Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo.

The development of effector and memory CD4 cell populations depends upon both T cell receptor (TCR) engagement of peptide/major histocompatibility complex (MHC) class II complexes and ligation of costimulatory molecules with counter receptors on antigen-presenting cells (APCs). We showed previously that sustained interactions with APCs could be crucial for optimal expansion of CD4 cells and for development of effectors that secrete cytokines associated with Th2 cells. Using an adoptive transfer model with TCR transgenic CD4 cells, we now show that responses of CD4 cells primed in B cell-deficient mice become aborted, but are fully restored upon the transfer of activated B cells. Although B cells have the capacity to secrete multiple cytokines that could affect CD4 priming, including IL-4, we were unable to distinguish a role for cytokines that are secreted by B cells. However, B cell costimulation via the OX40L/OX40 pathway that has been implicated in CD4 cell expansion, survival, and Th2 development was required. Th2 but not Th1 responses were impaired in OX40L-deficient recipients and normal responses were restored with OX40L sufficient B cells. The results suggest that without engagement of OX40L on B cells, CD4 cell responses to many protein Ag would be dominated by Th1 cytokines. These data have important implications for strategies to achieve optimal priming of CD4 subsets.

Availability of antigen-presenting cells can determine the extent of CD4 effector expansion and priming for secretion of Th2 cytokines in vivo.

Like dendritic cells (DC), activated B cells are effective antigen-presenting cells (APC) for naïve CD4 cells due to their expression of MHC class II and multiple costimulatory molecules. We showed previously that CD4 cells primed in B cell-deficient micro MT) mice undergo more limited expansion than in normal animals after immunization with keyhole limpet hemocyanin. Here we report that in the absence of B cells, priming of effectors with the capacity to produce the Th2 cytokines, IL-4, IL-5 and IL-13, was profoundly reduced whereas the development of effectors that secrete the Th1 cytokine IFN-gamma was much less affected. A blockade of IL-12 reduced priming of IFN-gamma-secreting effectors but did not reverse the IL-4, IL-5, or IL-13 deficiency of the response. CD4 cell expansion and priming for Th2 cytokines in micro MT mice was reconstituted by adoptive transfer of activated splenic B cells, which were present throughout the primary response. However, transfer of splenic DC from either control or micro MT mice also supported development of Th2 cytokine responses, indicating that an APC deficit rather than a unique contribution of B cells accounted for diminished effector priming. We conclude that CD4 cell expansion must be sustained via APC for the development of Th2 cytokine-secreting effectors in vivo and that in responses to protein antigen, B cells can be a crucial population to serve in this role. The results suggest that the level of APC engagement can not only determine the extent of effector expansion, but also the overall Th1/Th2 cytokine balance.

Parathyroid hormone-related protein response to hyperoxic lung injury.

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 +/- 710 pg/ml (mean +/- SE) at 48 h of hyperoxia. Levels fell to 299 +/- 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h (P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 +/- 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 +/- 153 pg/ml at 48 h of recovery (P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD(50), concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.

Withdrawal of stimulation may initiate the transition of effector to memory CD4 cells.

The initial steps that determine development of memory in CD4 cells are unknown. To distinguish an intrinsic capacity of effectors to become memory cells from contributions of as yet undefined survival factors, we analyzed the effects of withdrawal of signals via TCR, costimulation, and cytokines from Th1 or Th2 primary effectors induced in vitro from TCR-transgenic CD4 cells. Withdrawal of stimulation caused the transition of effectors to resting populations with a memory phenotype that did not undergo division following transfer to normal syngeneic recipients. The return of effectors to rest was accompanied by acquisition of the capacity to function as memory cells in vivo as defined by extended persistence and a more rapid response to Ag in vivo than naive cells in adoptive hosts. Upon challenge with Ag, these in vitro-rested Th1 and Th2 cells were similar to long-term in vivo-rested memory cells, but distinct from in vitro-generated primary effectors and in vivo-restimulated memory effectors by their ability to resist apoptosis. Cessation of stimulation may occur when activated CD4 cells exit lymphoid tissues after priming and transition to memory may be initiated if effectors either fail to gain access to Ag in peripheral tissues where restimulation can lead to activation-induced cell death or do not receive sufficient stimuli to continue a response. Our results suggest that the first stage leading to stable CD4 memory could occur stochastically and independently of instructional processes and as such, the development of memory may be a default pathway when signals that direct responses are not received.