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776–788) [designated HER2(776–788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-γ] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776–788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776–788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRVbeta6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HLA-DR, thus demonstrating that HER2(776–788) represents a naturally processed and presented epitope. Our data demonstrate that helper peptide HER2(776–788) represents a promiscuous epitope binding to at least three HLA-DR alleles, thus offering a broad population coverage. The use of antigenic peptides presented by major histocompatibility complex (MHC) class II in addition to those presented by class I may improve the therapeutic efficacy of active immunization.

Immunogenicity and safety of Advax™, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis B surface antigen: a randomized controlled Phase 1 study.

There is a need for additional safe and effective human vaccine adjuvants. Advax™ is a novel adjuvant produced from semi-crystalline particles of delta inulin. In animal studies Advax enhanced humoral and cellular immunity to hepatitis B surface antigen (HBsAg) without inducing local or systemic reactogenicity. This first-in-man Phase 1 clinical trial tested the safety and tolerability of three intramuscular doses of HBsAg formulated with Advax in a group of healthy adult subjects. Advax was well tolerated with injection site pain scores not significantly different to subjects receiving HBsAg alone and no adverse events were reported in subjects that received Advax. Seroprotection and HBsAb geometric mean titers (GMT) after three immunizations were higher in the Advax 5mg (seroprotection 5/6, 83.3%, GMT 40.7, 95% CI 11.9-139.1) and 10mg (seroprotection 4/5, 80%, GMT 51.6, 95% CI 10.0-266.2) groups versus HBsAg alone (seroprotection 1/5, 20%, GMT 4.1, 95% CI 1.3-12.8). Similarly the proportion of subjects with positive CD4 T-cell responses to HBsAg was higher in the Advax 5mg (4/6, 67%) and Advax 10mg (4/5, 80%) groups versus HBsAg alone (1/5, 20%). These results confirm the safety, tolerability and immunogenicity of Advax adjuvant observed in preclinical studies. Advax may represent a suitable replacement for alum adjuvants in prophylactic human vaccines subject to confirmation of current results in larger studies. Australia and New Zealand Clinical Trial Registry: ACTRN12607000598482.

Mutually exclusive regulation of T cell survival by IL-7R and antigen receptor-induced signals

Two major processes govern T cell proliferation and survival: interleukin-7-mediated homeostasis and antigen-induced selection. How cells transit between the two states is unknown. Here we show that T cell receptor ligation actively inhibits homeostatic survival signals while initiating a new, dominant survival programme. This switch is mediated by a change in the expression of pro- and anti-apoptosis proteins through the downregulation of Bcl-2 and the induction of Bim, A1 and Bcl-xL. Calcineurin inhibitors prevent the initiation of the new survival programme, while permitting the dominant repression of Bcl-2. Thus, in the presence of these drugs the response to antigen receptor ligation is cell death. Our results identify a molecular switch that can serve as an attractive target for inducing antigen-specific tolerance in treating autoimmune disease patients and transplant recipients.

Stretched cell cycle model for proliferating lymphocytes

Significance Cell division is essential for an effective immune response. Estimates of rates of division are often based on DNA measurements interpreted with an appropriate model for internal cell cycle steps. Here we use time-lapse microscopy and single cell tracking of T and B lymphocytes from reporter mice to measure times spent in cell cycle phases. These data led us to a stretched cell cycle model, a novel and improved mathematical description of cell cycle progression for proliferating lymphocytes. Our model can be used to deduce cell cycle parameters for lymphocytes from DNA and BrdU labeling and will be useful when comparing the effects of different stimuli, or therapeutic treatments on immune responses, or to understand molecular pathways controlling cell division. Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G1 phase, and many mathematical models assume a constant time for traversing the S/G2/M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G2/M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

The pro-survival proteins of the BCL-2 family regulate the survival of all cells, and genetic deletion models for these proteins have revealed which specific BCL-2 family member(s) is/are critical for the survival of particular cell types. A1 is a pro-survival BCL-2-like protein that is expressed predominantly in haematopoietic cells, and here we describe the characterisation of a novel mouse strain that lacks all three functional isoforms of A1 (A1-a, A1-b and A1-d). Surprisingly, complete loss of A1 caused only minor defects, with significant, although relatively small, decreases in γδTCR T cells, antigen-experienced conventional as well as regulatory CD4 T cells and conventional dendritic cells (cDCs). When examining these cell types in tissue culture, only cDC survival was significantly impaired by the loss of A1. Therefore, A1 appears to be a surprisingly redundant pro-survival protein in the haematopoietic system and other tissues, suggesting that its targeting in cancer may be readily tolerated.

The self peptide annexin II (208-223) presented by dendritic cells sensitizes autologous CD4+ T lymphocytes to recognize melanoma cells.

Abstract Annexin II is known to be over-expressed in different types of tumours. We show here that annexin II protein is expressed by melanoma cell lines in various amounts, consistent with previous findings that an annexin II (208–223) peptide could be eluted from isolated HLA-DR molecules of a constitutively MHC class II-positive melanoma line. T cells sensitized to annexin II (208–223) in vitro using peptide-pulsed autologous dendritic cells responded only to the lines which over-expressed annexin II, in a peptide-specific, HLA-DR-restricted fashion. These CD4+ T cells proliferated strongly and secreted large amounts of type 1 cytokines in response to annexin II (208–223) peptide or annexin II protein-positive melanoma cell lines. These results demonstrate that the annexin II (208–223) peptide, corresponding to a non-mutated sequence of a normal protein, induces antigen-specific T cells which can respond to melanoma cells over-expressing the annexin II molecule. This peptide may therefore be useful in immunotherapy for recruiting CD4+ type 1 helper cells active locally in the tumour environment.

T-cell stimuli independently sum to regulate an inherited clonal division fate.

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities.

The regulation of lymphocyte activation and proliferation

Activation induced proliferation and clonal expansion of antigen specific lymphocytes is a hallmark of the adaptive immune response to pathogens. Recent studies identify two distinct control phases. In the first T and B lymphocytes integrate antigen and additional costimuli to motivate a programmed proliferative burst that ceases with a return to cell quiescence and eventual death. This proliferative burst is autonomously timed, ensuring an appropriate response magnitude whilst preventing uncontrolled expansion. This initial response is subject to further modification and extension by a range of signals that modify, expand and direct the emergence of a rich array of new cell types. Thus, both robust clonal expansion of a small number of antigen specific T cells, and the concurrent emergence of extensive cellular diversity, confers immunity to a vast array of different pathogens. The in vivo response to a given pathogen is made up by the sum of all responding clones and is reproducible and pathogen specific. Thus, a precise description of the regulatory principles governing lymphocyte proliferation, differentiation and survival is essential to a unified understanding of the immune system.

Epigenetic targeting of Notch1-driven transcription using the HDACi panobinostat is a potential therapy against T-cell acute lymphoblastic leukemia

Epigenetic targeting of Notch1-driven transcription using the HDACi panobinostat is a potential therapy against T-cell acute lymphoblastic leukemia