WO1999051253A2 - Immune response modulation - Google Patents

Abstract

A method of modulating an immune response in a patient in need of such modulation, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof, or an effective amount of an antagonist or of an agonist of leptin.

Description

IMMUNE RESPONSE

The present invention relates to modulation of the immune response, in particular to its modulation during nutritional deprivation.

Nutritional deprivation suppresses immune function^1"3. The recent cloning of the ob gene and identification of its protein product leptin⁴ has provided fundamental insight into the regulation of body weight^5,6. Circulating levels of this adipocyte-derived hormone are proportional to fat mass^6,7 but may be lowered rapidly by fasting^8,9 or increased in response to inflammatory mediators^10,11. In addition to the hypothalamus, where it mediates the central actions of leptin⁶, its receptor has also been found in lymph nodes¹². The impaired T cell immunity of obese mice^13,14, now known to be defective in leptin (pb/obf or its receptor (db/db)^nΛ5, has never been explained. Impaired cell-mediated immunity^1"3 and reduced leptin levels⁷ are both features of low bodyweight in humans. Indeed, malnutrition predisposes to death from infectious diseases¹⁶.

Leptin is structurally homologous to interleukin-2 and it signals through a class I cytokine receptor. It is a 16 kDa adipocyte-derived hormone (Zhang, Y., Proenca, R. , Maffei, M., Barone, M., Leopold, L., & Friedman, J.M. (1994) Nature (London) 372: 425-432) whose plasma concentrations are proportional to fat mass (Considine et al. (1996) N. Engl. J Med 334:292-295; Maffei, M et al (1995) Nat. Med (London) 2:1155-1161) but lowered rapidly by fasting in both rodents and humans (Ahima, R.S et al (1996) Nature (London) 382:250-252; Boden, G., et al (1996) J Clin. Endocrinol Metab. 81:3419-3423). Indeed, there is increasing evidence that leptin is a key signal of nutritional status regulating several aspects of metabolism and neuroendocrine function as part of an adaptive response to starvation (Ahima, R.S et al (1996) Nature (London) 382:250-252; Flier, J.S. (1998) J Clin. Endocrinol Metab.

83: 1407-1413).

Lymphoid atrophy is a well recognised consequence of nutritional deprivation in animals, including man (Simon, J. (1845) in A Physiological Essay on the Thymus Gland. (London: Renshaw); Jackson, CM. (1925) in The Effects of Inanition and Malnutrition upon Growth and Structure. (Philadelphia. : Blakiston's Sons and Co); Smythe, P.M et al (1971) Lancet II 506-508; Martinez, D et al (1975) / Natl. Cancer Inst. 55:935-939; Chandra, R.K. (1991) Am. J. Clin. Nutr. 53:1087- 1101). This disproportionate loss of lymphoid tissue with starvation is particularly pronounced in the thymus, which has been designated "the barometer of malnutrition" (Simon, J. (1845) in A Physiological Essay on the Thymus Gland. (London: Renshaw)). The thymus is crucial to T-cell development; it provides the specialised microenvironment required for T- cell maturation and the generation of a diverse T-cell receptor repertoire (Blackman, M., Kappler, J., & Marrack, P. (1990) Science 248: 1335- 1341; von-Boehmer, H. (1994) Cell 76:219-228; Ashton Rickardt, P.G et al (1994) Cell 76:651-663; Ritter, M.A., & Boyd R.L. (1993) Immunol. Today 14:462-469; Shortman, K et al (1990) Semin. Immunol. 2:3-12).

Acute starvation causes a significant reduction in circulating leptin levels (Ahima, R.S et al (1996) Nature (London) 382:250-252; Boden, G et al (1996) J Clin. Endocrinol Metab. 81:3419-3423). Measurements of leptin levels have been made in patients suffering from conditions associated with changes in body mass. For example, low circulating leptin levels were found in protein-energy malnourished chronically ill elderly patients by Cederholm et al (1997) / Intern Med 242(5), 377-382. Serum leptin levels were low and appeared not to be directly associated with fat and muscle depletion.

Grunfeld et al (1996) J Clin Endocrinol Metab 81(12), 4342-4346 report the measurement of serum leptin measurements in patients with aquired immunodeficiency syndrome (AIDS) and found no increase in leptin levels relative to body fat in patients who were anorectic, were losing weight or had a history of weight loss, suggesting to the authors that elevations in leptin do not play a key role in these symptoms. Yarasheki et al (1997) Metabolism 46(3), 303-305 also report that circulating leptin concentrations reflected adipose tissue mass in HIV-infected men with low body fat content and suggest that low leptin levels do not stimulate food intake in HIV-infected individuals.

Simons et al (1997) Clin Sci Colch 93(3), 273-277 investigated plasma concentrations of total leptin in patients with lung cancer. Those patients with higher leptin levels had less weight loss, were less underweight and had a higher fat mass. The conclusion was elevated leptin levels were not involved in the development of cachexia, but that the response to low leptin levels may be abnormal.

We determined the effect of leptin on the weight and cellularity of lymphoid and non-lymphoid tissues in mice fasted for 48 hours and compared this with the effects observed in PBS-treated starved mice and ad libitum fed controls. We also looked at the effect of starvation with and without leptin repletion on specific thymocyte and splenocyte subpopulations and thymic histology. In order to examine the effect of leptin replacement on lymphoid tissue in a chronically leptin deficient model, we administered leptin to ob/ob mice. These mice are unable to produce functional leptin due to a mutation in the obese gene (Zhang, Y et al (1994) Nature (London) 372: 425-432).

We report here that leptin has a specific effect on human T lymphocyte responses, differentially regulating the proliferation of naive and memory cells. Proliferation by naive, but not memory cells, in response to allogeneic stimulator cells was increased five-fold by the addition of physiological concentrations of leptin. In contrast, memory, but not naive T cells secreted ten-fold more of the pro-inflammatory cytokine IFN-γ when stimulated in the presence of leptin, skewing the response towards a Thl phenotype. These effects were not observed when using T cells from db/db mice, demonstrating their specificity. Leptin caused cellular clumping and upregulation of key adhesion molecules ICAM-1 and VLA-2 on T cells, a phenomenon which would increase the cell-cell contact required for their activation. Thus, leptin increased both production of pro-inflammatory cytokines and the expression of adhesion molecules. Leptin also increases the proliferation of immature T cells. In vivo, administration of leptin to mice, reversed the immunosuppressive effects of acute starvation.

Our findings suggest a novel role for leptin in linking nutritional status to cognate cellular immune function, and provide a molecular mechanism to account for the immune dysfunction observed in starvation. A first aspect of the invention provides a method of modulating an immune response in a patient in need of such modulation, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof, or an effective amount of an antagonist or of an agonist of leptin.

Leptin is a protein which, in the mouse, is expressed from the ob gene by adipocytes and other cells. The amino acid sequence of mouse leptin and the cDNA sequence of mouse leptin mRNA is given in Zhang et al (1994) Nature 372, 425-432, as is the amino acid sequence of human leptin and the cDNA sequence of human leptin mRNA.

Suitable quantities of leptin can be made using standard recombinant DNA methods, for example, see Erickson et al (1996) Nature 381, 415-421 and Chehab et al (1996) Nature Genet. 12, 318-320. Leptin may be produced in any suitable host cell such as a bacterial or yeast or insect or mammalian cell. It is particularly preferred if the leptin is produced in and obtained from a mammalian cell, particularly one which has been transfected or infected with a nucleic acid which encodes and expresses leptin. The methods described herein for the expression of leptin receptor may also be used for the expression of leptin.

By "functional derivative of leptin" we include (a) variants of leptin which, for example, have amino acid replacements compared to wild-type leptin; (b) portions or fragments of leptin whether or not they also have amino acid replacements compared to the wild-type leptin sequence; and (c) fusions of leptin with other peptide sequences or fusions of variants or fragments of leptin. In all cases, the derivative is a functional derivative in the sense that it retains substantially the same activity towards the immune system as herein disclosed for leptin. Amino acid replacements in variant leptins are, typically, of a conservative nature. By "conservative" we include replacements within the groups Ala, Gly; Phe, Tyr; He, Leu, Val; Glu, Asp and Gin, Asn. Further functional derivatives include leptin any of the above-mentioned derivatives modified so as to improve their stability or pharmaco-kinetic properties. Modifications of proteins that do this, such as PEGylation (polyethylene glycol treatment) are well known in the art. Such modifications may improve the circulating half-life of leptin following administration to the patient.

It will be appreciated that it is preferred if the leptin is from the same species as the patient to be treated. For example, it is particularly preferred if, when the patient is a human patient the leptin in human leptin or a derivative thereof.

By an "agonist of leptin" we include a molecule which binds to the leptin receptor, preferably human leptin receptor, and upon binding the target cell exhibits a response which qualitatively is substantially the same as upon binding leptin itself. It will be appreciated that agonists of leptin may induce a leptin-like response at a higher concentration than does leptin (in which case the agonist is less potent than leptin itself) or the agonist may induce a leptin-like response at a lower concentration than does leptin (in which case the agonist is more potent than leptin itself). For the avoidance of doubt a functional derivative or leptin may be a leptin agonist, but the term agonist also includes other molecules which bind to the leptin receptor; for example, it is believed that certain anti- leptin receptor antibodies may act as leptin agonists. By an "antagonist of leptin" we include a molecule which binds to the leptin receptor, preferably human leptin receptor, with no intrinsic leptin activity but with sufficient affinity to block the binding, at physiological concentrations, of endogenous leptin and thereby prevent its biological action. In the context of the invention the antagonist of leptin is one which prevents the biological action of leptin on the T cells of the immune system as herein disclosed. In man the reported circulating levels of leptin are from around 12 ± 4 ng.ml^"1 for lean individuals and around 42 ± 9 ng.ml^"1 for obese individuals.

A leptin antagonist (human leptin mutant R128Q) is described in Verploegen et al (1997) FEBS Lett. 405, 237-240, incorporated herein by reference.

It will be appreciated that it is preferred that the antagonist antagonises the leptin from the species who is to be treated.

Preferably, when the patient to be treated is human the antagonist is an antagonist of human leptin.

By "compound that substantially prevents leptin having its biological effect on the leptin receptor" we include any such compound that does this and specifically include compounds which bind leptin. Preferably, the leptin receptor is a leptin receptor of the type found in T cells of the immune system as herein disclosed. Preferably, the antagonist of leptin is an anti-leptin antibody or fragment or derivative thereof including any protein engineered or synthetic antibody or derivative or fragment thereof which can bind leptin. Monoclonal antibodies are preferred. An agonist or antagonist of leptin may be an anti-leptin receptor antibody.

Monoclonal antibodies which will bind to leptin are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens including leptin receptor. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982).

Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

Suitably prepared non-human antibodies can be "humanized" in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.

The variable heavy (V_H) and variable light (V_L) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V_H and V_L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293- 299.

By "ScFv molecules" we mean molecules wherein the V_H and V_L partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments. Whole antibodies, and F(ab')₂ fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab')₂ fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.

The human and mouse leptin receptors cDNA have been cloned by Tartaglia et al (1995) Cell 83, 1263-1271, incorporated herein by reference.

The sequence of the human leptin receptor cDNA is found in the GenBank/EMBL databank under GenBank Accession No U43168. Preferably, the leptin receptor is a human leptin receptor.

By "compound which binds to the leptin receptor" we mean a compound which binds selectively to the leptin receptor and preferably not to other components on the cell surface.

Preferably, the compound binds to the leptin receptor with a dissociation constant of at least 10^"2 M, more preferably at least 10^"4 M, still more preferably at least 10^"5 M or at least 10^"6 M or at least 10^"8 M. It will be appreciated that strong binding to the leptin receptor is preferred. Compounds with a relatively low affinity for the leptin receptor are less preferred since a higher concentration of such compounds would be required in order to achieve a desirable suppression of unwanted immune response.

Conveniently, the compound is identified by determining whether it can compete with leptin for binding to the leptin receptor. Suitably, when the human leptin receptor is used, human leptin is used to compete with the compound. Receptor binding assays, and in particular competition assays to identify antagonists which bind to a receptor are well known in the art.

It will be appreciated, as discussed above, that for competitive binding assays the "leptin receptor" may be any leptin receptor or any leptin- binding portion of the leptin receptor.

Suitably, the compound may also be identified by determining whether it can block the action of leptin by binding to the leptin receptor in a non- competitive fashion.

Both competitive leptin antagonists and non-competitive leptin antagonists are useful in the modulation of an immune response.

Methods of identifying non-competitive antagonists which bind to a receptor are well known in the art. The putative antagonist would be tested for its ability suppress the immune response.

Agonists of leptin may also be identified using methods disclosed herein.

Although whole cells which express the leptin receptor can be used in the identification of a compound which binds to the leptin receptor (and competes with leptin for binding), it is more convenient to use a component of a cell which comprises the said receptor. It is particularly preferred if a cell membrane component of the cell is used. Thus, a preferred embodiment of the invention provides a cell which expresses a leptin receptor (or any leptin-binding portion of a leptin receptor) or a component of said cell comprising said receptor or said portion. Conveniently, the cell which expresses the leptin receptor is a cell comprising a recombinant genetic construct encoding leptin receptor. The advantage of such cells is that they can be engineered to express the leptin receptor to a desirable level. A further advantage is that cells which do not normally express leptin receptor can be engineered to do so.

Genetic constructs which encode and express leptin receptor in a host cell can be made using methods well known in the art. Leptin receptor cDNA is available, and can be cloned into suitable vectors.

The leptin receptor DNA is expressed in a suitable host to produce a leptin receptor. Thus, the DNA encoding the leptin receptor may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of leptin receptor. Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to

Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7

July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al,

4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference. The DNA encoding leptin receptor may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell. Alternatively, the gene for such selectable trait can be on another vector, which is used to co- transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be used in the identification of a compound which binds to the leptin receptor. It is preferred if the host cell is a eukaryotic cell, most preferably a mammalian cell.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.

An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).

A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules. Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.

A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell lines. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

Electroporation is also useful for transforming cells and is well known in the art for transforming yeast cell and vertebrate cells. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.

It will be appreciated that isolated natural T cells which express leptin receptor may be used in place of genetically engineered cells.

Once it has been determined that a compound binds to said leptin receptor it is preferred then to determine whether said compound is an antagonist of leptin and substantially suppresses an unwanted immune response, or whether said compound is an agonist of leptin and enhances a desirable immune response.

It will be appreciated that it is preferred that compounds which compete with leptin for binding to a leptin receptor are identified fust before testing whether said compounds are able to substantially prevent an unwanted immune response or substantially enhance a desirable immune response.

By "administering an effective amount of leptin or a functional derivative thereof, or an effective amount of an antagonist or of an agonist of leptin" is specifically included, in the case where these compounds are proteins, the possibility that what is administered to the patient is a means for production of said compounds. Thus, the invention includes administering to the patient a nucleic acid which encodes leptin or a functional derivative thereof, or an agonist or antagonist of leptin. The nucleic acid suitably comprises a gene or cDNA encoding the protein. The nucleic acid may be naked DNA or it may conveniently be comprised in a nucleic acid delivery system such as a liposome or virus or the like. Liposomes or other lipid- based systems which can delivery nucleic acid to a cell in a body are known in the art. Suitable viral delivery systems include retroviral systems which are RNA based, or DNA based viral systems such as adenovirus or adeno-associated virus.

Other suitable methods of delivering protein-encoding nucleic acid to a patient are known in the art.

In a further embodiment, the patient may conveniently be administered a cell which has been engineered to produce leptin or a functional derivative thereof or an agonist or antagonist of leptin. Mammalian, including human, cells may readily be engineered to produce these polypeptides by introduction of a suitable protein-encoding nucleic acid. Conveniently, cells to be used in this embodiment are removed from the patient to be treated, the protein-encoding nucleic acid is introduced into the cell using methods well known in the art, some of which are disclosed herein, and the modified cell is reintroduced into the patient.

The patient in need of modulation of an immune response may be any such patient. The immune response may be enhanced or the immune response may be suppressed but, in either case the amount of leptin or a functional derivative thereof that is administered, or the amount of an agonist or antagonist of leptin that is administered, is an amount effective to have a desirable effect in the patient. Thus, the amount of leptin or a functional derivative thereof or agonist of leptin will be sufficient to enhance the immune response to a therapeutically useful extent, or the amount of an antagonist of leptin will be effective to suppress the immune response to a therapeutically useful extent.

It will be appreciated that modulation of an immune response may be effected via a change in the number or type of cells available to participate in the immune response, or by a change (quantitative or qualitative) in the activity of the cells available to participate in the immune response. Thus, modulation of an immune response may be effected, for example, by a change in the number or relative proportions of different classes of T cells, for example naive, memory or various classes of immature T cells, as described in Examples 1 and 2. Naive and memory T cells may be present in the circulation or peripheral tissue. Immature (maturing) T cells are found mainly in the thymus or spleen; thus a deficiency in the formation, maturation or survival of immature T cells may be manifested in a smaller than normal thymus. Naive and memory T cells are derived from immature T cells; thus, a deficiency in the formation, maturation or survival of immature T cells may result in a deficiency in the number of naive and memory T cells. CD4+CD8+T cells may be immature T cells, as discussed in Example 2.

Modulation of an immune response may also be effected by a change in the production of proinflammatory cytokines by T cells on stimulation, as described in Example 1.

It will be appreciated that modulation of an immune response may be effected by changes that are only apparent in the presence of a stimulus, for example major histocompatibility complex incompatible (allogeneic) stimulator cells, as described in Example 1, for example changes in the production of proinflammatory cytokines or cellular proliferation following stimulation, or it may be effected by changes that are apparent in the absence of a stimulus, for example a change in the number or relative proportions of T cells of different classes or at different stages of develompent, as described in Example 2, in particular a change in the number of CD4+CD8+ immature thymocytes.

A second aspect of the invention provides a method of enhancing an immune response in a patient in need of such enhancement, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin.

It is preferred if the dose of leptin or a functional derivative thereof or an agonist of leptin administered is enough to give a serum concentration in the patient of between 10^"10 M to 10^"5 M, preferably between 10^"9 M to 10^"6 M.

In a preferred embodiment of the invention the patient has been, is, or will be in a state of starvation. The state of starvation is likely to be one of chronic malnourishment and includes any reduction of food intake which subsequently reduces cell-mediated immune responses. Reduction of cell- mediated immune responses can be determined by methods well known in the art and include the measurement of reduced response to infectious agents and a delayed type hypersensitivity (DTH) response as described in the Examples. Determining the CD4⁺ T cell count may also be useful. Without prejudice to the scope or practice of the invention, we believe that administration of leptin may be useful in substantially restoring or replacing the loss of immune function (immune deficiency or dysfunction) in a patient who is in a state of starvation or malnourishment. It will be appreciated that administration of leptin may be useful in encouraging the development of improved immune function in undernourished patients, for example undernourished babies, children or elderly patients.

The patient may have below normal body weight or below normal proportion of body weight as fat. Normal body weight ranges and proportions of body weight as fat are well know to those skilled in the art and may be expressed in terms of a body mass index, calculated as weight (kg) divided by (height (metres))²; W/H². Normal ranges may be given, for example, in Blacks Medical Dictionary. Triceps skin fold measurements may be used in assessing body fat, as described, for example, in Cederholm et al (1997) / Intern Med 242(5), 377-382.

The patient may be suffering from or at risk of cachexia, for example as a result of heart or kidney failure or cancer.

The patient may have thymic atrophy and/or splenic atrophy. Thymic atrophy may occur in cachexic patients or undernourished patients, as discussed above. Thymic atrophy may be diagnosed or indicated, for example, by measurements of peripheral T cell numbers, or by other methods well known to those skilled in the art.

Thymic function and/or size reduces progressively following puberty. Without prejudice to the scope or practice of the invention, we believe that administration of leptin may be useful in substantially restoring or replacing the loss of immune function (immune deficiency or dysfunction) in an elderly patient. Thus, the patient may be an elderly patient, for example a human patient over the age of 60, in particular a malnourished elderly patient and/or with low body mass.

In a further preferred embodiment, the patient has or is at risk of a microbial infection. By "microbial infection" we include infection with microbial pathogens such as bacteria, viruses and fungi. The microbial pathogens typically are those that trigger a cell-mediated immune response and include, in particular, intracellular pathogens including viruses (particularly HIV) and intracellular bacteria such as Mycobacteria and fungi such as Pneumocystis and Aspergillus. The patient to be treated may suitably be suffering from an acute infection in which antibiotics or other anti-microbial agents may not be sufficient to clear the pathogen from the patient. The patient to be treated may also suitably be infected with antibiotic-resistant bacteria or have a viral infection. In all of these cases, we believe that treatment of the patient with an effective amount of leptin or a functional derivative thereof will be useful in boosting the patient's immune response and will be useful in combating the disease caused by the microbial agent.

A particular group of patients who are believed to be likely to benefit from treatment with leptin, or a functional derivative thereof, are patients with HIV infection and particularly those with acquired immune deficiency syndrome (AIDS). AIDS patients do not show the normal increased leptin concentration which is seen in non-AIDS patients during acute infections and so it is preferred if leptin or a functional derivative thereof is administered to a patient with AIDS especially during times of acute infection, for example, by an opportunistic pathogen, or as prophylaxis against such infection. It will be appreciated that leptin or a functional derivative thereof may be useful in promoting the development of immature and mature T cells; it may promote generation of a new T cell repertoire.

It is known that starvation significantly impairs vaccine efficiency; thus, vaccination of people who have a poor diet, such as in third world countries, leads to less than optimal protection. In a further embodiment of the invention, a patient to be vaccinated is also administered an effective amount of leptin or a functional derivative thereof or an agonist of leptin to boost or enhance the immune response to the vaccine. The leptin or a functional derivative thereof or agonist of leptin may usefully be given before, during or after vaccination.

Preferably, the leptin or functional derivative thereof or agonist of leptin is administered within about 7 days either side of the vaccination. In one embodiment, it is administered for seven consecutive days around the time of vaccination. Vaccines with which it is believed to be useful to co- administer leptin or functional derivatives thereof or agonists of leptin include BCG, tetanus, malaria, yellow fever, mumps, measles, and rubella vaccines.

The leptin or functional derivative thereof or agonist of leptin may be used as an adjuvant.

A further preferred embodiment of the invention includes the administration of leptin or a functional derivative or an agonist of leptin thereof to a patient who will be, is being or has been treated with an anti- tumour vaccine.

Preferably, the leptin or functional derivative thereof or agonist of leptin is administered within about 7 days either side of the vaccination. In one embodiment it is administered for 7 consecutive days around the time of vaccination. The anti-tumour vaccine may be given prophylactically but, as is well known in the art, anti-tumour (anti-cancer vaccines) are often used therapeutically. Anti-tumour vaccines include vaccine systems which are based on raising an immune response towards tumour-associated antigens and include anti-tumour vaccines which make use of MAGE tumour-associated antigen and p53.

A suitable anti-tumour vaccination according to the practice of the invention includes the injection into the patient of his own (syngeneic) dendritic cells which have been transfected with a nucleic acid expressing MAGE or p53.

Clearly, the basis of the anti-tumour vaccine is determined on the type of tumour to be treated or prevented.

A third aspect of the invention provides a method of suppressing an immune response in a patient in need of such suppression, the method comprising administering to the patient an effective amount of an antagonist of leptin.

A suitable dose of leptin antagonist may be between about 1-100 ng/ml final concentration in the serum of the patient. Suppression of an immune response in a patient is desirable in a number of circumstances, such as in the prevention of transplant rejection and in the treatment of autoimmune diseases, allergy and in certain other biological therapies which have an effect on the immune system.

We have found that leptin has a significant effect on naive T cell responses and it is these which are considered to be the initial cause of damage to transplanted tissue in a transplant patient. Antagonists of leptin (including, as noted above, antibodies which block leptin action) are believed to be useful in the treatment of patients at the time of allograft transplantation in order to block unwanted immune responses and to allow tolerance to develop towards the allograft. Thus, in a preferred embodiment of the invention, the patient who is in need of immune suppression and who is administered an effective amount of an antagonist of leptin is a patient who has been, is being, or will be treated by allograft transplantation.

The term "allograft" includes "xenograft". A xenograft is a type of allograft in which the cell or tissue or organ to be transplanted is from a different species to the recipient. For example, organs from pigs may be transplanted into a human recipient. Suitably, the pig may be an engineered pig in which certain cell surface antigens are human antigens (ie expressed from human-derived genes) and not pig antigens.

Allograft transplantations include transplantations of heart, kidney, lung, liver, small bowel, pancreas and, also, skin grafts. It is known that caloric restriction (a reduction in food intake) can prevent the development of autoimmune disease in murine models. We believe that blocking the leptin-T-cell axis may be useful in the treatment of autoimmune diseases. Thus, in a further embodiment of the invention, the patient who is in need of immune suppression and who is administered an effective amount of an antagonist of leptin is a patient who has or is at risk of an autoimmune disease.

Autoimmune diseases include rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, Hashimoto's thyroiditis, coeliac disease, myasthenia gravis, pemphigus vulgaris, systemic lupus erythromatosus, Grave's disease and systemic vasculitis.

A fourth aspect of the invention provides the use of leptin or a functional derivative thereof, or an antagonist or an agonist of leptin, in the manufacture of a medicament for modulating an immune response in a patient in need of such modulation.

A fifth aspect of the invention provides a method of treating a patient with or at risk of a microbial infection the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or antagonist of leptin and an anti-microbial agent. Preferably, the patient to be treated is an AIDS patient.

A sixth aspect of the invention provides a method of treating a patient with or at risk of cancer the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin and an anti-tumour vaccine. Patients at risk of cancer are those with a familial predisposition to cancer (such as breast cancer, ovarian cancer or bowel cancer) or those patients who have been identified as having a pre-cancerous lesion. The anti- tumour vaccine is tailored according to the cancer.

A seventh aspect of the invention provides a method of improving vaccine efficacy in a subject to be vaccinated, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof before, during or after administration of the vaccine. Preferably, the subject has been, is or will be in a state of starvation.

An eighth aspect of the invention provides a method of improving the prospects of a successful allograft transplantation in a patient, the method comprising administering to the patient an effective amount of a leptin antagonist before, during or after the transplantation. Suitably, the administration of leptin antagonist reduces the risk of transplant rejection to a clinically useful extent. It will be appreciated that the leptin antagonist may usefully be administered with other immunosuppressive reagents such as those well known in the art, such as cyclosporin.

A ninth aspect of the invention provides a method of treating an autoimmune disease in a patient, the method comprising administering to the patient an effective amount of a leptin antagonist.

A tenth aspect of the invention provides a method of treating an inflammatory condition the method comprising administering to the patient an effective amount of an antagonist of leptin. An eleventh aspect of the invention provides a method of determining the immune levels of a subject, the method comprising determining the leptin level in the patient.

A twelfth aspect of the invention provides a pharmaceutical composition comprising, in combination but not necessarily for contemporaneous administration, leptin or a functional derivative thereof or an agonist of leptin and any one of an anti-microbial agent or an anti-tumour vaccine or a vaccine.

Anti-tumour vaccines and conventional vaccines have been described above.

Antibiotics such as anti-bacterial agents, for example natural and synthetic penicillins and cephalosporins, sulphonamides, erythromycin, kanomycin, tetracycline, chloramphenicol, rifampicin and including gentamicin, ampicillin, benzypenicillin, benemamine penicillin, benzathine penicillin, phenethicillin, phenoxy-methyl penicillin, procaine penicillin, cloxacillin, flucloxacillm, methicillin sodium, amoxiciilin, bacampicillin hydrochloride, ciclacillin, mezlocillin, pivampicillin, talampicillin hydrochloride, carfecillin sodium, piperacillin, ticarcillin, mecillinam, pirmecillinan, cefaclor, cefadroxil, cefotaxime, cefoxitin, cefsulodin sodium, ceftazidime, ceftizoxime, cefuroxime, cephalexin, cephalothin, cephamandole, cephazolin, cephradine, latamoxef disodium, aztreonam, chlortetracyclme hydrochloride, clomocycline sodium, demeclocydine hydrochloride, doxycycline, lymecycline, minocycline, oxy tetracycline, amikacin, framycetin sulphate, neomycin sulphate, netilmicin, tobramycin, colistin, sodium fusidate, polymyxin B sulphate, spectinomycin, vancomycin, calcium sulphaloxate, sulfametopyrazine, sulphadiazine, sulphadimidine, sulphaguanidine, sulphaurea, capreomycin, metronidazole, tinidazole, cinoxacin, ciprofloxacin, nitrofurantoin, hexamine, streptomycin, carbenicillin, colistimethate, polymyxin B, furazolidone, nalidixic acid, trimethoprim-sulfamethoxazole, clindamycin, lincomycin, cycloserine, isoniazid, ethambutol, ethionamide, pyrazinamide and the like; anti-fungal agents, for example miconazole, ketoconazole, itraconazole, fluconazole, amphotericin, flucytosine, griseofulvin, natamycin, nystatin, and the like; and anti-viral agents such as acyclovir, AZT, ddl, amantadine hydrochloride, inosine pranobex, vidarabine, and the like, may be used.

A thirteenth aspect of the invention provides a pharmaceutical composition comprising, in combination but not necessarily for contemporaneous administration, an antagonist of leptin and an immunosuppressive reagent. The immunosuppressive reagent may be any suitable reagent, for example cyclosporin or other such drugs in clinical use.

The components of the composition may be mixed together in a form ready for administration, or they may be present separately for separate administration. In the latter case, the pharmaceutical composition could be considered to be a package of medicines or a kit of parts.

Suitably, the leptin or functional derivative thereof or an agonist of leptin is kept freeze-dried and is only reconstituted prior to use.

The aforementioned compounds for use in the methods of the invention (leptin or a functional derivative thereof or agonist of leptin; or an antagonist of leptin) or a formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.

Whilst it is possible for a compound to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

The invention will now be described in more detail by reference to the following Example and Figures wherein

Figure 1 shows that the incubation of cells in the presence of leptin increased the T cell alloresponse in human and murine systems. This effect was not seen when the db/db mouse was used as a source of responder cells, a, b, Thymidine incorporation in the presence of leptin in an MLR performed using human PBL as responder and MHC- mismatched PBMC as stimulator cells (a) or highly purified CD4⁺ T cells as responders at a responder/stimulator ratio of 1:1 (b) (6 experiments). c, Preincubation of either responder CD4⁺ T cells or irradiated stimulator allogeneic monocytes with 10^"8 M leptin for 12 hours, prior to co-culture; the data are expressed as percentage increase of proliferation in the MLR above baseline proliferation in the absence of leptin (2 x 10⁴ cpm) d, MLR using murine heterozygous dbl+ (H-2^d) and homozygous db/db (H-2^d) splenocytes as responders with irradiated C57BL/6 (H-2^b) allogeneic splenocytes (3 experiments). All data are expressed as mean + s.e.m. Figure 2 shows that the long signalling form of the leptin receptor (ObRb) mRNA is expressed in highly purified human CD4⁺ T cells, a, RT-PCR with primers specific for the long intracytoplasmic tail, only expressed in ObRb: lane 1 hypothalamus; lane 2 no RNA control; lane 3 PBMC; lane 4 CD4⁺ T cells; lane 5 monocytes; lane 6 renal mesangial cells, b, Southern blot analysis of the PCR product confirmed the specificity of the PCR product, c, RT-PCR with primers specific for β-actin.

Figure 3 shows that leptin differentially regulates the proliferation of naive and memory T cells, a, Depletion of CD45RO⁺ but not of

CD45RA⁺ cells from human PBMC led to a marked increase in proliferation in an MLR against allogeneic PBMC (4 experiments), b,

Preincubation for 12 hours with leptin (10⁸ M), increased proliferation of

CD4⁺ CD45RA⁺ T cells but had little effect on CD4⁺CD45RO⁺ T cells in an MLR against irradiated allogeneic PBMC; the data are expressed as percentage increase of proliferation in the MLR after pretreatment with leptin (3 experiments), c, MLR using human umbilical cord blood responder cells (UCB) against irradiated allogeneic PBMC (6 experiments), d, An adult T cell recall antigen response to tetanus toxoid showing minimal effect of added leptin (3 experiments). All data are expressed as mean ± s.e.m.

Figure 4 shows the differential cytokine production in the presence or absence of leptin using different responder cell populations in an MLR against allogeneic irradiated PBMC. CD45RO⁺ cells showed a marked increase in IFN-γ (a), but not IL-2 production (b), whereas CD45RA⁺ cells and PBMC showed increased production of both cytokines. UCB cells showed minimal increase in IFN-γ production and a marked stimulation of IL-2 production (a, b). Only CD45RO⁺ cells show detectable IL-4 production, which is completely suppressed by leptin (c) (3 experiments). All data are expressed as mean ± s.e.m.

Figure 5 shows that leptin led to increased intercellular adhesion and upregulation of adhesion molecules, a, b, Human PBMC cultured for 36 hours with (a) or without (b) leptin (5 experiments), c, Two colour flow cytometric analysis of CD4⁺ T cells after 36 hours culture with allogeneic irradiated PBMC showed an increase in VLA-2 (CD49b) and ICAM-1 (CD54). VLA-2 and ICAM-1 had been dowmegulated to baseline levels by 80 hours of incubation (3 experiments). The figure shows the percentage of CD4⁺ T cells that expressed these molecules as detected by PE-linked mAbs. d, A representative flow cytometric profile showing the population of cells expressing VLA-2 after the addition of leptin. A similar profile was observed with ICAM-1.

Figure 6 shows that leptin reversed the starvation-induced immunosuppression in normal mice, a, 48 hour food deprivation caused a 28% reduction in body weight of male C57BL/6 mice. The degree of weight loss was identical in the PBS and leptin treated groups. There was no statistical difference in the mean body weights between groups at the beginning of the study or at the time of challenge or reading of the DTH response.

b, Ad libitum fed mice primed with oxazolone had a significant DTH recall response to oxazolone compared to ad libitum fed mice primed with vehicle (sham DTH). Starvation at the time of priming abrogated the DTH recall response to oxazolone. This effect was reversed by leptin replacement during starvation, to a level not statistically different to that seen in ad libitum fed mice.

All data are expressed as mean % increase in ear thickness + s.e.m. (n = 6 per group). * P < 0.0005, ** P = 0.00001, one-way analysis of variance with the Bonferroni adjustment for multiple group testing.

Figure 7 shows the effect of leptin administration during starvation on lymphoid (a,b,e,f) and non-lymphoid tissues (c,d) in C57BL/6 mice, (a) thymic weight (b) splenic weight (c) liver weight (d) kidney weight (e) thymocyte subpopulations (f) splenic subpopulations. Values represent means ± SEM. * P < 0.05 compared with ad libitum fed controls, * * P < 0.05, * * * P < 0.0001 compared with both ad libitum fed controls and lep tin-treated starved mice.

Figure 8 shows the effect of leptin administration during starvation on thymic histology in C57BL/6 mice. (a,b) ad libitum fed controls, (c,d) PBS-treated starved mice, (e,f) leptin-treated starved mice. Sections are stained with H & E and are shown at 25 X (a,c,e) and 250 X (b,d,f) magnification, c = cortex, m = medulla, a = apoptotic thymocytes.

Figure 9 shows the effect of chronic leptin administration on thymocyte subpopulations in ob/ob mice. Values represent means ± SEM. ** P < 0.05, *** P < 0.0005 compared with both ad libitum and pair-fed control mice. Figure 10 shows representative flow cytometric analyses of thymocytes stained for CD4 and CD8 from (a) wildtype, (b) ad libitum fed ob/ob, (c) pair fed ob/ob and (d) leptin-treated ob/ob mice.

Example 1: Leptin modulates the human T cell immune response and reverses starvation-induced immunosuppression in mice

Many reports suggest that nutritional status influences immune function^1"3. We have performed a series of in vitro and in vivo experiments to explore the hypothesis that leptin has immunoregulatory effects, and may therefore account for the connection between nutrition and immunity. Most immune responses are orchestrated by CD4⁺ helper T cells (Th). The strength and nature of an immune response is determined by the cytokines released by these cells¹⁷. We first tested the effect of leptin on Th responses in the context of the mixed lymphocyte reaction (MLR) resulting from the culture of T cells with major histocompatibility complex (MHC) incompatible (allogeneic) stimulator cells. The doses of leptin used in these experiments were chosen to incorporate the range of serum levels measured in humans¹⁸. Leptin induced a marked dose-dependent increase in the proliferative response of peripheral blood lymphocytes (PBL) in response to allogeneic peripheral blood mononuclear cells (PBMC) (Figure la). The alloproliferative response was augmented in a similar fashion when the experiment was repeated using highly purified CD4⁺ T cells as responders, suggesting that T cells, rather than accessory cells were the target of leptin action (Figure lb). To address directly whether it was the responder or the stimulator cells that were being affected by leptin, either responder or stimulator populations were preincubated for 12 hours with leptin and then washed extensively prior to co-culture in the absence of leptin. This showed that preincubation of the responder CD4⁺ T cells with leptin produced far greater enhancement of proliferation in the subsequent MLR than preincubation of the stimulator cells (Figure lc). The augmentation of proliferation was still seen despite the fact that the leptin was only present for 12 hours during preincubation rather than for the duration of the MLR (5 days) as in the previous experiments. Incubation of CD4⁺ T cells with leptin, in the absence of allogeneic stimulator cells, produced no increase in proliferation (data not shown), suggesting that cognate recognition by the T cell receptor (TCR) was required before leptin could have its effect.

To confirm the specificity of these effects, we compared the responses of T cells from leptin receptor-defective db/db mice with those of their littermate controls (dbl+). Several splice variants of the leptin receptor (ObR) are expressed in vivo. Although all of the receptor isoforms bind leptin, the long isofoπn of the receptor (ObRb) appears to be of prime importance in signal transduction^{12, 15}. A point mutation within the ObR gene of the diabetic (db) mice generates a new splice donor site that dramatically reduces the expression of the long isoform (ObRb) in homozygous db/db mice^12,15 and renders them resistant to the weight lowering effects of endogenous and exogenous leptin⁵. The alloproliferative response of splenocytes from homozygous db/db mice was similar to that of heterozygous dbl+ mice in the absence of leptin, consistent with reports that these cells can function normally ex vivo¹². However, the leptin-mediated augmentation of proliferation in response to allogeneic splenocytes seen with responder cells from db/+ mice was not seen with db/db responder cells, despite the higher doses of leptin used (Figure ld). Similar results were obtained when MHC class II transfected murine fibroblasts were used as allogeneic stimulators (data not shown). These results indicate that the observed enhancement of proliferation by leptin was due to a specific effect of leptin receptor signalling rather than to a non-specific mitogenic stimulus.

In agreement with previous reports, using long isoform specific primers, we were able to detect the expression of ObRb mRNA by the reverse transcriptase-polymerase chain reaction (RT-PCR) in human hypothalamus and PBMC¹⁹, but not in kidney¹⁵. Consistent with our functional data, we detected ObRb mRNA in highly purified resting human CD4⁺ T cells but not in resting monocytes (Figure 2). Taken together, these data suggest that leptin enhances T cell responses primarily by binding to its receptor on the T cells, rather than through a direct effect on the stimulator cell.

The MLR provokes a strong proliferative response by both naive and memory T cells²⁰. In time course studies, the effect of leptin in an MLR was maximal at day 7, a kinetic characteristic of a primary (naive) immune response (data not shown). We therefore investigated the possibility that leptin had differential effects on naive (CD45RA⁺) and memory (CD45RO⁺) CD4⁺ T cells. This hypothesis was supported by the observation that depletion of CD45RO⁺ rather than CD45RA⁺ cells from the responding population of PBMC in an MLR enhanced the response to leptin (Figure 3a). Proliferative responses were also determined in an MLR after pre-incubating the responder cell populations of highly purified naive or memory T cells for 12 hours with leptin and then washing the cells extensively prior to co-culture with stimulator cells. The effect of leptin was much more pronounced on the CD4+CD45RA + T cells than on CD4⁺CD45RO⁺ T cells (Figure 3b). Further evidence that leptin amplifies primary T cell responses was provided by the use of umbilical cord blood T cells (UCB), the most pure preparation of naive T cells. As shown in Figure 3c, proliferation was increased ten-fold in the presence of leptin. The reduced effect of leptin on the memory phenotype T cells in the MLR was paralleled by the finding that leptin had little effect on recall responses of previously immunised individuals to tetanus toxoid (Figure 3d), or on proliferation of a panel of established human T cell clones (data not shown).

CD4⁺ T cells (Th) determine the nature of immune effector function according to the pattern of cytokines that they secrete¹⁷. This cell population can be divided into two subsets, Thl cells that secrete pro- inflammatory cytokines (such as IL-2 and IFN-γ) and Th2 cells that secrete cytokines with predominantly regulatory functions (such as IL-4). Several factors have been identified that polarise Th cells into these subsets¹⁷. The possibility that leptin influenced Th cytokine production was examined by the measurement of IL-2, IFN-γ and IL-4 in MLR experiments (Figure 4). Leptin led to an increase in IL-2 production by all the T cell populations studied except for the CD45RO⁺ cells which, despite a lack of proliferation and IL-2 production, showed a substantial increase in IFN-γ secretion (Figure 4a, b). Only the CD45RO⁺ population secreted measurable amounts of IL-4, and this was completely inhibited by the addition of leptin (Figure 4c). These data suggest that leptin may bias T cell responses towards a pro-inflammatory phenotype (Thl).

Proinflammatory cytokines such as IFN-γ can upregulate adhesion molecules²¹ and certain accessory molecules can bias a T cell response towards the production of specific cytokines²². Morphological examination of cultures of human PBMC (Figure 5a, b) or murine splenocytes (data not shown) showed that leptin induced marked cellular clumping at 36 hours by both human and dbl+ cells, but not by db/db cells. Immunofluourescent microscopy demonstrated that the cells in these clumps were CD4⁺ T cells and CD14⁺ monocytes (data not shown), suggesting that increased intercellular adhesion may underlie some of the effects of leptin by bringing responder Th cells into close proximity with antigen presenting cells. Indeed, in an MLR, leptin induced the expression of the adhesion molecules ICAM-1 (CD54) and VLA-2 (CD49b) on CD4⁺ T cells (Figure 5c), but had no effect on the expression of other molecules that mediate adhesion (CD49a, c, d, f, CD50 or CD62L). The proportion of cells induced to express ICAM-1 and VLA-2 is in keeping with the predicted proportion of alloreactive T cells in an unselected population (Figure 5d). However, these accessory molecules are expressed on T cells after activation in certain circumstances and their upregulation may simply parallel the augmented T cell response seen in the presence of leptin. These molecules were not expressed by T cells in an MLR in the absence of leptin at any time point tested.

Nutritional deprivation affects immune function and also rapidly reduces circulating leptin levels in mice and humans^8,9. In order to determine the in vivo relevance of our in vitro findings, we investigated the capacity of exogenous leptin replacement to reverse the inhibitory effects of starvation on T cell priming as measured by a delayed-type hypersensitivity (DTH) response. Mice were primed by application of oxazolone to flank skin and challenged five days later by painting oxazolone on the ear. Starvation of mice for 48 hours led to a 69% reduction in the mean DTH response, as measured by ear swelling. This inhibition was completely reversed by the twice daily injection of leptin during the period of starvation (Figure 6).

Until now, leptin has primarily been implicated in the regulation of body weight⁶ and reproductive function⁹. However recent reports of its effect on the pancreas²³ and haemopoiesis^24,25, together with the detection of peripherally distributed receptors¹², suggest a wider role. It is well recognised that starvation is associated with a higher frequency of infectious diseases¹⁶. In this Example, we show that leptin is able to modulate specific aspects of T cell function, including intercellular adhesion, proliferation, and cytokine production, with differential effects on naive and memory T cells. In addition, the exogenous administration of leptin during acute starvation was able to reverse the fasting-induced immune deficiency in mice. The extent to which this observation is mediated by a direct action of leptin on the CD4⁺ T cell rather than an indirect effect on the hypothalamic-piruitary-adrenal axis remains to be determined. However, this regime of leptin repletion in mice has been shown to only partially suppress the fasting-induced rise in corticosterone and is without effect on the starvation-induced fall in plasma insulin and glucose levels⁹.

These findings may help to explain the occurrence of immune dysfunction in humans with low body weight^1"3 and the observation that caloric restriction is able to abrogate autoimmune disease in several animal models²⁶. It seems increasingly likely that a falling leptin concentration acts as a peripheral signal of starvation that serves to conserve energy in the face of limited reserves⁹. In this situation, energy must be preserved for vital functions such as central nervous system metabolism, whilst 'luxury items' not immediately required, such as the reproductive axis and a finely tuned cognate immune system are inhibited. The data presented here demonstrate that adipose tissue modulates cognate T cell immune function and suggest that leptin may be the key link between nutritional status and an optimal immune response.

Methods

Reagents. The human and murine recombinant leptin was purchased from R&D, Minneapolis, MN and from Peprotech, London, GB. The recombinant leptin used was > 97% pure as judged by SDS-PAGE analysis and contained < 0.1 ng/mg LPS as determined by the Limulus amebocyte lysate method. Addition of LPS in these concentrations had no effect on the observed responses (data not shown). The tetanus toxoid was from Evans Medical Ltd, Leatherhead, UK. The following mAbs were used for purifying the cells for the proliferation and cytokine assays: L243 (anti-HLA DR; American Type Culture Collection (ATCC), Rockville, MD), Leu-19 (anti-CD56; Becton Dickinson), UCHT4 (anti-CD8; ATCC), BU12 (anti-CD 19; ATCC), UCHM1 (anti-CD 14; ATCC), UCHM1 (anti-CD14; ATCC), Leu-llb (anti-CD 16; ATCC), Leu-M9 (anti-CD33; ATCC), SN130 (anti-CD45RA; gift from G. Janossy, Royal Free Hospital, London), UCHL1 (anti-CD45RO; gift from P. Beverley, ICRF). The following directly conjugated mAbs were used for flow cytometric analysis: anti-DR, anti-CD3, anti-CD4, anti-CDllb, anti-CD71 (Sigma, Dorset, GB); anti-CD2, anti-CD62L (Pharmingen, San Diego, CA); anti-CD49a-f, anti-CD50, anti-CD54, anti-CD58, (Serotec, Oxford, GB). Preparation of cells. The blood was obtained from healthy adult volunteers or from the umbilical cords (UCB) of neonates (Queen Charlotte's Hospital, London, GB). Peripheral-blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque (Pharmacia, Sweden) density centrifugation. PBMC were depleted of adherent cells by two 45 minute rounds of adherence to plastic on tissue culture dishes (Falcon) at 37°C. The CD4⁺, CD4⁺CD45RA⁺ or CD4⁺CD45RO⁺ T cell subsets were prepared from the nonadherent population by immunomagnetic negative selection as previously described²⁷. The purity of the separated T cell subsets was assessed by flow-cytometric analysis (results not shown) using a FACScan (Becton Dickinson, Mountain View, CA) and was always > 98% . All the purified T cells were unresponsive to 72 hours culture with PHA (2μg/ml), confirming their functional purity. For the depletion experiments, PBMCs, were depleted by two rounds of immunomagnetic negative selection with saturating concentration of SN130 or UCHL1 mAbs. Monocytes were purified by taking adherent cells from plastic and depleting with anti-CD3 and anti-CD 19 mAbs. After depletion, the purity was assessed by flow cytometry ( > 98% free of contaminating cells). Mouse splenocytes from the various mouse strains used were prepared as previously described²⁸.

Mice. The leptin receptor mutant db/db mouse (C57BL/Ks-db) (H-2^d), the heterozygous dbl⁺ (C57BL/Ks) (H-2^d) and the C57BL/6 (H-2^b) were from Harlan Labs, Bicester, Oxford, UK, and were used between 6-8 weeks of age.

For the in vivo experiments, 6 week old male C57BL/6 mice were housed in pairs under controlled temperature (21-23°C) and a 12 hour light/dark cycle (lights on 07.00 hours). Animals were handled twice daily at 09.00 h and 18.00 hours and food intake and body weight were recorded daily. Animals comprised four groups (n = 6 per group). Two groups were allowed continuous access to laboratory chow and two groups were starved for 48 hours during which they received twice daily intraperitoneal injections of either PBS or recombinant murine leptin (lμg/g initial body weight). Before and after the period of starvation mice were allowed to feed freely. Cellular immune function was assessed by the delayed-type hypersensitivity (DTH) response. Measurement of the DTH response was performed essentially as described²⁹. Briefly, mice were primed on two consecutive days by application of 50μl 3 % oxazolone in acetone/olive oil (4:1 vol/vol) to the shaved flank. Five days later, mice were challenged with 10 μl 1 % oxazolone applied to the ear. Ear thickness was measured at 24 hours using an electronic micrometer. Mice undergoing 'sham DTH' were primed with 50 μl vehicle. Control challenge with 10 μl vehicle was performed on the contralateral ear of all mice.

Proliferation assays. The human mixed lymphocyte reactions (MLRs) were performed using various purified cell populations as responders (10⁵ cells/well) and the same number of allogeneic irradiated (30 Gy) PBMC or monocytes unless otherwise indicated, in 96- well plates (Nunc) in triplicate for 5 days in a final volume of 200 μl. All proliferation experiments were performed in RPMI medium (Life Technologies Ltd., Paisley, Scotland) supplemented with 10% heat-inactivated FCS (Globepharm, GB) or human AB serum (North London BTS, Colindale, GB), 2mM glutamine, lmM sodium pyruvate, 0.1 mM non-essential amino acids, 50 IU/ml penicillin, 50 μg/ml streptomycin. The cells purified for the preincubation experiments were incubated for 12 hrs in the presence or absence of leptin (10⁸ M), washed four times, counted and plated in triplicate in 96- well plates with 10⁵ irradiated allogeneic PBMC or monocytes for 5 days. The cells were then pulsed with 0.5 μCi 3H- Thymidine (TdR) (Amersham) for an additional 12 hrs and harvested onto glass fibre filters (Wallac). Proliferation was measured as 3H-TdR incorporation by liquid scintillation spectroscopy. The antigen recall response was performed with PBMC (10⁵ cells/well) with different doses of tetanus toxoid and harvested after 5 days.

The mouse MLRs were performed as previously described²⁸.

Flow cytometric analysis. Two-colour flow cytometric analysis was performed using FITC-conjugated anti-CD4 mAb as the first colour and a variety of PE-conjugated mAbs as the second colour in detecting all the molecules described above. Analysis of cells in MLRs was performed at 0, 12, 36, 48 and 80 hours, using purified CD4⁺ (10⁵ cells/well) as responders and as stimulators the same number of irradiated (30 Gy) allogeneic PBMC. Flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA).

Cytokine analysis. The production of IL-2, IL-4 and IFN-gamma was measured by ELISA (Amersham Life Science, Buckinghamshire, GB) in supernatants collected after 72 hrs using irradiated allogeneic PBMC as stimulators, in the presence or absence of leptin (10^"8M).

RT-PCR and Southern Blotting. Total cellular RNA was extracted using RNAzol reagent (AMS Biotechnology, Oxon, UK). 5μg of total RNA was reverse transcribed in a 20μl reaction volume using oligo(deoxythymidine)^12"18 primer, as previously described³⁰. Primer selection and PCR amplification were performed as described¹⁹ to generate a product specific for the long form (ObRb) of the human leptin receptor (nucleotide positions 2831-3719). The first round primers were: 5'- AAGATGTTCCGAACCCCAAG-3' (forward) and 5'

C A AATTTGGACTCTGGTTTCT-3 '(reverse). The second round primers were: 5'-AATTGTTCCTGGGCACAAGG-3' (forward) and 5'- CACAAATCTGAAGGTTTCTTC-3' (reverse). Agarose gel electrophoresis in the presence of ethidium bromide confirmed the presence of the expected 888 bp PCR product the nature of which was further verified by Southern blot analysis³⁰ using an oligonucleotide probe 5'-TATCAGATCAGCATCCCAACAT-3' (nucleotide positions 3309- 3330 on human leptin receptor cDNA). cDNA quality was determined by the relative amplification of the human β-actin gene. The β-actin primers were 5'-GTGGGGCGCCCCAGGCACCA-3' (forward) and 5' GAAATCGTGCGTGACATT AAGGAG-3' (reverse) which generated a 540 bp product. The CD4⁺ T cells used in this experiment were extensively purified and were > 99.5% pure on flow cytometric staining and unresponsive to PHA. The remaining 0.5% were negative for B cell, monocyte, NK, and CD8 surface markers.

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Example 2: Leptin Protects Mice from Starvation-Induced Lymphoid Atrophy and Increases Thymic Cellularity in ob/ob Mice

Thymic atrophy is a prominent feature of malnutrition. Forty-eight hours starvation of normal mice reduced the total thymocyte count to 13 % of that observed in freely fed controls, predominantly due to a diminution in the CD4+CD8+ thymocyte subpopulation. Histologically, starvation caused a profound depletion in cortical thymocytes leading to loss of corticomedullary differentiation. Prevention of the fasting induced fall in the level of the adipocyte-derived hormone leptin, by administration of erogenous recombinant leptin, protected mice from these starvation- induced thymic changes. The ob/ob mouse, which is unable to produce functional leptin due to a mutation in the obese gene, has impaired cellular immunity together with a marked reduction in the size and cellularity of the thymus. We found that the ratio of CD4+CD8+ (cortical) to CD4- CD8- (precursor) thymocytes was 4-fold lower in ob/ob compared with wildtype mice. Peripheral administration of recombinant leptin to ob/ob mice caused an 18-fold increase in thymic cellularity and raised the CD4 + CD8 + : CD4-CD8- ratio. In contrast, a comparable weight loss in pair-fed PBS-treated ob/ob mice had no impact on thymocyte number. These data indicate that the reduced circulating leptin concentration is pivotal in the pathogenesis of starvation- induced lymphoid atrophy. Materials and Methods

Mice.- 10 week old male C57BL/6 wildtype and ob/ob mice (Harlan, Oxford, UK) were housed in pairs at 22-23°C with a 12 hour dark/light cycle (lights on at 07:00 hours).

Starvation experiment: Mice comprised three groups («=8 per group). There was no significant difference in body weight between groups of mice at the start of the experiment (21.6 ± 0.4, 21.7 ± 0.5, 21.4 ± 0.5g; P = NS). One group was allowed ad libitum access to laboratory chow and received intraperitoneal (ip) injections of 0.2ml PBS at 09:00 and 18:00 hours twice daily for two days. Two groups of mice were deprived of chow for 48 hours and received ip injections of either 0.2ml PBS or recombinant murine leptin (1 μg/g initial body weight) at 09:00 and 18:00 hours. All mice were allowed continuous access to water. This experimental paradigm of leptin administration during starvation has been shown to achieve circulating leptin concentrations 6 and 12 hours following injection similar to that of ad libitum fed control mice and has been considered as replacement therapy (4,6,17). At the end of 48 hours, mice were killed by CO₂ inhalation and blood was collected by terminal cardiac puncture between 09.00 and 10.00 hours, immediately centrifuged and the plasma was separated and stored at -20°C until assayed for glucose, insulin and corticosterone.

Ob/ob mice. - Mice comprised three groups (n=6 per group). One group was allowed to feed ad libitum, the second group was injected with recombinant murine leptin (1 μg/g initial body weight twice daily ip) and the third group was pair-fed to the food intake of the leptin-treated mice and received twice daily injections of PBS. All mice were weighed and their food intake recorded daily. On the morning following 10 days of leptin administration, mice were killed and blood collected as above. All the experiments were performed according to British Home Office regulations.

Assays: Glucose was measured by the glucose oxidase method (YSI 2300 glucose analyser; YSI Bloanalytical Products, Yellow Springs, OH). Plasma insulin was measured by RIA as previously described (23). Plasma corticosterone concentration was measured using a commercially available RIA (ICN Biomedicals, Inc. Costa Mesa, CA).

Organ weight /cellularity: Organs were removed from euthanased mice and liver and kidney weights were determined to the nearest O.Olg. Spleens were removed and weighed to the nearest O. lmg. A single cell suspension of splenocytes was prepared using the blunt end of a syringe and passage through nylon gauze. Red blood cells were lysed with ammonium chloride lysing buffer. All thymic tissue was carefully removed using fine forceps after exposure of the chest cavity and weighed to the nearest O. lmg. Thymic weight was not determined in the ob/ob mice as it was not possible to completely dissect this organ free of its surrounding connective tissue. A single cell suspension of thymocytes was prepared by teasing the thymuses with sterile needles on a petri dish. Red cells were lysed with ammonium chloride lysing buffer. Splenocyte and thymocyte numbers were determined by counting cells three times from different areas of a hemocytometer and the mean cell counts calculated. Cell subpopulations were analysed by two-colour flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA) with the appropriate FITC or PE conjugated purified monoclonal antibodies (anti-CD4, anti- CD8; Pharmingen, San Diego, CA).

Histology: The acute starvation experiment was repeated using the protocol above in age-matched mice. On this occasion intact thymuses were removed, formalin-fixed, paraffin-embedded, sectioned and stained with hematoxylin and eosin (H & E) for histological evaluation of thymic architecture.

Statistical analyses: Analyses were performed using one-way ANOVA with Tukey post hoc testing for normally distributed data or by Kruskal- Wallis analysis of variance for skewed data. Results are expressed as mean ± SEM. P < 0.05 was considered to be statistically significant.

Results

Acute starvation led to a disproportionate fall in lymphoid mass Forty-eight hours starvation of normal male mice reduced mean body weight by 20% in both PBS and leptin-treated mice (17.5 ± 0.4 vs. 17.0 ± 0.3g; P = NS). The greatest effect of acute starvation was on the thymus (Fig. 7). Mice treated with leptin during the period of starvation were completely protected against the profound thymic atrophy induced by the 48 hour fast (Fig. 7a). Although not as dramatic as the changes observed in the thymus, starvation for 48 hours also caused a significant reduction in splenic weight. The effect of starvation on splenic atrophy was significantly blunted when exogenous leptin was administered during the period of starvation (Fig. 7b). In contrast to the effect of leptin on lymphoid tissue, leptin administration during fasting had no impact on non-lymphoid tissue mass (Fig 7c, d).

The effects of acute starvation on thymocyte subpopulations were reversed by leptin

Acute starvation caused a dramatic fall in the mean total thymocyte count to 13 % of that observed in control mice (Fig 7e). This reduction in total thymic cellularity was mirrored by a reduction in the absolute numbers of all thymocyte subsets examined. The greatest effect of fasting was seen in the double positive CD4+CD8+ thymocyte subpopulation. Starvation caused a reduction in both the absolute cell number (Fig 7e) and also the overall relative proportion of CD4+CD8+ thymocytes (66.0 ± 1.26%) compared with that observed in ad libitum fed mice (76.2 + 1.32%; P < 0. 001). Although acute starvation caused a relative increase in the overall percentage of single positive CD4+CD8- thymocytes (17.0 ± 0.7% (fasted, PBS) vs. 11.1 ± 0.69% (fed); P < 0.005), there was still a significant reduction in absolute numbers of CD4+ CD8- thymocytes in the PBS-treated starved group compared with the ad libitum fed controls. Leptin was able to completely protect against these starvation-induced changes in both thymocyte number (Fig 7e) and subpopulation proportions (%CD4+CD8+ thymocytes: 76.6 ± 1.92 (fasted, leptin) vs. 76.2 ± 1.32 (fed); %CD4+CD8- thymocytes: 10.8 ± 1.39 (fasted, leptin) vs. 11.1 _+ 0.69 (fed); P = NS). The relative proportions of CD4-CD8- or CD4-CD8+ thymocytes were not significantly affected by starvation or leptin treatment. Leptin spared splenic T-cells from the effects of acute starvation Forty-eight hour starvation resulted in a 42 % reduction in the total number of splenocytes (Fig If) . The absolute number of splenocytes negative for both CD4 and CD8 (non T-cells) was particularly reduced by acute starvation, although leptin was only able to partially prevent the fasting- induced changes in this subpopulation. The greatest effect of leptin was seen in the CD4+ splenic T-cell subpopulation. Starvation reduced the number of CD4+ splenocytes to 56% that of fed controls, an effect that was completely abrogated by the administration of exogenous leptin. A similar trend was seen in the CD8 + subset (Fig 7f).

Leptin protected against the starvation-induced loss of cortical thymocytes Representative histological sections of thymuses from each group are shown in Figure 8. The normal corticomedullary differentiation of the thymus (Fig. 8a,b) was lost following starvation, reflecting a marked reduction in the number of cortical thymocytes with relative preservation of the medullary architecture (Fig. 8c). In contrast, the thymuses from starved mice treated with exogenous leptin during starvation retained corticomedullary differentiation and cortical thymocyte density (Fig. 8e,f). In the PBS-treated starved thymuses, there were abundant small, darkly stained apoptotic thymocytes visible throughout the remaining cortex (Fig. 8d). Although occasional apoptotic thymocytes were seen in leptin-treated starved thymuses, they were far less prevalent than in thymuses from the PBS-treated starvation group.

In vitro, leptin may reduce apoptosis of thymocytes. The starvation-induced changes in plasma insulin, glucose and corticosterone were not significantly affected by leptin Starvation of both PBS and leptin-treated mice caused a similar significant reduction in both plasma glucose (6.3 ± 0.7 (fasted, PBS) and 5.1 + 0.7 (fasted, leptin) mmol liter^"1; P=NS, vs. 14.6 ± 1.5 mmol liter^"1 (fed) ; P < 0.01) and insulin concentrations (30.1 ± 12.5 (fasted, PBS) and 41.5 ± 16.6 (fasted, leptin) pmol liter^"1; P=NS, vs. 99.0 ± 12.4 pmol liter^"1 (fed); P < 0.01). Fasting was associated with a significant increase in plasma corticosterone concentrations (85.6 ± 13.5 (fed) vs. 221.1 ± 53.5 (fasted, PBS) ng/ml; P < 0.05). Although leptin treatment during fasting somewhat blunted the starvation-induced rise in plasma corticosterone, this was not statistically significant (221.1 ± 53.5 (fasted, PBS) vs. 178.2 ± 33.5 (fasted, leptin) ng/ml; P = NS).

The thymus of the obese (ob/ob) C57BL/6 mouse is markedly hypocellular Details of the organ weight and cellularity in 10 week old C57BL/6 ob/ob mice and age-matched wildtype controls are shown in Table 1. As expected, body and liver weights were significantly greater in the ob/ob mice in comparison with the wildtype mice. Splenic weights significantly were significantly lower in the ob/ob mice compared with the wildtype controls, although total splenocyte number was similar. Indeed, there was no significant difference in the relative proportion of the different splenocyte subpopulations examined between ob/ob and wildtype. In contrast, thymic cellularity was dramatically reduced in the ob/ob mice in comparison with wildtype mice with a significant alteration in the relative proportions of the examined thymocyte subpopulations between the two groups. Ob/ob mice had a significantly lower percentage of CD4 + CD8 + thymocytes and significantly higher percentage of CD4-CD8- and CD4+CD8- thymocytes compared with wildtype mice. Indeed, there was a greater than four-fold reduction in the ratio of CD4+CD8+ to CD4- CD8- thymocytes in the ob/ob mice compared with wildtype (Table 1).

Leptin administration dramatically increased thymic cellularity in ob/ob mice

The effect of administration of recombinant leptin for 10 days on organ weight, cellularity and metabolic parameters in ob/ob mice are shown in Table 2. Leptin administration led to a similar reduction in body weight and liver mass in both food-restricted and leptin-treated mice. Although splenic weight was significantly higher in leptin-treated mice than both ad libitum and food restricted controls, there was no statistical difference when total splenocyte numbers or splenocyte subpopulations were compared between groups. As with acute starvation of normal mice, leptin treatment of ob/ob mice had a considerable impact on the thymus. Whereas food restriction alone had no significant effect on total thymocyte count, leptin treatment in ob/ob mice caused an 18-fold increase in total thymic cellularity (Fig. 9). This leptin- induced increase in thymocyte number was seen in all examined thymocyte subpopulations. The greatest increment, however, was found in the number of CD4+CD8 + thymocytes, with a relative reduction in the number of CD4-CD8- thymocytes. Indeed, there was a three-fold increase in the proportion of CD4+CD8+ to CD4-CD8- thymocytes following chronic leptin administration (10.2 + 1.78) whilst this ratio remained similar in the pair- fed and ad libitum fed mice (3.93 + 1.57 and 3.39 ± 1.67 respectively; R=NS). Representative flow cytometric analyses with CD4 and CD8 staining of thymocytes from wildtype mice and ob/ob mice are shown in Fig. 10. The metabolic effects of leptin administered peripherally for 10 days to ob/ob mice are shown in Table 2. There was a similar reduction in plasma glucose concentration in the food restricted and the leptin-treated mice. Although leptin treatment reduced plasma insulin levels to a greater extent than that observed by a similar degree of food restriction and weight loss, this effect did not reach statistical significance. Following 10 days of exogenous leptin administration the mean plasma corticosterone concentration was not statistically different between any of the groups of ob/ob mice (Table 2).

Discussion

Lymphoid atrophy has long been recognised as a prominent feature of starvation in animals and man (7-11). Our findings now suggest that the reduction in plasma leptin concentration with fasting is of prime importance in its pathogenesis. Consistent with previous reports (7,9-11), the thymus underwent the most profound reduction in weight and cellularity with starvation. Whereas the mass of non-lymphoid organs (kidney and liver) were unaffected by leptin treatment during fasting, we have shown that exogenous leptin, administered only during the period of fasting, was able to completely protect against the thymic atrophy induced by acute starvation. Similar results were observed for the spleen, although the effects of both fasting and the response to exogenous leptin were less dramatic in this organ.

Thymocyte subpopulations at different stages of maturation are found in distinct parts of the thymus and can be distinguished by their expression of cell surface molecules, the most important of which are the CD4, CD8 and T-cell receptor (TCR) molecules. The greatest loss in thymocyte number with acute starvation was in the double positive (CD4+CD8+) thymocyte subpopulation. Leptin treatment during starvation completely protected against the fasting-induced loss of these immature thymocytes. As the double positive thymocyte is the major cell-type in the thymic cortex, this finding is consistent with the dramatic reduction in cortical cellularity and loss of corticomedullary differentiation observed histologically following starvation. In addition, many of the remaining cortical thymocytes in the PBS-treated starved thymuses appeared apoptotic. In contrast, in the thymuses taken from mice that had been treated with leptin during starvation, the cortex was well preserved and apoptotic thymocytes were sparse. The more mature single positive thymocytes reside in the thymic medulla. The observed increase in the overall proportion of single positive CD4+ CD8- thymocytes remaining in the PBS-treated starved thymuses is, therefore, likely to reflect the relative preservation of the thymic medulla over the cortex during starvation. However, as a small proportion of the earliest T-lineage precursors is also CD4 + CD8- in mice (24), it is feasible that an interruption of early thymocyte maturation could contribute to the observed increase in the relative proportion of this subset. The absolute number of single positive CD4+CD8- thymocytes was, nevertheless, still significantly reduced by starvation. Again, these fasting-induced changes in thymocyte subpopulation number and proportion were not seen if exogenous leptin was administered during the period of starvation.

Ob/ob mice display a number of the neuroendocrine abnormalities similar to those seen in chronic starvation (Bray, G.A et al (1990) Front. Neuroendocrinology 11: 128-18 1 ; Schwartz, M.W et al (1995) Am. J Physiol. 269:R949-57) and have evidence consistent with impaired cell- mediated immunity and lymphoid atrophy (Chandra, R.K. (1980) Am. J Clin. Nutr. 33: 13-16; Meade, C.J et al (1979) Int. Arch. Allergy Appl. Immunol. 58: 121-127) which may be analogous to that found in long- standing human undernutrition (Smythe, P.M et al (1971) Lancet II 506- 508; Chandra, R.K. (1991) Am. J. Clin. Nutr. 53:1087-1101; Pertschuk, M.J. , Crosby, L.O. , Barot, L., & Mullen, J.L. 1982. Am. J Clin. Nutr. 35:968-972).

Leptin was partially able to protect against the effect of acute starvation in reducing the total number of splenocytes in wildtype mice. However, it was only in the CD4+ splenic T-cell subpopulation that leptin was found to completely abrogate the effect of acute starvation. Splenocytes negative for both CD4 and CD8 comprise a heterogeneous population of cell types including B cells, NK cells and macrophages. Collectively, leptin treatment was only able to partially protect these cells against the effects of acute starvation. These findings would suggest that factor(s) in addition to the fasting-induced fall in leptin are responsible for the overall reduction in this splenocyte subpopulation with starvation. Whether leptin has differential effects on the survival of the individual cell types that comprise this subpopulation remains to be determined.

Like others, we found that the spleens of ob/ob mice were significantly smaller than those of age-matched wildtype mice (20,21). Although the absolute splenocyte number and relative proportions of the various splenocyte subpopulations expressing CD4 and CD8 markers tended to be lower in the ob/ob than wildtype mice, these differences were not statistically significant. Although treatment of ob/ob mice for 10 days with recombinant leptin significantly increased spleen size, it was without a significant impact on total splenocyte number or subpopulation proportions. In chronic leptin deficiency, unlike acute starvation where circulating leptin is rapidly lowered, the number of splenocytes expressing CD4 or CD8 markers appears to be relatively well maintained.

In marked contrast to our findings in the spleen, the cellularity of the thymus was considerably lower in ob/ob compared with wildtype mice. In addition, the ob/ob mice had a significantly lower percentage of double positive and higher percentage of double negative and CD4+CD8- thymocytes compared with age-matched wildtype controls. Whereas in wildtype mice where food restriction reduced thymocyte number, food restriction in ob/ob mice had no further suppressive effect on the already reduced thymic cellularity. As was the case when exogenous leptin was administered to fasted wildtype mice, leptin administration to ob/ob mice had a dramatic effect on thymic cellularity. After only 10 days thymocyte number had increased by over 18-fold in the leptin-treated ob/ob mice. This augmentation of thymocyte number was seen in all thymocyte subpopulations. However, as we observed with leptin treatment during acute starvation, this overall improvement was largely due to an increase in the double positive thymocyte number. The abnormally low ratio of double positive to double negative thymocytes we had observed in ob/ob compared with wildtype mice was also improved by chronic leptin administration. The double positive thymocytes are by far the most numerous cells in the normal thymus and any reduction in their number would certainly be expected to have a major impact on total thymic cellularity. It therefore seems likely that the effect of leptin in protecting against thymic atrophy may be due to its influence on the survival of, or entry into, this immature thymocyte subpopulation.

Starvation is accompanied by significant metabolic and endocrine changes that could potentially contribute to the observed lymphoid atrophy. However, the starvation-induced fall in plasma insulin and glucose concentrations was not significantly affected by leptin administered during 48 hours of starvation in wildtype mice, consistent with a previous report (4). As anticipated, leptin treatment of ob/ob mice was found to reduce body weight and plasma insulin and glucose concentrations (25-28). Similar reductions in these parameters were observed in both the leptin- treated and the food-restricted ob/ob mice. It therefore seems unlikely, at least in these models, that leptin' s effect on thymic cellularity is secondary to changes in insulin or glucose. Activation of the hypothalamic-pituitary- adrenal (HPA) axis is a feature of nutritional deprivation (4,19). Administration of recombinant leptin during fasting has been shown to blunt the starvation-induced rise in plasma corticosterone in mice (4). We found that leptin did not have a statistically significant effect on the fasting-induced rise in plasma corticosterone but it was able to completely reverse the effects of starvation on thymic involution. In addition, there was a marked increase in thymic cellularity following just 10 days of leptin administration to ob/ob mice over which time period plasma corticosterone concentrations were not significantly reduced by leptin treatment. The effects of glucocorticoids on the thymus are complex; intrathymic production of glucocorticoids is well established and their importance in antigen-specific thymocyte development and survival is becoming increasingly recognised (29-31). However, as glucocorticoids are also well known to have lympholytic effects (32-34) with an effect on the immature cortical thymocyte subpopulation in particular (34), it is still possible that suppression of the HPA axis may contribute to the observed lymphoprotective effect of leptin in vivo. As leptin administration has been shown to blunt the suppressive effects of fasting on the thyroid, gonadal and growth hormone axes (4,35) a potential contribution of other hormones to the lymphoprotective action of leptin in vivo cannot be excluded.

Since its first description in 1810, starvation has been recognised to cause a disproportionate depletion of lymphoid tissue compared with non- lymphoid tissue. These changes affect the thymus in particular (7-11). We have found that prevention of the starvation-induced reduction in plasma leptin concentration by exogenous administration was able to protect normal mice from this "nutritional fhymectomy". In addition, leptin administered chronically to ob/ob mice dramatically improved their marked thymic hypocellularity. Since the majority of thymocytes (over 95%) die within the thymus as a result of mechanisms designed to delete autoreactive, non-self restricted cells (12-16), the thymus is an energy- expensive organ (36). Our findings are consistent with the hypothesis that the physiological role of leptin is as a signal of starvation (4,6,17). We propose that a reduced leptin concentration is pivotal to the pathogenesis of starvation-induced lymphoid atrophy. References

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34. Ishdate, M. , & Metcalf, D. , (1963) Aust. J Exp. Biol. Med Sci. 41:637-649. 35. Carro, E., Senaris, R., Considine, R.V., Casanueva, F.F., & Dieguez, C (1997) Endocrinology 138:2203-2206. 36. George, A.J.T., & Ritter, M.A. (1996) Immunology Today 17:267- 272. Table 1. Characteristics of Lean (+/+) and Obese (ob/ob) Mice

Lean ( + / + ) Obese (ob/ob)

Body weight (g) 21.6±0.4 43.8±0.8*

Liver weight (g) 1.24±0.05 3.18+0.20*

Liver/ lOOg body weight 5.71±0.14 7.39±0.37*

Spleen weight (mg) 64.5±4.0 37.4±2.7*

Spleen/lOOmg body weight 0.30±0.02 0.09+0.01*

Total splenocyte count (xlO⁶) 33.0±2.88 26.7±3.00^§

CD4-CD8-splenocytes (% total) 66.6± 1.13 66.7+2.32^§

CD4 + splenocytes (% total) 21.7±0.40 22.1±2.21^§

CD8 + splenocytes (% total) 10.7±0.77 l l.l+0.70^§

Total thymocyte count (xlO⁶) 76.3±5.57 0.42±0.17*

CD4 +CD8+ thymocytes (% total) 76.2± 1.32 49.3±7.23^*

CD4-CD8- thymocytes (% total) 5.41±0.93 21.2+7.31^*

CD4+CD8- thymocytes (% total) 11.1±0.69 23.0±0.76^*

CD4-CD8+ thymocytes (% total) 5.16±1.49 6.51±0.71^§

Ratio CD4+ CD + : CD4-CD8- 16.7+3.21 3.39+ 1.67^*

Values represent means + SEM.

^*P<0.05, *P <0.005, *P<0.001, ^§P=NS.

Table 2. The Effect of Leptin Administration in ob/ob Mice

Ad libitum fed Food restricted Leptin-treated

Initial body weight (g) 43.4±0.9^§ 44.1 + 1.3 43.6±0.9^§

Final body weight (g) 43.0±0.9 38.4+1.0* 36.8±l.l^t§

Liver weight (g) 3.18+0.17 2.12+0.29* 1.93±0.16*^§

Liver/ lOOg body weight 7.39±0.37 5.45±0.67^* 5.22±0.20*^§

Spleen weight (mg) 37.4±2.7^§ 34.0±3.7* 56.1±5.9^*^

Spleen/ lOOg body weight 0.09+0.01^§ 0.09+0.01 0.16±0.02*¹

Splenocyte count (x 10⁶) 26.7±3.0^§ 3.17+5.9 39.6±3.2^§

Thymocyte count (x 10⁶) 0.42±0.17^§ 1.01±0.05 7.88±0.94^φ|

Plasma glucose (mmol liter¹) 36.4+2.6 14.8± 1.06* 17.1±1.4*^§

Plasma insulin (pmol liter ¹) 3701+347 1559+201* 712±169*^§

Plasma corticosterone (ng/ml) 213.1 +48.3^§ 197.4+40.6 123.0+53.8^§

Values represent means ± SEM.

^*P<0.05, *P < 0.001 compared with ad libitum fed controls. ^§P=NS, ¹P< 0.01, ^!P< 0.001 compared with food restricted controls.

Claims

1. A method of modulating an immune response in a patient in need of such modulation, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof, or an effective amount of an antagonist or of an agonist of leptin.

2. A method of enhancing an immune response in a patient in need of such enhancement, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin.

3. A method of suppressing an immune response in a patient in need of such suppression, the method comprising administering to the patient an effective amount of an antagonist of leptin.

4. A method according to Claim 1 or 2 wherein the patient has been, is or will be in a state of starvation.

5. A method according to claim 4 wherein the patient is an undernourished baby or child.

6. A method according to any one of claims 1, 2, 4 to 5 wherein the patient has a sub-normal body mass index.

7. A method according to claim 6 wherein the patient has cachexia.

8. A method according to any one of claims 1, 2, 4 to 7 wherein the patient has thymic atrophy.

9. A method according to any one of claims 1 , 2, 4 to 8 wherein the patient is over 60 years old.

10. A method according to any one of claims 1, 2, 4 to 9 wherein the patient has or is at risk of a microbial infection.

11. A method according to Claim 1, 2, 4 to 10 wherein the patient has or may develop AIDS.

12. A method according to any one of Claims 1, 2, 4 to 9 wherein the patient has been, is being or will be administered a vaccine.

13. A method according to any one of Claims 1, 2, 4 to 9 wherein the patient has been, is being or will be administered an anti-tumour vaccine.

14. A method according to Claim 1 or 3 wherein the patient has been, is being, or will be treated by allograft transplantation.

15. A method according to Claim 1 or 3 wherein the patient has or is at risk of an autoimmune disease.

16. Use of leptin or a functional derivative thereof, or an antagonist or agonist of leptin, in the manufacture of a medicament for modulating an immune response in a patient in need of such modulation.

17. A method of treating a patient with or at risk of a microbial infection the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin and an anti-microbial agent.

18. A method according to Claim 17 wherein the patient has, or is at risk of, AIDS.

19. A method of treating a patient with or at risk of cancer the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin and an anti- tumour vaccine.

20. A method of improving vaccine efficacy in a subject to be vaccinated, the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin before, during or after administration of the vaccine.

21. A method according to Claim 20 wherein the subject has been, is or will be in a state of starvation.

22. A method of treating a patient with or at risk of cachexia the method comprising administering to the patient an effective amount of leptin or a functional derivative thereof or an agonist of leptin.

23. A method of improving the prospects of a successful allograft transplantation in a patient, the method comprising administering to the patient an effective amount of a leptin antagonist before, during or after the transplantation.

24. A method of treating an autoimmune disease in a patient, the method comprising administering to the patient an effective amount of a leptin antagonist.

25. A method of treating an inflammatory condition the method comprising administering to the patient an effective amount of an antagonist of leptin.

26. A method of determining the immune levels of a subject, the method comprising determining the leptin level in the patient.

27. A pharmaceutical composition comprising, in combination but not necessarily for contemporaneous administration, leptin or a functional derivative thereof or an agonist of leptin and any one of an anti-microbial agent or an anti-tumour vaccine or a vaccine.

28. A pharmaceutical composition comprising, in combination but not necessarily for contemporaneous administration, an antagonist of leptin and an immunosuppressive agent.

29. Any novel method of treating an immune system dysfunction as herein disclosed.