Stella C. Knight

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques.

Abstract The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at −196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, v v ) was assessed from the [3H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, v v ) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, v v ) followed by gentle centrifugation (50g). Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.

Use of different cooling rates during freezing to separate populations of human peripheral blood lymphocytes

Human peripheral lymphocytes have been frozen to −196 °C at different cooling rates in dimethylsulfoxide (DMSO) to see whether subpopulations could be separated by different susceptibilities to freezing damage. Recovery of populations was assessed by the incorporation of 3H-thymidine in response to stimulation by nonspecific mitogens phytohemagglutinin (PHA), concanavalin A (CON A), or pokeweed mitogen (PWM) or by a specific antigen (old tuberculin, PPD) during culture in a microplate after thawing. Any intrinsic differences between the susceptibilities of these populations to damage during freezing were too small to allow separation. This was judged by the optimal 3H-thymidine incorporation in response to the different mitogens being recovered at the same cooling rate for a given concentration of DMSO. The cooling rate allowing the optimal uptake of 3H-thymidine was increased as the concentration of DMSO was lowered. It was found that the susceptibility of a population of cells to freezing damage could be altered by activating that population by incubation with a mitogen (CON A) or an antigen (PPD) before freezing. In general this allowed optimal 3H-thymidine incorporation to be recovered at a cooling rate slower than that required by the unactivated lymphocytes. The concentration of mitogen to which the cells were exposed before freezing was an important factor in the relationship between thymidine uptake and cooling rate. These results suggest the use of cryobiological techniques for the separation of populations of cells. We have demonstrated in this work that the state of activation of lymphocytes can be a major factor in determining their susceptibility to freezing damage.

Ultrastructural appearance of freeze-substituted lymphocytes frozen by interrupting rapid cooling with a period at −26°C

Summary Human peripheral lymphocytes in dimethylsulphoxide (5% v/v) were cooled rapidly to either −196° or −26°C for 5 or 60 min before −196°C. Samples were freeze-substituted at −80°C for four weeks before examination with the electron microscope. Aliquots were thawed and tested for function using a lymphocyte culture method. There was good correlation between functional survival on thawing and the shrinkage of the cells at −26°C. Cells that had insufficient time to shrink contained intracellular ice cavities on freeze-substitution and showed little or no survival. The results suggest that protection at a subzero holding temperature may be caused by the same factors that allow cells to survive freezing at optimal conventional cooling rates.

Effect of Human Immunodeficiency Virus on Dendritic Cells Isolated from Human Peripheral Blood

Antigens, including viral antigens, are presented to the immune system on antigen-presenting dendritic cells (DC). For example, human DC pulsed with influenza virus in vitro subsequently initiated proliferation of autologous lymphocytes. The interaction between HIV and human DC was therefore examined. Low density mononuclear cells (5 × 105, around 25% DC plus macrophages (MO) were cultured with 102–103 TC ID50 units of virus (H9-IIIB) and then examined by electron microscopy. After three to five days of culture with virus, DC but not MO from five out of 12 individuals showed HIV budding from the surface of cells. Immunogold labeling experiments showed that some DC expressed low levels of CD4 (T4) antigen. Occasionally the level of class II antigens appeared reduced on infected cell populations. An infection by HIV in vivo might therefore affect the initiation of immune responses by compromising the antigen-presenting function of DC.

HIV Infection of Peripheral Blood Dendritic Cells

Dendritic cells (DC) are important antigen-presenting cells, particularly in the initiation of immune responses in resting T cells and our studies are providing increasing evidence for their involvement in the immunosuppression seen in AIDS. Peripheral blood DC expressed low levels of the CD4 HIV receptor which was up-regulated by exposure in vitro to gamma interferon. Infection of DC with HIV, following in vitro exposure to virus, was demonstrated by electron microscopy (EM) and by a combined in situ hybridization and immunolabelling technique which facilitated identification of infected DC at the light microscope level. Results of in situ hybridization to detect viral RNA and DNA suggest that DC may become latently infected or express very low levels of virus. Thus they could serve as reservoir of potential infection which may escape immune surveillance. DC taken from HIV-infected individuals also contained HIV genome, as identified by the combined in situ hybridization and immunolabelling method. In addition, a fall in the number of DC was observed in infected individuals which preceded the decrease in the number of lymphocytes. Functional studies, on both DC infected in vitro and on DC from infected individuals, revealed a deficiency in antigen presentation. Since depletion and dysfunction of DC precedes the appearance of T-cell defects, this may be a functional lesion in the development of disease.

Chapter 30 – Mixed Leukocyte Reactions

This chapter discusses some of the practical issues of interpretation of the mixed leukocyte reactions (MLR). The MLR can be used to assess histocompatibility differences between two individuals by the recognition of histocompatibility antigens differentially expressed on dendritic cell (DC) of stimulator and responder type. The test also assesses in vitro the ability of DC to initiate primary immune responses either directly or indirectly. The MLR has become the standard functional test to identify DC; DCs are the only cells known to be potent stimulators of primary T cell responses. The experimental assessment of primary stimulation using the MLR will indicate the functional capacity of DC that is in turn affected by many genetic and environmental aspects of the DC. Factors affecting stimulatory capacity of DC include their state of maturity; immature DC may be poor stimulators of allogeneic T cells and may even have tolerogenic effects. The subtype of DC, the types of antigens to which they are exposed and the consequent cytokine profile of the stimulatory DC, all influence the outcome of primary stimulation by DC. Thus, the MLR can be used to assess the basic capacities of DC, including those produced for various forms of immunotherapy, to stimulate primary immune responses.