Methods for augmenting immunological responses through the administration of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEA-S)

Methods for augmenting immune responses in immunodeficient individuals are disclosed. The methods utilize steroid hormones, particularly DHEA, its prohormones (particularly DHEA-S), and DHEA-cogeners. Additional embodiments of the invention include pharmaceutical compositions for use in the methods.

TECHNICAL FIELD 
The invention relates to methods and compositions used for the augmentation 
of an immune response in vivo and in vitro by the use of steroid hormones, 
more specifically, by the use of dehydropepiandrosterone (DHEA), 
dehydroepiandrosterone sulfate (DHEA-S), and analogs thereof. 
BACKGROUND 
Immunosuppression in animals can result from a depressed capacity to 
produce species of lymphokines which are essential to the development of 
protective forms of immunity. Imbalances between various types of 
lymphokines, where species of lymphokines capable of promoting one form of 
immune response exhibit enhanced production, while those lymphokines 
needed to promote protective forms of immunity are suppressed, can also 
lead to immunosuppression. Individuals may be immunosuppressed as a 
consequence of endogenous elevations in adrenal glucocorticosteroid (GCS) 
levels. This condition could result from viral infections, from certain 
bacterial infections, certain parasitic infections, cancer, some 
autoimmune syndromes, and stress and trauma, or as a secondary consequence 
to any clinical condition that causes an elevated production of 
interleukin-1 (IL-1). Plasma glucocorticoid steroid levels also can be 
elevated exogenously as a consequence of therapeutic treatment for a 
variety of clinical conditions. In addition to the above, it is also well 
known that certain essential functions of the immune system decline with 
age, a situation which correlates with elevations in adrenal output of 
glucocorticoid steroids and depressions in production of other types of 
adrenal steroid hormones. 
It is known that lymphocytes exported from the thymus undergo a series of 
differentiation events which confer upon them the capacity to recognize 
and respond to specific peptide antigens presented appropriately in the 
context of self major histocompatibility complex (MHC) molecules. 
Mechanistically, thymic maturation is a complex process which includes an 
irreversible rearrangement of T cell receptor genes, the cell surface 
expression of these gene products as disulfide-linked heterodimers, 
positive and negative selection processes to provide appropriate 
restriction and avoidance of self-reactivity, and the synthesis and 
expression of CD4 or CD8 as accessory adhesion molecules. 
Microenvironmental influences within the thymus play an essential role in 
the fidelity of this process. 
Subsequent to leaving the thymic microenvironment, mature T lymphocytes 
gain access to the recirculating T cell pool where they move freely via 
the blood between mucosal and nonmucosal lymphoid compartments in the 
mammalian host (Hamann et al. (1989), Immunol. Rev. 108:19). T-lymphocyte 
expression of lymphoid tissue-specific homing receptors, which are 
complementary for vascular addressins on high endothelial venules present 
in Peyer's patches and peripheral lymph nodes, provide a biochemical means 
for selectivity to this recirculation process (id.). Non-activated 
lymphocytes can move freely between mucosal and nonmucosal lymphoid 
tissues due to the presence of both types of homing receptors on their 
plasma membranes (Pals et al. (1989), Immunol. Rev. 108:111). Effector 
lymphocytes, and antigen-activated immunoblasts which are stimulated in a 
particular site in the body, however, exhibit a far more selective 
migratory behavior. These cells move primarily to tissues originally 
involved in antigen exposure and cellular activation (Hamann et al. 
(1989), supra; Pals et al. (1989), supra.). 
An immune response is initiated following T cell recognition of antigen 
peptides in the context of self MHC molecules and generally takes place in 
one of the host's secondary lymphoid compartments. Cellular activation is 
triggered by the binding of antigen to the T cell receptor (TCR), forming 
an antigen/TCR complex which transduces the antigen-specific extracellular 
stimulation across the plasma membrane, and generates intracellular 
signals which include the activation of protein kinase C and the increases 
in intracellular calcium. While signal transduction can lead to T cell 
unresponsiveness, positive signal transduction events trigger a series of 
additional biochemical processes. One consequence of this activation is 
the stimulated production of a number of biologically active molecules 
which are collectively termed lymphokines. (See, Alcover et al. (1987), 
Immunol. Rev. 95:5; Gelfand et al (1987), Immunol. Rev. 95:59). 
The lymphokines, many of which function primarily through autocrine and 
paracrine mechanisms, serve to mediate numerous effector functions 
controlled by T cells through their capacity to regulate cellular 
proliferation, differentiation, and maturation events in lymphocytes, plus 
other hematopoietic and somatic tissue cells (Paul (1989), Cell 57:521). 
Each of the various types of lymphokines exhibit pleiotropic activities, 
dependent upon the specific type of cellular targets being stimulated. The 
biological evaluation of recombinant forms of specific lymphokines has 
determined that individual species can possess both distinct and 
overlapping cellular activities (Paul (1989), supra). Interleukin-2 (IL-2) 
and interleukin-4 (IL-4), for example, share the capacity to facilitate T 
cell growth but are disparate in their relative contribution to cellular 
and humoral immune responses. Cloned T cell lines, restricted in their 
capacity to produce individual species of lymphokines, have been described 
which demonstrate unique capabilities in serving as effector cells or 
helper cells for various immune responses (Paul (1989), supra; Hayakawa et 
al. (1988), J. Exp. Med. 168:1825; Mossman et al. (1989), Ann. Rev. 
Immunol. 7:145). 
Treatment of individuals for immunosuppression has been focused on the use 
of purified lymphokines, usually IL-2, to restore normal propagation of T 
cells. Illustrative of this are the disclosures of U.S. Pat. No. 4,661,447 
(issued Apr. 28, 1987 to Fabricus et al.), U.S. Pat. No. 4,780,313 (issued 
Oct. 25, 1988 to Koichiro et al.), and U.S. Pat. No. 4,789,658 (issued 
Dec. 6, 1988 to Yoshimoto et al.). However, the systemic administration of 
IL-2 for therapeutic purposes has numerous side effects. These side 
effects include fever, hypotension, hepatic and renal failure, myocardial 
infarctions, capillary leak syndrome, and massive edema (Dinatello et al. 
(1987), New England J. Med. 317:940. 
Applicants' invention embodies methods for treating immunosuppression which 
are without the side-effects found with the purified lymphokines. These 
methods utilize the androgen steroid hormones, more specifically 
dehydroepiandrosterone (DHEA), the sulfated derivative thereof (DHEA-S), 
and analogs thereof. 
DHEA is steroid hormone that has been extensively studied for many years. 
It has been reported to be involved in a wide variety of physiologic, 
immunologic, and pathologic conditions (for reviews, see Regelson et al, 
(1988), Ann. N.Y. Acad. Sci. 521:260; Gordon et al. (1986), Adv. Enzyme 
Reg. 26:355-382). Most endocrinologists believe that the primary function 
of DHEA is to serve as a precursor for the synthesis of testosterone and 
the estrogens by the gonads. The biosynthetic relationship of DHEA to 
other steroid hormones is shown in FIG. 1 (taken from Cook and Beastall in 
Steroid Hormones, A Practical Approach (Green and Leake, eds., IRL Press 
Limited, 1987). Prior to its release into the bloodstream, the vast 
majority of newly synthesized DHEA becomes sulfated. The conjugated 
steroid DHEA-S (shown in FIG. 2), is a secretory product of the adrenal 
gland in man and certain primates. DHEA-S represents the major steroid 
hormone in the circulation of humans, and is converted to DHEA via a 
sulfatase. 
Therapeutic uses for DHEA and certain analogs have been reported for 
diabetes, dry skin, ocular hypertension, obesity, and retroviral 
infections. Illustrative of these reports are the disclosures of U.S. Pat. 
No. 4,395,408 (issued Jul. 26, 1983 to Torelli et al.), U.S. Pat. No. 
4,518,595 (issued May 21, 1985 to Coleman et al.), U.S. Pat. No. 4,542,129 
(issued Sep. 17, 1985 to Orentreich), U.S. Pat. No. 4,617,299 (issued Oct. 
14, 1986 to Knepper), U.S. Pat. No. 4,628,052 (issued Dec. 9, 1986 to 
Peat), U.S. Pat. No. 4,666,898 (issued May 19, 1987 to Coleman et al.), 
European Patent Application No. 0 133 995 A2 dated Feb. 8, 1984 (inventor, 
Schwartz et al.), and UK Patent Application No. GB 2 204 237 A dated Apr. 
14, 1988 (inventor, Prendergast). 
SUMMARY OF THE INVENTION 
It is an objective of the invention to provide a method for enhancing the 
biosynthesis of selected lymphokines by activated T cells. Another 
objective of the invention is to enhance immune functions in warm blooded 
animals by restoring their capacity to naturally produce physiological 
concentrations of these lymphokines with a minimization of side effects. 
Further objectives of the invention are to provide applications of the 
method for clinically diagnosing deficiencies of interleukin production, 
maintaining in vitro tissue cultures of T cells, and overcoming certain 
types of immunosuppression associated with elevated GCS levels, caused by 
endogenous production or exogenous administration. Final objectives of the 
invention are to provide applications of the method as a vaccine adjuvant 
to selectively direct the vaccine-induced immune response down a 
protective, rather than a potentially pathologic or non-protective, 
immunologic pathway, as a treatment for naturally occurring ageing-related 
decreases in immune function, as a treatment for stress or trauma-induced 
decreases in immune function, and as a means to facilitate desensitization 
to agents to which a warm-blooded animal is allergic. 
DHEA-S is a prohormone which is naturally converted to DHEA in the 
peripheral lymph nodes of animals with normal immune function. The DHEA 
produced then influences the T lymphocytes within the lymph node and 
exerts controlling influences on their ability to respond when activated. 
This provides a means to regulate the potential of T cells by fluctuating 
the degree to which a particular steroid hormone exists within a 
particular tissue. Old individuals and/or stressed individuals, including 
humans, lose the capacity to produce DHEA-S, resulting in altered T-cell 
responsiveness. Various embodiments of the invention restore the 
metabolite produced from DHEA-S in the anatomic compartment in which 
T-cell responsiveness is required for normal immune responses to 
T-cell-dependent antigens. 
Accordingly, one aspect of the invention is a method for treating naturally 
occurring age-related decline in immune function, comprising administering 
to a warm blooded animal at least one steroid hormone which enhances T 
cell lymphokine production, wherein the steroid hormone is selected from 
the group consisting of DHEA and DHEA cogeners. 
Another aspect of the invention is a method for treating naturally 
occurring age-related decline in immune function, comprising administering 
to a warm blooded animal at least one steroid hormone which enhances T 
cell lymphokine production, wherein the steroid hormone is a DHEA 
prohormone. 
Yet another aspect of the invention is a method for augmenting in an 
immunodeficient individual an immune response comprising administering to 
the individual a pharmaceutical composition comprised of a prohormone of 
DHEA. 
An additional aspect of the invention is a method for augmenting in an 
immunodeficient individual an immune response comprising administering to 
the individual DHEA, and wherein the immunodeficiency is due to trauma. 
Still another aspect of the invention is a method for augmenting in an 
immunodeficient individual an immune response to an antigen comprising 
administering to the individual a steroid selected from DHEA and a DHEA 
cogener, wherein the administration is such that the DHEA and antigen will 
drain to the same lymph node.

DETAILED DESCRIPTION OF THE INVENTION 
The most important function of the immune system is to provide its host 
with protection against diseases. To carry out these tasks, a large and 
diverse array of effector mechanisms have evolved, the majority of which 
exhibit antigen specificity. Each individual effector mechanism possesses 
a degree of uniqueness with respect to its ability to influence the rate 
of progression, to detoxify, or to promote the elimination of microbial 
pathogens or tumor cells. Such a diversity in available mechanisms is 
absolutely essential since no single effector response can effectively 
deal with all forms of pathogenic insults. Furthermore, to protect normal 
function of the various non-lymphoid organ systems and tissues of the body 
requires careful selection, activation, and compartmentalization of the 
most appropriate types of immune effector mechanisms. Equally important is 
the simultaneous capacity to down-regulate the development of other types 
of responses. Immunologic effector responses must, therefore, be both 
effective and practical, and at the same time be appropriately regulated 
anatomically to reduce the risk of pathologic consequences. 
The nonlymphoid tissues and organs of the body, which work collectively to 
sustain the life of the host, must also be capable of providing regulatory 
information to cells of the immune system. This information, mediated 
through the activities of inflammation-induced tissue cytokines, 
prostaglandins, plus other types of biological response modifiers, becomes 
integrated into the complex equation to control the mechanisms which 
regulate effector response selection. 
T cells, through their capacity to produce a number of lymphokines in 
response to activation, play a central role in guiding the development of 
immune effector responses. Mechanisms which operate to control the 
synthesis and secretion of these pleiotropic biologic response modifiers, 
therefore, directly influence the quantitative and qualitative nature of 
immunity. The lymphokines and cytokines provide important information, not 
only to cells of the immune system, but also to cells of the other tissue 
and organ systems. For this information to be meaningful, it is essential 
that lymphokine production remains tightly controlled at the levels of 
both cellular source and duration. Autocrine and paracrine effects by 
lymphokines and cytokines should be the norm, since only a few species of 
lymphokines and cytokines are capable of working effectively when provided 
via endocrine routes. These essential anatomic restrictions, therefore, 
cannot be adequately provided by bolus injection of recombinant 
lymphokines and/or cytokines, and may explain the limited success 
associated with this form of therapy. 
The vast majority of the T cells in the peripheral circulation are known to 
reside within the recirculating T cell pool. These cells continuously 
enter and exit secondary lymphoid organs throughout the body, maintaining 
residence within any particular site for only finite periods of time. Over 
the lifespan of any individual mature T cell, therefore, it has probably 
taken up temporary residence in most of a host's secondary lymphoid 
organs. T-cell recirculation provides the immune system with a means for 
clonally-restricted T cells to provide a level of surveillance over all 
the tissue and organ systems. 
It is universally accepted that most T cells acquire their specificity for 
antigen, and a self-MHC-restricting element, during processes which occur 
during their ontogeny within the thymus. However, the extent to which 
intrathymic maturation confers genetic restrictions upon individual T 
cells that regulate their potential for immunologic involvement has not 
been delineated. 
A general concept which explains the results in the Examples, but which is 
not intended to limit our invention, is that the genetic programs of 
resting recirculating T cells are continuously being altered by extrinsic 
environmental influences. The steroid hormones, either presented in their 
active forms systemically (e.g. glucocorticosteroids (GCS)), or being 
provided to T cells only within discrete microenvironments as a 
consequence of end-organ metabolism e.g., DHEA, DHT, OR 1,25(OH).sub.2 
D.sub.3 ! perform important roles in this process. The basal regulation of 
the immune system at the level of the T cell requires the continual 
presence of the needed substrates (prohormones). The anatomic 
compartmentalization of functional potential for T cells, therefore, would 
be dependent on the cellular source of the steroid metabolizing enzymes 
able to convert the steroid hormone substrates to their bioactive species. 
Our studies show that macrophages can contain each of these enzymes. 
More specifically, DHEA-S is naturally converted to DHEA in the peripheral 
lymph nodes of animals with normal immune function. The DHEA produced then 
influences the T lymphocytes within the lymph node and exerts controlling 
influences on their ability to respond when activated. This provides a 
means to regulate the potential of T cells by fluctuating the degree to 
which a particular steroid hormone exists within a particular tissue. Old 
individuals and/or stressed individuals, including humans, lose the 
capacity to produce DHEA-S, resulting in altered T-cell responsiveness. 
The invention in its various embodiments restores the metabolite produced 
from DHEA-S in the anatomic compartment in which T-cell responsiveness is 
required for normal immune responses to T-cell-dependent antigens. 
As used herein, the term "individual" refers to a vertebrate and preferably 
to a member of a species which exhibits DHEA-S sulfatase activity, and 
includes but is not limited to domestic animals, sports animals, and 
primates, including humans. 
The term "effective amount" refers to an amount of DHEA-S, DHEA, or DHEA 
cogener sufficient to restore normal immune responsiveness in an 
immunodeficient subject to which it is administered, i.e., it restores 
DHEA in the anatomic compartment in which T-cell responsiveness is 
required to a level for normal immune responses to T-cell-dependent 
antigens. The exact amount necessary will vary from subject to subject, 
depending on the species, age, and general condition of the subject, the 
severity of the condition being treated, the mode of administration, etc. 
Thus, it is not possible to specify an exact effective amount. However, 
the appropriate effective amount may be determined by one of ordinary 
skill in the art using only routine experimentation. 
As used herein, the term "immunodeficient individual" means an individual 
whose response to immune stimulation to a foreign antigen is significantly 
less than that of the average of normal individuals of the same species. 
Methods of determining "immunodeficiency" are known in the art, and 
include, for example, an examination of lymphokine production by activated 
T cells; the ability of the individual to demonstrate contact 
hypersensitivity; the ability of the individual to raise a humoral 
response to antigen challenge, or the resistance of the individual to 
infection by microorganisms. 
"Treatment" refers to the administration of a composition to an individual 
which yields a protective immune response, and includes prophylaxis and/or 
therapy. 
An "antigen" refers to a molecule containing one or more epitopes that will 
stimulate a host's immune system to make a secretory, humoral and/or 
cellular antigen-specific response. The term is also used interchangeably 
with "immunogen". 
An "immunological response" to a composition or vaccine comprised of an 
antigen is the development in the host of a cellular and/or 
antibody-mediated immune response to the composition or vaccine of 
interest. Usually, such a response consists of the subject producing 
antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic 
T cells directed specifically to an antigen or antigens included in the 
composition or vaccine of interest. 
By "vaccine composition" or "vaccine" is meant an agent used to stimulate 
the immune system of an individual so that current harm is alleviated, or 
protection against future harm is provided. 
"Immunization" refers to the process of inducing a continuing high level of 
antibody and/or cellular immune response which is directed against an 
antigen to which the organism has been previously exposed. 
As used herein, the term "prohormone" pertains to water soluble precursors 
of DHEA, i.e., DHEA derivatives from which DHEA may be synthesized in 
vivo, for example, DHEA-S (and other precursors known in the art). 
As used herein, a "pharmacologic dose" is one which gives a desired 
physiological effect. 
The practice of the present invention will employ, unless otherwise 
indicated, conventional techniques of molecular biology, microbiology, 
biochemistry, and immunology, which are within the skill of the art. Such 
techniques are explained fully in the literature. See, e.g., ANIMAL CELL 
CULTURE (R.I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL 
Press, 1986; the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.), 
IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, 
London), Scopes, (1987); and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volumes 
I-IV, (D. M. Weir and C. C. Blackwell, eds., 1986.) All patents, patent 
applications, and publications mentioned herein, both supra and infra, are 
hereby incorporated herein by reference. 
One embodiment of the invention is a method for enhancing or maximizing the 
production of T cell lymphokines which are correlated with protective 
immunity. The method comprises exposing T cell lymphocytes which have a 
potential to make selected T cell lymphokines to appropriate 
concentrations of particular steroid hormones prior to activation. If the 
exposure is in vitro, the particular steroid hormone to which the T cell 
lymphocyte is exposed depends upon the lymphokine which is selected for 
enhancement or maximized production. If the exposure is in vivo, a 
pharmaceutical composition comprised of the steroid hormone is 
administered to the individual, particularly to an immunodeficient 
individual. Immunodeficiency may be for a variety of reasons, for example, 
age, i.e., very young (e.g., neonate) or aged, stress, or trauma. The 
administration to the individual is by techniques known in the art, 
including, for example, parenteral, transdermal, or transmucosal. 
In accordance with the invention, exposing T cell lymphocytes which have a 
potential to make selected T cell lymphokines to DHEA or a DHEA cogener 
prior to activation enhances the production of IL-2, IL-3, .gamma.-IFN, 
and GM-CSF. DHEA cogeners which are useful in the invention have the 
following structure: 
##STR1## 
in which R is hydrogen in alpha or beta configuration or nothing, 
resulting in a double bond between carbon atoms 5 and 6; R.sub.1 is 
hydrogen or a halogen in .alpha.-configuration; and R2 is oxygen or methyl 
ketone (--COCH.sub.3); or 
##STR2## 
in which R is hydrogen in alpha or beta configuration or nothing, 
resulting in a double bond between carbon atoms 5 and 6; R.sub.1 is 
hydrogen or a halogen in .alpha.-configuration; R2 is oxygen or methyl 
ketone (--COCH.sub.3); and R.sub.3 is OH or sulfate. 
Alternatively, and particularly in vivo, the selected steroid hormones may 
be administered to individuals through precursor substances which are then 
metabolized to DHEA or its metabolites. For example, the sulfonated form 
of DHEA, DHEA-S, can be administered provided that the administration is 
to an individual that can metabolize the prohormone to DHEA by 
tissue-associated DHEA-sulfatases. 
The simultaneous enhancement or maximization of the production of more than 
one T cell lymphokine may be achieved by exposing the T cell lymphocyte to 
more than one steroid hormone prior to activation. The exposure to more 
than one steroid hormone can be simultaneous or sequential. The 
concentration of each of the steroid hormones should be balanced to 
achieve the desired enhancing effects. For example, if it were desirous to 
enhance the production of IL-2, .gamma.-IFN, and IL-4, the T cell 
lymphocytes could be exposed to physiologic or pharmacologic levels of 
DHEA and a physiologic level of GCS. This would avoid the IL-2 and 
.gamma.-IFN depression which is characteristic of a pharmacologic level of 
GCS. 
Evidence derived from experimental and clinical observations indicates that 
immunologic reactions elicited to either simple or complex antigens often 
manifest as a balanced heterogenous blend of both cellular and humoral 
components, with the fractional contribution of any individual type of 
effector mechanism oftentimes dominating the overall response. This level 
of heterogeneity is essential to the development of a protective immune 
response. Alterations to this natural balance, whether caused by genetic 
or physiologic changes associated with age or stress or trauma, can lead 
to a depressed capacity to elicit protective immune responses, and might 
also lead to immunologic responses having pathologic consequences. 
Administration of steroid hormones, particularly DHEA-S or DHEA or 
DHEA-cogeners in accordance with the invention would be useful in treating 
such immune system imbalances in individuals. For example, 
immunosuppression (a form of immunodeficiency) in warm blooded animals may 
be mediated by elevated GCS levels. These elevated levels can result from 
a variety of causes including, but not limited to, stress and trauma 
(including, for example, post-surgical trauma, burn trauma), as a 
secondary consequence to any clinical condition which causes an elevated 
production of IL-1, or therapeutic treatment for a variety of clinical 
conditions. The elevated GCS levels can result in an imbalance in the 
production of essential interleukins. The normal balance of essential 
interleukin production may be restored by therapeutic administration of 
DHEA, DHEA-S, or its cogeners. 
Additionally, if it were known that elevated GCS levels were the result of 
certain behavior or maladies, administration of DHEA-S, DHEA, or DHEA 
cogeners could be used as a prophylaxis prior to the onset of the 
elevation in GCS levels and resultant immunosuppression. In these cases, 
where the administration is chronic, it is advisable to use the prohormone 
form (e.g., DHEA-S) to prevent side effects associated with the 
administration of large doses of DHEA. For instance, there is a bovine 
malady commonly known as "Shipping Fever" which has a high rate of 
morbidity and mortality associated with the stress induced by long 
distance shipment. This stress is associated with chronic increased levels 
of GCS. Prophylactic administration of prohormones (e.g., DHEA-S) in 
accordance with the invention prior to and/or during bovine shipment may 
counteract the immunosuppressive effects of the chronically elevated GCS 
levels, reducing the risk of these animals to infectious agents and weight 
loss. 
The invention may also be used as a diagnostic tool in evaluating 
lymphokine production deficiency. In this application T cell lymphokine 
production of a first group of T-cell lymphocytes which have a potential 
to make selected T cell lymphokines after T cell lymphocyte activation is 
measured. A second group of the same type of T cell lymphocytes is exposed 
to a particular steroid hormone prior to T cell activation. The selected T 
cell lymphokine is then measured after activation. The amount of T cell 
lymphokine production of the two groups of T cell lymphokines are 
compared. The sensitivity of the diagnostic tool is maximized when the 
amount of the particular steroid hormone to which the second group of T 
cell lymphocytes is exposed is sufficient to maximize the production of 
the T cell lymphokines which the particular steroid hormone enhances. For 
example, if the T cell lymphokine is IL-2, or IL-3, or .gamma.-IFN, or 
GM-CSF, the preferred steroid hormone may be selected from the group DHEA 
or a DHEA cogener having the structure recited above. 
Another application of the invention is to treat naturally occurring 
age-related decreases in immune function, which correlate with a decrease 
in circulating DHEA-S levels. Associated with age related decline in 
immune function is a decrease in the production of certain lymphokines. 
Treatment of aging, warm blooded animals with steroid hormones, preferably 
DHEA-S or DHEA-cogeners, substantially restores the production of the 
selected lymphokines involved in the cascade leading to immunologic 
competence. 
Generally, the person in charge of the administration of the steroids, 
DHEA, DHEA-S, or DHEA-cogeners will choose the appropriate form of the 
steroid based upon the compartmentalization effect and metabolic products 
resulting therefrom. For example, if the indication for administration is 
prophylaxis or chronic therapeutic treatment, the prohormone DHEA-S is 
preferred to escape the side effects associated with of the administration 
of chronic high levels of DHEA. In this case the level of DHEA-S may be in 
the range of about 5 to about 100 mg per day, preferably may be in the 
range of about 10 to about 80 mg per day, and even more preferably may be 
in the range of about 15 to about 60 mg per day. 
Alternatively, if the indication for treatment is acute trauma or stress, 
it may be preferable to treat with a bolus administration of DHEA. The 
bolus administration may be in the range of about 1 to about 20 mg per kg 
of body weight, more usually may be in the range of about 2 to about 10 mg 
per kg of body weight, and preferably may be in the range of about 3 to 
about 8 mg per kg of body weight. 
The compounds of the present invention can be administered to the 
immunologically deficient individual in a variety of forms adapted to the 
chosen route of administration, for example, orally, intravenously, 
intramuscularly, or via subcutaneous, topical, or inhalation routes. 
Pharmaceutical compositions made up of formulations comprised of the 
steroids (particularly DHEA, DHEA-S, and DHEA-cogeners) and suitable for 
the administration by each of these routes may be prepared by one of 
ordinary skill in the art. See, for example, Remington's Pharmaceutical 
Sciences, 17th Edition (1985, Mack Publishing Company, Easton, Penn.). For 
example, the pharmaceutical composition containing the steroid may also 
contain a carrier or solid or be encapsulated in a material that is 
non-toxic to the inoculated animal and is compatible with the steroid. 
Suitable pharmaceutical carriers include liquid carriers, such as normal 
saline and other non-toxic salts at or near physiological concentrations, 
and solid carriers not used for humans, such as talc or sucrose, also feed 
for farm animals. When used for administering via the bronchial tubes, the 
steroid hormone is preferably presented in the form of an aerosol. 
Another application of the invention is to use a pharmaceutical composition 
containing the steroid, preferably DHEA or a DHEA cogener, as a vaccine 
adjuvant to augment or selectively direct the vaccine-induced immune 
response down a protective immunologic pathway. When the individuals are 
immunized with an immunizing agent, administration of the steroid may be 
prior to or contemporaneously with the vaccination. Typical methods of 
administering the steroid hormone include implants, mixing the steroid 
hormone with the immunizing agent, or topically applying the steroid 
hormone composition to skin sites which drain to the same lymph nodes as 
the antigen of the vaccine. This latter method is preferably used with 
individuals who are immunologically deficient due to low levels of DHEA-S 
and/or DHEA and in whom one wishes to augment the immune response, for 
example, the aged or neonates or individuals who are therapeutically 
immunosuppressed. 
Described below are examples of the present invention which are provided 
only for illustrative purposes, and not to limit the scope of the present 
invention. In light of the present disclosure, numerous embodiments within 
the scope of the claims will be apparent to those of ordinary skill in the 
art. 
EXAMPLES 
Example 1 
DHEA enhances IL-2 Production by Activated Murine T Cells 
In this experiment the capacity of DHEA and DHEA-S to alter the production 
of IL-2 and IL-4 following in vitro lymphocyte treatment or exposure was 
evaluated. DHEA significantly enhanced the production of IL-2 over a wide 
dose range, and DHEA-S, over the same dose range, had no effect on IL-2 
and IL-4 production. FIG. 3A is the dose response curve of DHEA and FIG. 
3B is the dose response curve of DHEA-S developed in this experiment. 
Spleen cells obtained from normal BALB/c mice were prepared as a single 
cell suspension at a concentration of 1.times.10.sup.7 cells/ml in RPMI 
1640 supplemented with 2 mM L-glutamine, 5.times.10.sup.-5 M 
2-mercaptoethanol, 20 .mu.g/ml gentamycin-sulfate, and 1% nutridoma-NS 
(Boehringer-Mannheim). Individual aliquots of cells were then pulsed for 
30 minutes at 37.degree. C. with the indicated concentrations of DHEA or 
DHEA-S. After pulsing, the cells were washed several times in balanced 
salt solution, resuspended in fresh medium, and then dispensed into 
24-well culture plates with a stimulatory concentration of anti-CD3 (Leo 
et al. (1987), Proc. Natl. Acad. Sci. USA 84:1374). After a 24-hour 
incubation period, culture supernatants were harvested for assessment of 
IL-2 and IL-4 activity using the method of Mossman (J. Immunol. Meth. 
(1983)). In this experiment, 100% control titers of IL-2 and IL-4 from 
normal stimulated splenocytes in FIG. 3A were 640 and 160 units/ml, 
respectively. For control splenocytes from FIG. 3B, 100% control titers of 
IL-2 and IL-4 were 2560 and 320 units/ml, respectively. 
This same experiment was repeated to assay for .gamma.-IFN production. A 
dose response curve similar to that reported in FIG. 3A for DHEA was 
obtained for .gamma.-IFN. 
This same experiment was performed using the DHEA cogener 16-alpha-bromo 
DHEA in place of DHEA. A dose response curve similar to that reported in 
FIG. 3A was obtained for 16-alpha-bromo DHEA. 
Example 2 
DHEA enhances IL-2 Production in GCS-Treated Normal Splenocytes and Cloned 
T Cell Lines 
The capacity of DHEA to facilitate a reversal of glucocorticoid-induced 
suppression of IL-2 production by either normal murine lymphocytes, or 
cloned T cell lines with similarities to either Th1-type or Th2-type 
helper T cells was evaluated. 
Single cell suspensions of normal murine spleen cells were prepared in 
Nutridoma-supplemented complete RPMI at 10.sup.7 cells/ml. They were then 
pulsed with 10.sup.-7 M corticosterone and/or 10.sup.-8 M DHEA as 
described in FIG. 4A. After several washes, the cells were stimulated with 
anti-CD3. The enhancement of IL-2 production by DHEA exposed normal 
splenocytes is shown in FIG. 4A. FIG. 4B and FIG. 4C show the regulation 
of lymphokine production by two ovalbumin (OVA)-specific cloned T cell 
lines. The OVA-specific T cell clones were derived from nylon-wool 
enriched splenic T cells from OVA-immunized (C3H.times.C57/B6) F.sub.1 
mice using the method of Berzofsky (1985), J. Immunol. 35:2628. OVA/3 and 
OVA/2 cell lines were derived from different clonings, each having 
distinct patterns of lymphokine production. Culture conditions and assay 
procedures for IL-2 and IL-4 are as in Example 1. 
Referring to FIG. 4A, exposure of splenocytes to the effects of 
corticosterone (10.sup.-7 M) greatly reduced the capacity of cells to 
produce IL-2 subsequent to activation with anti-CD3. DHEA treatment alone 
augmented IL-2 production. Lymphocytes exposed to corticosterone and DHEA, 
followed by their activation in vitro, produced normal or enhanced levels 
of IL-2 and enhanced levels of IL-4. 
Referring to FIG. 4B and FIG. 4C, OVA/2 an ovalbumin (OVA)-specific cloned 
T cell line with characteristics similar to Th2-type cells) and OVA/3 (a 
cloned T cell line with characteristics similar to Th1-type cells), were 
exposed in vitro to the effects of DHEA and/or glucocorticoids prior to 
their culture with antigen and syngeneic antigen-presenting cells. As 
shown in FIG. 4C, DHEA treatment of OVA/3 greatly augmented the capacity 
of this cell line to produce IL-2, while exposure to DEX resulted in an 
IL-4 dominant phenotype, similar to what is observed with Th2-type clones. 
Treatment of OVA/3 with DEX followed by DHEA, resulted in a marked 
elevation in IL-2 production with only a minimal enhancement of IL-4. As 
shown in FIG. 4B, the effects of steroid treatment on the capacity of 
OVA/2 to produce TCGF gave comparable results. DHEA exposure of this T 
cell clone was capable of shifting the pattern of TCGF production from a 
Th2-like to a Th1-like phenotype (IL-2 dominant), while DEX treatment 
alone augmented IL-4 production following activation in vitro with OVA. 
Treatment of OVA/2 with both DEX and DHEA caused an enhanced capacity to 
produce both IL-2 and IL-4. 
Example 3 
A Single Injection of Mice with DHEA or DHEAS Enhanced the Biosynthesis of 
IL-2 by Activated Lymphoid Cells 
This example demonstrates the effects of in vivo administration of DHEA and 
DHEA-S on IL-2 and IL-4 biosynthesis. 
Groups of (C3H.times.BL/6)F1 mice were given a single intraperitoneal 
injection of 100 .mu.g DHEA or DHEA-S. After three days, spleen cells from 
the treated groups, plus spleen cells from an untreated age-matched 
control group, were prepared for culture as described in Example 1. The 
relative titers of IL-2 and IL-4 in the 24-hour culture supernatants were 
determined in the presence of anti-IL-2, or anti-IL-4, or both anti-IL-2 
and anti-IL-4, or no blocking antibodies. The assay was read visually. 
FIG. 5 presents the results of the study. Non-activated cultured lymphoid 
cells produced undetectable (less than 2 units) of either IL-2 or IL-4. 
Example 4 
DHEA Enhances IL-2 Production in Splenocytes from Corticosterone-Treated 
Mice 
The reversal of the inhibitory effects caused by chronic glucocorticoid 
administration to normal mice in vivo on the capacity of their T cells to 
produce IL-2 was demonstrated as follows. 
Biodegradable pellets (Innovative Research, Inc.) containing corticosterone 
and designed to deliver the steroid at a dose of 5 .mu.g/hr were implanted 
subcutaneously into (C3H.times.BL/6) F.sub.1 mice. The splenocytes from 
the mice were harvested 72 hours after the implantation. Prior to 
activation, the splenocytes were pulsed with a short pulse of DHEA 
(10.sup.-8 M). Culture and assay procedures for IL-2 and IL-4 were as 
described in Example 1. The results are presented in FIG. 6. 
As seen in the figure, the DHEA pulse caused a significant enhancement of 
IL-2 production. Under these conditions, the glucocorticoid-induced 
augmentation in IL-4 synthesis was not affected, resulting in a population 
of lymphoid cells capable of producing high levels of both IL-2 and IL-4. 
Example 5 
DHEA in vivo Enhances IL-2 Production in Mice with and without 
Corticosterone Treatment 
This example demonstrates that DHEA administered in vivo influences the 
profile of T cell growth factors (TCGF) produced by splenocytes isolated 
from treated animals. 
Biodegradable pellets (Innovative Research, Inc.) containing corticosterone 
or DHEA that deliver the steroids at 5 .mu.g/hr were implanted 
subcutaneously into three separate groups of BALB/c mice 72 hours prior to 
harvesting and preparation of spleen cells. Single cell preparations of 
splenocytes from each group were cultured as described in Example 1, and 
stimulated with the polyclonal T cell mitogen, anti-CD3. After 24 hours, 
culture supernatants were collected and assayed for IL-2 and IL-4 activity 
as described in Example 1. 
As seen in FIG. 7, the stimulation of splenocytes isolated from normal 
animals consistently gave a standard pattern of TCGF production where IL-2 
dominated over IL-4. Lymphocytes isolated from corticosterone-treated 
animals demonstrated a marked reversal of this pattern; IL-4 consistently 
represented the dominant TCGF. Similar to what is observed following an in 
vitro treatment with this androgen steroid, activated splenocytes from the 
DHEA-treated animals exhibited an enhancement in IL-2 production. Under 
conditions where both steroids were elevated in vivo, it was found that 
isolated splenocytes from these animals produced enhanced levels of both 
IL-2 and IL-4 subsequent to their activation with anti-CD3 in vitro. 
Example 6 
The Effect of DHEA and 1,25-dihydroxyvitamin D3 in vivo on IL-2 and IL-4 
Production in vitro 
CH3 mice received implants of biodegradable DHEA or 1,25(OH).sub.2 D.sub.3 
pellets designed to deliver steroid at a rate of 5 and 1.25 .mu.g/hr, 
respectively. Three days after implantation, both the steroid treated 
groups and a normal control group of mice were immunized in the hind 
footpads with 100 .mu.g OVA in CFA. Ten days after immunization, the 
draining lymph nodes and spleens from all groups were prepared for 
culture. Lymph node cells were stimulated with 100 .mu.g OVA. Culture 
supernatants were assayed for IL-2 and IL-4 activity after 24 hours using 
the HT-2 bioassay. The results are shown in FIG. 8. From the figure it may 
be seen that DHEA administration caused approximately a four-fold increase 
in IL-2 production, and no stimulation of IL-4 production. In contrast, 
1,25(OH).sub.2 D.sub.3 administration caused an approximate eight-fold 
increase in IL-4 production, but did not stimulate IL-2 production. 
Similar alterations in the ability of antigen-activated T cells to produce 
IL-2 and IL-4 were observed when the steroid hormone was mixed with the 
immunizing antigen, or was topically applied to skin sites above the site 
of vaccination. 
Example 7 
The Reversal of Age-related Decline in IL-2 and .gamma.-IFN Production 
This example demonstrates age-related decline in the production of certain 
lymphokines, and restoration by steroid hormone treatment. The lymphokines 
assayed are IL-2, IL-4, and .gamma.-IFN; the steroid hormone administered 
is DHEA. 
Age associated changes in lymphokine production are shown in FIG. 9. 
(CH3.times.BL/6)F.sub.1 mice of the indicated ages were sacrificed and 
their spleen cells prepared for culture with mitogen, anti-CD3. Culture 
supernatants were harvested and evaluated for the relative contribution of 
IL-2 and IL-4 using the HT-2 bioassay. As seen in the figure, aged mice 
(13 and 17 months) produced significantly less IL-2 and significantly more 
IL-4 than did younger mature mice (3 and 7 months). Non-activated cells 
produced less than 1 unit of either IL-2 or IL-4. 
A reversal of the aging effect on IL-2 production by DHEA is shown in FIG. 
10. In the study, both young (6 mos.) and old (16 mos.) mice were 
implanted with DHEA pellets delivering a dose of 5 .mu.g/hr. After three 
days, DHEA groups and control age-matched groups were sacrificed and their 
spleen cells prepared for culture with the mitogen anti-CD3. Culture 
supernatants were harvested and evaluated for the relative contribution of 
IL-2 and IL-4 using the HT-2 bioassay, and for .gamma.-IFN using the assay 
of Green. Non-activated cells produce less than 1 unit of either IL-2 or 
IL-4 and no detectable .gamma.-IFN. 
Similar enhancement in the capacity of lymphocytes derived from old mice to 
produce IL-2 was observed following a direct exposure of the splenocytes 
in vitro to DHEA (10.sup.-9 to 10.sup.-7 M). 
Example 8 
Activated T cells from Aged donors Produce an Altered Pattern of 
Lymphokines Compared to Normal 
Using a serum-free culture system which allows in vitro activation of 
lymphocytes under conditions devoid of the restrictive regulatory 
influences by platelet-derived growth factor and other serum-associated 
modulators of cellular activity, lymphocytes from mature adult and aged 
mice were compared for lymphokine production following T-cell activation. 
Splenocyte cultures were either stimulated with 1 .mu.g/ml 
anti-CD3.epsilon. or left unstimulated to control for any spontaneous 
lymphokine production. After a 24-hour incubation period, cell-free 
culture supernatants were analyzed for lymphokine content. The materials 
and methods used in these studies were as follows. 
The BALB/c mice used were bred from breeding stock originally purchased 
from the National Cancer Institute. The source of aged mice for these 
experiments was retired breeders from our own colony. Age and sex-matched 
mice, ranging in age from 13 to 39 weeks for mature adult, and 112-120 
weeks for aged mice were used. 
Monoclonal antibody reagents were prepared from culture supernatants of 
B-cell hybridomas adapted to growth under serum-free conditions. The 
hybridoma clones secreting rat anti-murine .gamma.-IFN (XMG1.2), AND RAT 
ANTI-MURINEil-5 (TRFK4 and TRFK5) were obtained from DNAX (Palo Alto, 
Calif.). The hybridoma clone producing hamster anti-murine CD3.epsilon. 
monoclonal antibody, 1452C-11.2, was obtained from J. Bluestore 
(University of Chicago). The hybridoma producing antibody specific for 
murine IL-4 (11B11) and murine .gamma.-IFN (R46A2) was purchased from the 
ATCC. A number of purified rat anti-murine cytokine antibodies were 
purchased from PharMingen (San Diego, Calif.) and used for quantitation of 
specific murine cytokines by capture ELISA; these were anti-murine IL-3 
antibodies (cat. nos 18011D and 18022D), biotinylated anti-IL-4 (cat. no. 
18042D), anti-murine GM-CSF antibodies (cat. nos. 18091D and 18102D). 
Murine recombinant .gamma.-IFN was obtained from Genentech 
(5.times.10.sup.6 units/mg protein) and used as a reference in the 
.gamma.-IFN bioassays. Murine recombinant IL-2, IL-4 and IL-5, were 
derived from culture supernatants of X63Ag8-653 cells transfected with 
multiple copies of a single murine interleukin gene. After the relative 
concentration of each lymphokine in culture was determined by a comparison 
to a known recombinant standard, these reagents were used as reference 
lymphokines in both bioassays and capture ELISA. Other sources of 
purified, murine, reference lymphokines were IL-5 obtained as a gift from 
R. Coffman, DNAX, or IL-2 and IL-4 purchased from Collaborative Research 
Inc. (Bedford, Mass.). Ovalbumin (Sigma Chemicals, St. Louis, Mo.) was 
dissolved in double distilled water at a concentration of 20 mg/ml. The 
solution was filter sterilized and frozen in 3 ml aliquots at -20.degree. 
C. For immunization, ovalbumin was mixed with the commercial aluminum 
hydroxide preparation (Maalox). One hundred .mu.g in 25 microliters Maalox 
was injected into a single hind footpad. 
Single cell suspensions of lymphoid cells were prepared from appropriate 
lymphoid organs of normal mice, washed twice in sterile balanced salt 
solution and cultured at a density of 1.times.10.sup.7 cell/ml/well with a 
T-cell specific mitogen, routinely anti-CD3.epsilon., in a 24-well Cluster 
culture plate (Costar, Cambridge, Mass.) for a period of 24 hours to 
elicit lymphokine secretion. Cell-free culture supernatants were collected 
and stored at -20.degree. C. until assayed for lymphokine content. The 
culture period, cell concentrations, and culture medium, consisting of 
RPMI 1640 supplemented with 1% Nutridoma-SR (Boehringer-Mannheim), 
antibiotics, 200 mM L-glutamine and 5.times.10.sup.-5 M 2-mercaptoethanol, 
were all carefully evaluated to determine the optimal conditions for 
stimulating production of the lymphokines under evaluation. 
HT-2 cells were used as an indicator cell line for the bioassay of IL-2, 
using a modification of a colorimetric assay for cell viability. Each test 
supernatant is titrated in duplicate in Nutridoma-SR-supplemented media 
(referred to as serum-free) containing 4.times.10.sup.3 HT-2, and 
saturating amounts of anti-IL-4 monoclonal antibody. During the final 4 
hours of a 24-hour incubation, 5 .mu.g of 
3-4,5-Dimethylthiazole-2-yl!-2,5 diphenyl tetrazolium bromide (MTT) is 
added to each culture, followed by the addition of 100 microliters of a 
20% SDS/50% dimethylformamide solution to dissolve formazan crystals. 
Spectrophotometric readings are recorded at 570 nm-650 nm. One unit of 
activity in a test supernatant is equivalent to the O.D. of a half-maximal 
response of HT-2, relative to a standard recombinant source. 
Where indicated, the amount of other cytokines in test supernatants was 
quantitated by capture ELISA, adapted from the method of Schumacher. 
Briefly, 100 microliters of 2 .mu.g/ml capture antibody in 0.05M Tris-HCV 
(pH9.6) was adsorbed to the wells of a 96-well microtest plate, washed and 
blocked with PBS/1% BSA. Test supernatants and 2-fold serial dilutions of 
the appropriate reference cytokine (100 microliters/well) were dispensed 
and after sufficient incubation and washing, 100 microliters of 
biotinylated-detection antibody, 1 .mu.g/ml, was dispensed into each well. 
The ELISA was developed using avidin-HRP and ABTS-substrate. 
Spectrophotometric readings were recorded at 405 nM. The limit of 
detection for most of these cytokines is 15-30 pg/ml. 
Anti-Ovalbumin antibody ELISA was performed as follows. Ovalbumin (Sigma 
Chemical, St. Louis, Mo.) was diluted to a concentration of 20 .mu.g/ml in 
50 mM Tris-HCl (pH 9.6). 100 microliters/well of this solution was used to 
coat the wells of high protein binding, microtiter plates (Corning cat. 
no. 2581) following an overnight incubation at 4.degree. C. The plates 
were then blocked with 250 microliters PBS/10% FCS for 90 minutes at 
37.degree. C. and then rinsed multiple times with PBS/0.5% Tween 20. Serum 
test samples plus positive and negative control serum antibodies were 
titrated against PBS/10% FCS over eight 2-fold dilutions. After another 90 
minute incubation at 37.degree. C. and multiple washes in PBS/0.5% Tween 
20, 100 microliters of a 1:1000 dilution of HRPO-coupled goat anti-murine 
IgM and goat anti-murine IgG was dispensed into each well. This step was 
followed by an incubation at 37.degree. C. for 90 minutes, PBS/0.5% Tween 
20 washes, and addition of 100 microliters of an ABTS substrate for 
spectrophotometric detection of antibody activity in the assay. Readings 
from a spectrophotometer were recorded at 405 nM. The titer of specific 
antibody in a test serum was assigned as the inverse of the antibody 
dilution that was equivalent to a half-maximal response. Antibody activity 
of most sera was saturating at the lowest dilutions, implying a high level 
of efficiency in the capture and the detection of ovalbumin-specific 
immunoglobulin. 
FIGS. 11A-FIG. 11F are graphs showing the pattern of lymphokines produced 
by T cells from aged BALB/c donor mice and younger donor mice. In the 
study, splenocytes were prepared from groups of 3 mature adult (28 weeks 
of age) and 3 aged (112 weeks of age) BALB/c donor mice. 1.times.10.sup.7 
splenocytes were cultured under serum-free conditions in triplicate and 
activated with 1 .mu.g/ml CD3.epsilon.. Culture supernatants were analyzed 
for the level of IL-2 by quantitative bioassay, and for IL-4, IL-5, 
.gamma.-IFN, IL-3 and GM-CSF as described above. In the figure, bars 
represent the mean .+-.SD for the value of each lymphokine presented. 
As seen in FIGS. 11A-11F, the in vitro activation of splenocytes from aged 
mice under serum-free conditions resulted in a reduced production of some 
lymphokines and an enhanced production of others, compared to the pattern 
of lymphokines produced by activated T cells from mature adult mice. 
Activation-induced production of IL-2, IL-3, and GM-CSF were all 
significantly reduced in cell cultures from old donors, while the levels 
of IL-4, IL-5, and .gamma.-IFN were increased above normal adult levels. 
A comparison of lymphokine profiles between mature adult and aged mice has 
been performed using three strains of mice (BALB/c, C57BL/6 and C3H/HeN). 
The response of each of these strains was analyzed numerous times, and 
yielded similar results. 
Example 9 
Preservation of Normal Potential to Produce T-cell Lymphokines and Generate 
Humoral Immune Responses by Supplementation with DHEA Sulfate 
Circulating levels of DHEA sulfate declines markedly with advancing age in 
humans and other mammals. As shown above, direct treatment of T cells from 
aged or normal murine donors with DHEA prior to activation in vitro 
augmented their capacity to produce IL-2. In contrast, DHEA-S, the 
prohormone form of DHEA found principally in the circulation, was shown to 
have no direct effect on T-cell production of this lymphokine. When DHEA-S 
was administered to normal mature adults in vivo, it enhanced the 
potential for IL-2 production by T cells isolated from lymphoid organs 
having the greatest DHEA-sulfatose activity. The most active lymphoid 
organs are those having anatomic positions downstream from nonmucosal 
tissues. This example demonstrates that DHEA-S supplementation in vivo can 
influence the age-related changes in lymphokine production and humoral 
immune responses. 
Groups of adult BALB/c mice, between 35 and 39 weeks of age, were separated 
into two groups. One group was provided with 100 .mu.g/ml DHEA-S in their 
drinking water. The hormone was offered ad libitum to these animals. The 
other group was left untreated. Mice were maintained on oral DHEA-S 
supplementation until age 114 weeks when they were sacrificed and their 
spleens individually analyzed for the capacity to produce lymphokines 
following anti-CD3.epsilon. activation. The DHEA-S treated and untreated 
mice were evaluated by comparing their responses to the lymphokine profile 
produced by similarly activated splenocytes from mature adult mice (13 
weeks of age). 
More specifically, splenocytes were prepared from the following groups of 
BALB/c mice; 2 mature adult (13 weeks of age), 2 aged (114 weeks of age), 
and 2 aged (114 weeks) receiving 100 .mu.g/ml DHEA-S in their drinking 
water for the previous 61 weeks. 1.times.10.sup.7 splenocytes were 
cultured under serum-free conditions in triplicate and activated with 1 
.mu.g/ml CD3.epsilon.. Culture supernatants were analyzed for the level of 
IL-2 by a quantitative bioassay, and for IL-4, IL-5, .gamma.-IFN, IL-3 and 
GM-CSF by capture ELISA. 
FIGS. 12A-12F are graphs showing the results of DHEA-S supplementation on 
the capacity of T cells to produce a variety of lymphokines. In the FIGS. 
12A-12F, bars represent the mean .+-.SD for the value of each lymphokine 
presented. It may be seen from FIGS. 12A-12F that DHEA-S supplementation, 
administered prior to the onset of age-induced decline in 
immunocompetence, is accompanied by the preservation of normal lymphokine 
production and development of normal humoral immune responses. DHEA-S 
supplementation was not only able to preserve normal levels if IL-2, IL-3, 
and GM-CSF production by activated T cells, but was also able to prevent 
the age-related increase in .gamma.-IFN, IL-4, and IL-5 production seen in 
the cell supernatants from untreated aged donors. The results of this 
study demonstrate that a striking correlation exists between the 
age-related decline in endogenous DHEA production (plus its metabolites), 
and the age-associated alterations in T-cell production of lymphokines. 
The effect of DHEA-S supplementation on T cell function was also performed 
using BALB/c, C57BL/6 and C3H/HeN strains of mice. In each test of this 
experimental approach, lymphokine production by T cells from the treated 
aged donors had been preserved. 
In order to examine the effect of DHEA-S supplementation on the ability of 
old animals to mount immunologic responses to challenge with foreign 
protein antigens, the following procedure was used. Groups of 5 mature 
adult mice (13 weeks of age), 5 aged mice (114 weeks), and 5 aged mice 
(114 weeks) provided with chronic DHEA-S supplementation (100 .mu.g/ml 
DHEA-S in their drinking water for the previous 61 weeks, initiated at 8 
months of age), were footpad immunized with ovalbumin. The immunization 
was with 100 .mu.g ovalbumin in a 25 .mu.l volume of Maalox, administered 
in the hind footpads. All animals were bled on days 0, 3, 5, 7, 10, and 14 
post immunization, and individual serum samples analyzed for ovalbumin 
specific antibody titers by quantitative ELISA, using ovalbumin for 
capture and HRPO-coupled, goat anti-murine Ig detecting antibodies with 
specificity for IgM and IgG subclasses. Each ELISA assay was controlled 
with sera known to be positive or negative for anti-ovalbumin activity. 
The titer is the inverse of the antibody dilution equal to the 
half-maximal point on the titration curve. The results of the study, shown 
in the graph in FIG. 12G, demonstrate that old animals provided with 
chronic DHEA-S supplementation remain fully capable of rapidly mounting a 
significant humoral immune response to ovalbumin immunization, with 
kinetics, titers, and isotype profiles (data not shown), that are almost 
identical to mature adult controls. As expected, the untreated aged mice 
responded poorly to a similar antigen challenge, producing predominantly 
IgM. 
Example 10 
DHEA-S Administration to Aged Mice Can Reverse Age-Associated Changes in 
T-cell Lymphokine Production and Their Depressed Humoral Immune Responses 
to Protein Antigens 
As shown above, a direct exposure of lymphocytes from aged donors to DHEA 
in vitro, immediately altered the pattern of lymphokines produced 
following activation. In addition, we have found that nonmucosal tissue 
draining lymphoid organs possesses a far greater amount of DHEA sulfatase 
activity than mucosal tissue draining lymphoid organs. These findings led 
to the hypothesis that DHEA may be serving as an effector of positional 
information for lymphocytes residing in certain lymphoid compartments. Any 
changes in immune function caused by the depressed production of substrate 
DHEA-S might, therefore, be reversible if DHEA-S is reintroduced in situ. 
This was examined in the following studies. 
Splenocytes were isolated from equal sized groups of mature adult mice (25 
weeks of age), aged mice (120 weeks of age), and aged mice given a 
subcutaneous injection of DHEA-S (100 .mu.g in 100 .mu.l propylene glycol) 
24 hours previously. 1.times.10.sup.7 splenocytes were cultured under 
serum-free conditions in triplicate and activated with 1 .mu.g/ml 
anti-CD3.epsilon.. Twenty four hours later, culture supernatants from 
individual cell cultures were analyzed for the level of IL-2 by a 
quantitative bioassay, and for IL-4, IL-5, .gamma.-IFN, IL-3 and GM-CSF by 
capture ELISA. The results, shown in FIGS. 13A-13F, demonstrate that acute 
replacement therapy with DHEA-S to aged mice restores near normal patterns 
of T-cell lymphokines within 1 day of treatment. These results strongly 
suggest that lymphoid cells from old animals exhibit no intrinsic defects. 
Rather, some of the best documented functional changes to the immune 
system which accompany aging may be due to the reduced capacity to produce 
DHEA-S. 
A representative study showing that the administration of a bolus of DHEA-S 
to aged BALB/c mice restored the capacity of the mice to develop humoral 
responses is shown in FIG. 13G. In the study, groups of 5 mature adult (25 
weeks of age), 5 aged (120 weeks of age), and 5 aged (120 weeks) receiving 
100 .mu.g DHEA-S in 100 .mu.l propylene glycol by subcutaneous injection 
the previous 24 hours were immunized with 100 .mu.g ovalbumin in a 25 
.mu.l volume of Maalox, administered in the hind footpads. Sera from 
individual mice were collected on days 0, 3, 5, 7, 10 and 14 following 
primary immunization. The titer of anti-ovalbumin antibody was assessed by 
ELISA using ovalbumin for capture and HRPO-coupled, goat anti-murine Ig 
detecting antibodies with specificity for IgM and IgG subclasses. Each 
ELISA assay was controlled with sera known to be positive or negative for 
anti-ovalbumin activity. 
The results in FIG. 13G show that old animals provided with DHEA-S only 24 
hours prior to immunization with a foreign protein antigen responded even 
better than normal mature adults in the production of antibody. 
This method of reversing age-related decline in humoral responses has been 
evaluated twice using BALB/c mice and once with C3H/HeN strains of mice. 
Similar enhancements in antibody production were achieved in all groups of 
DHEA-S treated, aged groups of mice. 
The results discussed above support the concept that some of the 
age-associated changes in immune function are extrinsic in cause, and are 
mediated by the loss in endogenous production of an essential regulatory 
steroid prohormone. 
Example 11 
Topical Application of DHEA to Aged Animals Facilitates Changes in the 
Draining Lymph Node Microenvironment That are Conducive to Successful 
Immunization 
Groups of 5 mature adult (13 weeks of age) and 10 aged BALB/c mice (114 
weeks of age) were used in the study. All of the aged BALB/c mice received 
a topical application of 10 .mu.g DHEA in 3.5 .mu.l 95% ethanol to the 
right hind footpad, 3 hours prior to immunization with 100 .mu.g ovalbumin 
in a 25 .mu.l volume of Maalox. Five of the aged mice were immunized in 
the right hind footpad (site identical to the steroid application), and 
the other 5 immunized in the left hind footpad (site opposite to the 
steroid application). Sera from individual mice were collected on days 0, 
3, 5, 7, 10 and 14 following primary immunization. The titer of 
anti-ovalbumin antibody was assessed by ELISA using ovalbumin for capture 
and HRPO-coupled, goat anti-murine Ig detecting antibodies with 
specificity for IgM and IgG subclasses. Each ELISA assay was controlled 
with sera known to be positive or negative for anti-ovalbumin activity. 
The results, shown in FIG. 14, establish that a topical application of DHEA 
prior to immunization through the same skin site, provided the aged 
animals with the ability to generate completely normal humoral immune 
responses. The untreated group of aged animals, and aged animals provided 
with topical DHEA on the footpads opposite the site of immunization, 
responded quite poorly to immunization, with minimal antibody being 
observed. 
The results of reversing the age-related decline in humoral responses has 
been repeated with BALB/c mice, and with C3H/HeN strain of mice. 
These results establish that the pronounced lymphoid organ-specific changes 
in the types of lymphokines produced by T cells from aged animals given 
topical DHEA, can be paralleled by an equally dramatic enhancement in 
ability to generate potent humoral immune responses to challenge with a 
foreign antigen protein. 
Example 12 
Lymphokine Production and Contact Hypersensitivity Responses are Modulated 
in Thermally-Injured Mice 
Some of the most profound immunological changes that appear as a result of 
thermal injury are both rapid loss in the ability to develop cellular 
immune responses of several types and an inability of activated T 
lymphocytes to produce IL-2 and .gamma.-IFN. In order to establish the 
effect on IL-4, the production of this lymphokine by T lymphocytes from 
thermally-injured and control mice was examined. 
Six to eight 8 week old BALB/c mice were shaved on their dorsal surface 
surfaces as a preparation for receiving a thermal injury. Two days after 
removal of truncal fur, all of the experimental mice were anaesthetized, 
and half were given a 20% total body surface area (TBSA), full-thickness 
scald burn. Following revival from anesthesia, the burned mice were fluid 
resuscitated over a 3-day period using normal saline. Both 
thermally-injured and control groups of mice were allowed to feed and 
drink normally for 5 days, at which time all animals were sacrificed. 
Splenocytes from individual thermally-injured and control mice were 
prepared for culture in serum-free media. 1'10.sup.7 cells was dispensed, 
in triplicate, into 24-well culture plates with or without 1.5 .mu.g 
monoclonal anti-CD3.epsilon.. Culture plates were incubated in a 
38.degree. C., 10% CO.sub.2, humidified chamber for 24 hours prior to 
collection of cell-free supernatants for quantitative evaluation of IL-2, 
.gamma.-IFN, and IL-4. Assays for each of these lymphokines was as 
described above. 
The effect of the thermal injury on lymphokine production is shown in FIG. 
15A. The results on the depressed production of IL-2 and .gamma.-IFN 
agrees with other reports on humans and rodents. In contrast to this 
observed depression in IL-2 and .gamma.-IFN levels, activated T cells from 
thermally-injured mice were found to produce a greater amount of IL-4, as 
compared to activated splenocytes isolated from control mice. 
Because thermal injury is known to compromise development of contact 
hypersensitivity responses, groups of thermally-injured (20% TBSA) and 
control mice were contact sensitized to DNFB to demonstrate that the 
thermal injury protocol, in addition to reducing the capacity of activated 
T cells to produce IL-2 and .gamma.-IFN, also results in a reduced ability 
of injured mice to develop cellular immune responses. Five days after 
receiving a 20% TBSA, equivalent groups of normal and thermally-injured 
mice were sensitized by the application of DNFB to their shaved abdomens. 
All experimental animals were challenged 4 days later by topical 
applications of DNFB to the right hind footpad. The intensity of the 
hypersensitivity reaction was determined by quantitating the difference 
between thicknesses of the challenged and the unchallenged footpad. 
FIG. 15B shows the results of the DNFB challenge studies (the bars 
represent the mean.+-.SEM). The results confirm that the development of 
contact sensitivity responses and production of the lymphokines which are 
associated with promoting these responses are depressed in mice given a 
20% TBSA, full-thickness scald burn. Furthermore, the ability of T cells 
to produce the lymphokine, IL-4, which promotes B-cell differentiation, 
immunoglobulin isotype-switching, and also potent anti-inflammatory 
activity, is not apparently depressed as a result of thermal injury. These 
findings suggest that thermal injury is a selective modulator of T-cell 
function. 
Example 13 
Treatment in vitro of Splenocytes from Thermally-Injured Mice with DHEA 
Restores the Capacity to Produce Lymphokines 
The following study was performed in order to evaluate whether the exposure 
of T cells from thermally-injured mice to DHEA would influence their 
capacity to produce lymphokines subsequent to activation. Groups of 4 to 6 
BALB/c mice were either thermally injured with a 20% TBSA scald burn or 
were non-injured controls. Five days after thermal injury, the time when 
the "immunosuppression" is maximal, all surviving mice were sacrificed and 
splenocytes from individual mice were prepared for culture in serum-free 
media. Splenocytes were seeded into 24-well macroculture plates at 
1.times.10.sup.7 cells/ml/well. Parallel cultures from each mouse were 
sham or DHEA pulsed at 10.sup.-7 M for 60 minutes, washed multiple times 
to remove nonbound steroid, and then were incubated in serum-free medium 
and were either unstimulated or stimulated with 1.5 .mu.g 
anti-CD3.epsilon.. Following a 24-hour incubation period, cell-free 
supernatants were quantitatively analyzed for IL-2 using the standard HT-2 
bioassay, and for .gamma.-IFN, IL-4, IL-5, IL-3, and GM-CSF by capture 
Elisa. The results are shown in FIGS. 16A-16F, where the bars represent 
the .+-. standard deviation for each lymphokine. 
As seen from FIGS. 16A-16F, a comparison between lymphokines produced by 
activated splenocytes from thermally-injured and control animals indicates 
that thermal injury causes a depression in the capacity of activated T 
cells to secrete IL-2, .gamma.-IFN, IL-3, and GM-CSF. Only minimal changes 
(elevations) in the quantities of IL-4 and IL-5 were observed. The 
treatment of T cells from the thermally-injured animals with DHEA reversed 
the inhibitory effect on IL-2, .gamma.-IFN, IL-3, and GM-CSF production; 
the values of these lymphokines returned to near control levels. The DHEA 
treatment had no effect on IL-4 or IL-5 production. 
Example 14 
Treatment in vivo of Thermally Injured Mice with DHEA Preserves Normal 
Immune Function 
The following study illustrates that the direct administration of DHEA to 
mice shortly after thermal injury influences their levels of 
immunocompetence. 
Groups of 12 thermally-injured and 6 control BALB/c mice were established 
as described above. After subjecting the mice to a 20% TBSA scald burn, 
six of the thermally-injured mice were given a subcutaneous injection of 
100 .mu.g DHEA in a propylene glycol carrier. All remaining animals 
received the carrier alone. Five days later, all surviving mice were 
sacrificed and their splenocytes were individually prepared for culture, 
and activated with anti-CD3.epsilon. to induce lymphokine secretion. 
Culture supernatants were collected 24 hours after activation and 
evaluated for lymphokine content, as described above. The results of the 
study are presented in FIGS. 17A-17F, where the bars represent mean .+-.SD 
for each value. As seen in FIGS. 17A-17F, DHEA directly influences IL-2, 
.gamma.-IFN, IL-3, and GM-CSF production by T cells isolated from 
thermally-injured mice. The administration of a single bolus injection of 
DHEA (100 .mu.g) 1 hour after thermal injury was sufficient to preserve 
for at least 5 days a normal capacity by their lymphocytes to produce 
IL-2, .gamma.-IFN, IL-3, and GM-CSF following activation. No significant 
changes from normal were observed in the levels of IL-4 and IL-5 made by 
activated lymphoid cells from these animals. 
The effect of DHEA treatment in vivo on the animals' development of 
cellular immune responses was examined. Parallel groups of 
thermally-injured and control mice were either given 100 .mu.g DHEA in 
propylene glycol carrier or the carrier alone 1 hour post burn. These 
animals were contact sensitized 5 days later by administration of DNFB on 
the abdomen. Challenge doses of DNFB to the right footpads were applied 4 
days later. The differences in thickness between the right (challenged) 
and the left (unchallenged) footpads were used to quantitate the contact 
hypersensitivity responses. The results are shown in FIG. 18; the bars 
represent mean .+-.SD for each group of mice. As shown in FIG. 18, the 
intensity of the contact hypersensitivity responses elicited by 
thermally-injured mice are markedly depressed as compared to controls. The 
administration of DHEA to thermally-injured mice was found to completely 
preserve the ability of these animals to develop contact hypersensitivity 
responses of normal intensity. 
We conclude from these studies that DHEA treatment post burn is an 
effective therapy for preserving the capacity of T-lymphocytes from 
thermally-injured animals to produce normal quantities of a number of 
lymphokines, especially those that are essential for development of 
cellular immune responses. This finding is supported by the additional 
demonstration that DHEA-treated thermally-injured mice also retain their 
capacity to develop normal contact hypersensitivity responses. 
Example 15 
DHEA Treatment in vivo Promotes Resistance to Infection by L. monocytogenes 
in Thermally Injured Mice 
This study addresses the utility of DHEA therapy post burn in preserving 
resistance to a bacterial infection. C3H/HeN strain mice are inherently 
resistant to infection by the gram positive intracellular pathogen, L. 
monocytogenes. However, thermal injury results in an increased 
susceptibility to this pathogen. Therefore, a switch from "resistant" to a 
more "susceptible" phenotype provides a model system to evaluate the 
effect of DHEA on preserving the "resistant" phenotype in 
thermally-injured animals. 
Normal (control) and thermally-injured mice were prepared as described 
above. Half of the thermally-injured mice received a single bolus 
injection of 100 .mu.g DHEA subcutaneously within 1 hour after thermal 
injury. Three days later, all mice were infected with 2.times.10.sup.6 
viable L. monocytogenes organisms, and 3 days after infection the mice 
were sacrificed and homogenates of individual spleens were prepared. The 
number of colonies of L. monocytogenes per spleen were evaluated using 
standard methodology, and scored. The results are presented graphically in 
FIG. 19, where the bars represent means .+-.SEM for each treatment group. 
The results indicate that thermal injury enhances the susceptibility of 
the C3H strain mice to infection by L. monocytogenes. Of consequence, DHEA 
treatment of burned animals not only preserves the resistant phenotype, 
but surprisingly, the level of resistance to infection is actually 
enhanced by DHEA treatment over that observed in the control group. 
Example 16 
The Effect of 16.alpha.-bromo-DHEA and 16.alpha.-chloro-DHEA on 
Age-Associated Changes in T-cell Lymphokine Production 
In order to demonstrate the effectiveness of the cogeners of DHEA, 
16.alpha.-bromo-DHEA and 16.alpha.-chloro-DHEA on age-associated changes 
in T-cell lymphokine production, the following study was performed. 
Suspensions containing T cells were prepared from the mesenteric lymph 
node and from the spleen of groups of aged (old) mice. The T cell 
suspensions were exposed in vitro to DHEA, 16.alpha.-bromo-DHEA or 
16.alpha.-chloro-DHEA at a concentration of 10.sup.-7 M for 60 minutes. 
After activation with anti-CD3.epsilon., the IL-2 titers in the cell-free 
supernatants was measured by bioassay. The results, shown in FIG. 20, 
indicate that 16.alpha.-bromo-DHEA is as active as DHEA in restoring IL-2 
production by the activated T-cells. However, surprisingly, 
16.alpha.-chloro-DHEA had little, if any, effect on restoring IL-2 
production. 
Example 17 
The Effect of 16.alpha.-bromo-DHEA and 16.alpha.-chloro-DHEA on the 
Depressed Humoral Immune Responses of Aged Mice to Protein Antigens 
The effectiveness of the cogeners of DHEA, 16.alpha.-bromo-DHEA and 
16.alpha.-chloro-DHEA on age-associated changes in the humoral immune 
response is demonstrated in the following study. Groups of mature (young) 
and aged (old) mice were treated by topical administration of 10 .mu.g of 
DHEA, 16.alpha.-bromo-DHEA, or 16.alpha.-chloro-DHEA. Three hours 
subsequent to treatment, the treated and control animals were subjected to 
an ovalbumin (OVA) challenge as described in the Examples above, and the 
anti-ovalbumin antibody response was measured at 0, 3, 5, 7, 10, and 14 
days after immunization. The results, shown in FIG. 21, indicate that 
16.alpha.-bromo-DHEA (BrD) is as effective as DHEA in restoring humoral 
immune responsiveness. The chlorinated cogener, 16.alpha.-chloro-DHEA 
(ClD) yielded a lower, but significant effect on antibody production.